Novel immunomodulatory, neuromodulatory, osteogenic and anti-osteoporotic HKUOT-S2 proteins that enhance fracture repair and inhibit osteoporosis development

Abstract

Provided are compositions comprising a HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NO: 81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NO: 81-97. Methods of osteogenesis are provided, including, for example, methods of enhancing bone repair, modulating polarization of macrophages with proinflammatory and anti-inflammatory activity and bone resorption of osteoclasts, and methods of treating inflammatory diseases, spinal cord injury, liver and kidney diseases, cardiovascular diseases, diabetes, post-menopausal syndrome, infertility, or hematopoietic diseases, or enhancing wound healing.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 371,229, filed on August 12, 2022, which is incorporated herein by reference in its entirety, including any tables, figures, or drawings. Background Art

[0003] The incidence and prevalence of bone defects is a global challenge with serious public health and socioeconomic implications for populations and health authorities. [1-3] Fracture risk increases with age-related medical conditions such as osteoporosis and bone fragility that lead to frequent fractures, chronic pain, and hospitalizations. [4] By 2050, more than half of all osteoporosis-related hip fractures worldwide (equivalent to 3.25 billion cases) are expected to occur primarily in Asia. [5] It is estimated that the U.S. government spends more than $5 billion annually to provide medical interventions to treat various fractures. [6] However, some standard fracture treatment options (such as autologous bone transplantation) have been reported to have clinical disadvantages such as postoperative donor site morbidity and microbial infection. [6] Therefore, the application of tissue engineering and cell-based therapies to fracture treatment has been suggested to minimize some of the clinical disadvantages of these autograft and allograft fracture treatments. [6] However, it is very challenging to find osteogenic stimuli that can safely trigger fracture healing with minimal side effects. Understanding the key molecular and cellular regulatory mechanisms during bone injury and regeneration is essential for tissue engineering and cell-based therapies. [7,8] .

[0004] Bone-damaging stimuli trigger cellular and molecular mechanisms that regulate the sequence of events in a well-coordinated manner to gradually restore the damaged bone integrity [9] During bone injury, blood flow to the injury site is interrupted and recruited platelets go to work to help the blood clot.

[10] Neutrophils are the first immune cells to infiltrate the site of injury to perform phagocytic and antimicrobial functions.

[11] The activity of neutrophils and other cells triggers M1 and M2 macrophage-mediated inflammatory and anti-inflammatory responses, respectively, in a sequential manner. All these complex cellular events contribute to the stimulation of mesenchymal stem cell (MSC)-osteoblast differentiation [9,12] Molecules such as bone morphogenetic protein (BMP) stimulate MSC-osteoblast differentiation during fracture repair [13-16] Therefore, the entire fracture repair process can be divided into inflammation, repair and remodeling stages. [9,17] Bone remodeling involves tightly regulated bone resorption by osteoclasts and bone formation by osteoblasts

[18] During inflammatory, repair, or remodeling processes, homeostatic imbalances between these key cellular and molecular functions may compromise physiological and structural bone integrity, thereby increasing the risk of recurrent fractures.

[18] Homeostatic imbalance between osteoclast and osteoblast activity during bone remodeling leads to bone pathologies such as osteoporosis

[19] Therefore, fracture repair induction is very challenging for both patients and orthopedic surgeons because it is difficult to establish an optimal treatment regimen to restore the physiological balance between bone resorption and bone formation.

[0005] Advances in cell-based therapy and tissue engineering have provided strong evidence for the interactions between relevant cellular components to promote fracture repair.

[20] Polarized M2 macrophages have been shown to enhance MSC-osteoblast differentiation

[21] Alternatively, MSCs have been reported to promote M2 macrophage polarization for tissue regeneration

[22] It was also demonstrated that co-culture of M1 macrophages and MSCs enhanced osteoblast biomineralization

[23] All these evidences indicate that appropriate osteogenic stimuli and conditions are required to modulate key cellular and molecular components to enhance fracture repair. Therefore, cell-based therapies exploit the interactions and homeostatic coordination between key cellular and molecular components as therapeutic targets for bone regeneration. [20,24,25] The pro-inflammatory activity of M1 macrophages predominates in ovariectomized (OVX)-induced osteoporosis mice due to the persistent stimulation of M1 macrophage activity and the reduction of anti-inflammatory activity of M2 macrophages. [26,27] As a result, osteoclast-mediated bone resorption exceeds osteoblast-mediated bone formation, resulting in poor bone integrity.

[27] Most anti-osteoporosis drugs, such as bisphosphonates, inhibit osteoclast activity.

[28] The anti-osteoporotic drug (strontium ranelate) enhances osteoblast-mediated bone formation while inhibiting osteoclast-mediated bone resorption

[29] However, long-term use of these anti-osteoporotic drugs, which inhibit osteoclast anabolism and catabolism, may impair regulatory osteoblast-osteoclast interactions. Long-term use of bisphosphonates in osteoporotic patients is associated with an increased risk of atypical femoral fractures (AFFs)

[30] In a randomized controlled trial of osteoporotic Japanese women, negative side effects such as infections and gastrointestinal and musculoskeletal complaints were reported in both teriparatide and alendronate-treated osteoporotic patients.

[31] There is a growing body of work advocating the development of interventions that promote an optimal physiological and homeostatic balance between bone resorption and bone formation mechanisms to improve bone function and structural integrity with minimal side effects.

[32] .

[0006] Diosgenin from Dioscorea spp. enhances brain function in healthy human subjects and in a mouse model of Alzheimer's disease

[33] , inhibit bacterial and clinical fungal growth

[34] , and induced MC3T3-1E cells to differentiate into osteoblasts

[35] Dioscorin from Dioscorea spp. was reported to stimulate immunomodulatory functions in RAW264.7 cells and mice

[36] , and dispo85E also induces osteogenesis

[37] Estrogen protein from Dioscorea zingiberensis, named DOI, rescues osteoporosis

[38] However, the application of these Dioscorea proteins to modulate cellular components such as macrophage polarization and MSC-osteoblast differentiation to enhance bone defect repair and the mechanisms of action have not been fully explored.

[0007] Therefore, there remains a need for effective osteogenesis methods. Summary of the invention

[0008] The present invention relates to a composition comprising a HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97. The present invention further relates to an osteogenic method, which includes, for example, enhancing bone repair. In certain embodiments, the HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97 can induce macrophage polarization and human MSC (hMSC)-derived osteoblast mineralization. In certain embodiments, HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97 can promote the differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts by increasing estrogen receptor α (ERα), estrogen receptor β (ERβ) and ALP mRNA expression. In certain embodiments, HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97 can upregulate the expression of osteogenic genes ALP, COL1A1 and RUNX2. In certain embodiments, the HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity thereto, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity thereto can modulate the focal adhesion signaling pathway (Itgb2, Il16, Hck, Keap1, Fblim1, Epha2, Git1, Actn1, Sdc4, Lims1, Tns3, Klf11, Vcl, Lcp1, Efs, Sla, Enah,Mapre2, Itgb8, Il1rl1, Ptprc, Rsu1, Inpp5e, Ubox5, Src, Cpne3, Tln1, Mtf 2. Zyx, Vasp, Parva, Tnfsf13b, Dlc1, Irf2, Bcar1, Rexo2, Senp1, Phldb2, C.S lasp2, Cbl, Zfp384, Srcin1, Fes, Mapk3, Mapk1) and AMPK inhibitors (Pparg, Tsc2). Cd36, Cp, Gys1, Cpt1c, Akt3, Rps6kb1, Rab10, Ppp2r5e, Pfkp, Cab39l, Pdpk 1, Ppp2r5b, Fasn, Pfkfb4, Creb1, Stradb, Ppp2r5a, Rab14, Creb3l1, Pck1 Ccna2, Prkaa2, Pik3r3, Rheb, Mtor, Add1, Ulk1, Prkab1, Ppp2r1b, Pik3cb, E ef2k, Cab39, Scd1, Irs2, Pik3cd, Elavl1, Pik3r1, Ppp2r2d, Acacb, Ppp2r3 a, Foxo1, Foxo3, Scd4, Prkaa1, 9430076C15Rik, Irs4, Irs1, Lep, Mlycd, Cpt 1b) PI3K-Akt inhibitors (Ccnd2, Pdgfb, Col6a1, Col1a1, Rac1, Ccne1, Tsc2); Lamb1, Cdkn1b, Gys1, Il7r, Pgf, Prlr, Epor, Tek, Epha2, Bcl2l1, Pten, Csf 1. Atf6b, Akt3, Lama4, Kitl, Sgk1, Myb, Ddit4, Mdm2, Col6a2, Rps6kb1, Pik 3cg, Itgb3, Pik3r5, Ppp2r5e, Syk, Thbs4, Ptk2, Nr4a1, Cdkn1a, Thbs2, Vegf a, Pdpk1, Mlst8, Sos1, Pdgfrb, Csf1r, Ppp2r5b, Vegfb, Itgb8, Cdk2, Hras. Il6, Pdgfa, Sgk3, Creb1, Lamc1, Ppp2r5a, Itga4, Itgav, Creb3l1, Bcl2l11 Pck1, Nras, Ngf, Cd2, Il6ra, Efna1, Pdgfc, Ccne2, Tnc, Prkaa2, Jak1, Pik3 r3, Csf3r, Hgf, Casp9, Rheb, Mtor, Pdgfra, Spp1, Ibsp, Ereg, Col1a2, Gnb2.Kras, Cd19, Fgfr2, Fgf15, Angpt2, Col4a1, Col4a2, Fgfr1, Rbl2, Lpar2, Pdgfd, Ppp2r1b, Itga11, P ik3cb, Gng11, Tnxb, Ccnd3, Sos2, Lpar3, Lpar1, Tlr4, Chad, Pik3cd, Magi2, Pik3r1, Ppp2r2d, Itga1 , Reln, Ppp2r3a, Col6a6, Phlpp1, Magi1, Col6a3, F2r, Foxo3, Prkaa1, Lamb2, 9430076C15Rik, Fgfr3, Ghr, Irs1, Bcl2, Trp53, Kdr, Mapk3, Mapk1, Gng5, Itga10) mTOR signaling pathway (Mtor, Prkaa1, Prkaa2 and Tnfα ), BMP (Pparg, Vsir, Ube2o, Fstl1, Smad7, Skil, Tbx20, Trim33, Crb2, Smad6, Nbl1, Dlx1, Fzd1, Sorl1, Sox11, Numa1) and TGF-β signaling pathway (Ltbp1, Tgfb1, Tgfbr1, Id3, Bmp7, E2f4, Rps6kb1, Id2, Id4, Zfyve16 、Fst、Bmpr1a、Bmp1、Crebbp、Bambi、Tnf、Smad7、Acvr1c、Acvr1、Fbn1、Rbl1、Smad9、Smad1、Ppp2r1b、Neo1、Smad3、Tgfbr2、Smad6、Inhbb、Tfdp1、Smurf1、Nbl1、Fmod、Ep300、Mapk3、Mapk1、Bmpr2) enriched genes. In certain embodiments, HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs:81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs:81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs:81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs:81-97 induces genes involved in bone formation, musculoskeletal system development, tissue and organ morphogenesis. 、

[0009] In certain embodiments, HKUOT-S2 with a molecular weight of 32 kDa isolated from Dioscorea opposita Thunb was characterized using silver staining, MALDI-MS, LC-MS / MS, de novo peptide and N-terminal peptide sequencing techniques.

[0010] In certain embodiments, HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity thereto, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity thereto can be administered at about 0.01 mg kg -1 About 10 mg kg -1 or about 2.18 mg kg -1 The dose is safely and effectively administered to the subject.

[0011] In certain embodiments, HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97 can enhance bone defect repair by effectively regulating cellular functions (e.g., macrophage polarization regulating pro-inflammatory and anti-inflammatory activities, bone resorption by osteoclasts, and new bone formation by osteoblasts). In certain embodiments, HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97 can enhance osteogenic molecular function by differentially stimulating osteogenic gene expression that promotes biomineralization and increases BMD to promote bone defect repair.

[0012] In certain embodiments, the HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity with SEQ ID NOs: 81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity with SEQ ID NOs: 81-97 can induce biological functions and processes, such as osteoblast and osteoclast differentiation and musculoskeletal morphogenesis and development to produce desired new bone formation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0015] Figures 1A-1J .HKUOT-S2 significantly enhanced bone defect repair in vivo. Figure 1A-1B )μCT scans showed that HKUOT-S2 treatment gradually enhanced bone defect healing. Figure 1C-1G , Figure 1J )HKUOT-S2 treatment significantly increased BV / TV, BMD, TMD, Tb.th, Tb.N and BS / TV. Figure 1H )HKUOT-S2 treatment significantly reduced Tb.Sp. Fig. 1I ) HKUOT-S2 treatment had no significant effect on BS / BV. Statistical analysis was performed using Prism 5 software using one-way ANOVA followed by Turkey test post hoc comparison for normal distribution and Kruskal-Wallis post hoc comparison for nonparametric tests. Values ​​are shown as mean ± SEM, n = 5. X, 2X and 4X = 1.09 mgKg -1 , 2.18mgKg -1 and 4.36mgKg -1 (4X) HKUOT-S2 were treated respectively. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 were considered significant.

[0016] Figure 2A-2L .HKUOT-S2 significantly enhanced bone defect repair in vivo. Figure 2A-2B ) Fluorescent dye labeling showed that HKUOT-S2 treatment enhanced new bone formation. Figure 2C-2E )H&E and Giemsa Masson-Goldner trichrome staining showed that HKUOT-S2 treatment enhanced bone mineralization and fracture repair. Figure 2F-2G )2.18 and 4.36 mg kg -1 HKUOT-S2 significantly reduced TRAP+ cells in bone defect sites. Figure 2H-2I )2.18 and 4.36 mg kg -1 HKUOT-S2 treatment reduced TRAP+ cells but increased ALP+ cells at the growth plate. Figure 2J ) Low magnification (X900) TEM images showed normal cell morphology in all experimental groups. Figure 2K ) High-magnification TEM images (X5900) showed that in the HKUOT-S2-treated group, osteoblasts had normal ultrastructure and distinctly euchromatic nuclei. Figure 2L) High-magnification TEM images (X5900) showed that osteoclasts in the HKUOT-S2-treated group had normal ultrastructure and distinctly heterochromatic nuclei. Statistical analysis using Prism 5 software was performed using one-way ANOVA followed by Turkey test post hoc comparisons for normal distribution and Kruskal-Wallis post hoc comparisons for nonparametric tests. The values ​​are shown as mean ± SEM, X, 2X, and 4X = 1.09 mg / kg -1 , 2.18mgKg -1 and 4.36mgKg -1 (4X) HKUOT-S2 were treated separately. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 were considered significant. Histological image magnification = X20

[0017] Figures 3A-3H HKUOT-S2 significantly increased ALP and OCN levels to enhance bone defect repair. Figures 3A-3D )HKUOT-S2 treatment significantly increased the expression of Alp, Bglap1, Bglap1 and Runx2 in the defective femur. Figure 3E-3H ) HKUOT-S2 treatment significantly increased BALP and OCN protein levels in serum and bone lysates in vivo. Statistical analysis was performed using Prism 5 software using one-way ANOVA followed by Turkey test post hoc comparisons for normal distribution and Kruskal-Wallis post hoc comparisons for nonparametric tests. The values ​​are shown as mean ± SEM, X, 2X and 4X = 1.09 mg kg -1 , 2.18mgKg -1 and 4.36mgKg -1 (4X) HKUOT-S2 were treated separately. *p<0.05, **p<0.01 were considered significant

[0018] Figures 4A-4M Transcriptome analysis of HKUOT-S2-induced bone defect repair. Figure 4A ) Venn diagram of differentially expressed genes (left), differentially up-regulated genes (middle), and differentially down-regulated genes (right) induced by HKUOT-S2. Figure 4B ) Pie chart showing the distribution of differentially expressed genes induced by HKUOT-S2. Figure 4C ) Heat map of the most common differentially expressed genes induced by HKUOT-S2. The heat map was plotted based on the z-scores of the selected genes in the samples. Figure 4D Both )X and 2X HKUOT-S2 were significantly enriched in GO terms related to neutrophil differentiation, development, and function. Figure 4EBoth )X and 2X HKUOT-S2 were significantly enriched in GO terms related to monocyte and macrophage differentiation functions. Figure 4F ) All HKUOT-S2 treatments significantly enriched GO terms related to macrophage fusion. Figure 4G ) All HKUOT-S2 treatments significantly enriched GO terms related to osteoclast differentiation, development, and fusion. Figure 4H )X and 2X HKUOT-S2 treatments significantly enriched GO terms related to stem cell development, differentiation, and function. Fig. 4I ) All HKUOT-S2 treatments significantly enriched GO terms related to osteoblast proliferation, differentiation, and function. Figure 4J ) Significantly enriched KEGG pathways common to all HKUOT-S2 treated groups. Figure 4K )X and 2X HKUOT-S2 were significantly enriched in KEGG pathways. Figure 4L ) Significantly enriched GO terms common to all HKUOT-S2 treatments. Figure 4M )X and 2XHKUOT-S2-induced significantly enriched KEGG pathways related to the regulation of mTOR complexes and signaling pathways. Statistical analysis performed using Prism 5 software was performed using one-way ANOVA followed by Turkey test post hoc comparisons for normal distribution and Kruskal-Wallis post hoc comparisons for nonparametric tests. Values ​​are shown as mean ± SEM, n = 3. X, 2X and 4X = 1.09 mgKg -1 , 2.18mgKg -1 and 4.36mgKg -1 (4X) HKUOT-S2 were treated separately. *p<0.05 was considered significant

[0019] Figures 5A-5L .Verification of genes related to AMPK, mTOR and BMP signaling pathways. Figures 5A-5H )HKUOT-S2 treatment significantly increased Prkaa1, Prkaa2, Prkab1, Prkab2, Prkag1, Prkag2, Prkag3 and Mtor. Figures 5I-5L ) HKUOT-S2 treatment significantly increased BMP signaling-related genes such as Bmp2, Bmp7, and Bmpr2, but not Tgfβr2. Statistical analysis using Prism5 software was performed using one-way ANOVA followed by Turkey test post hoc comparison for normal distribution and Kruskal-Wallis post hoc comparison for nonparametric tests. The values ​​are shown as mean ± SEM, X, 2X, and 4X = 1.09 mg / kg -1 , 2.18mgKg -1 and 4.36mgKg-1 (4X) HKUOT-S2 were treated separately. *p<0.05, **p<0.01 were considered significant

[0020] Figures 6A-6L HKUOT-S2 enhanced the polarization and differentiation of hMSC-osteoblasts and macrophages. Fig. 6A )HKUOT-S2 enhances osteoblast differentiation. Figure 6B-6C )HKUOT-S2 enhanced M1 macrophage polarization. Figure 6D-6E )HKUOT-S2 enhanced M2 macrophage polarization. Fig. 6F ) HKUOT-S2 increased the double-stained CD206 and MGL-1 positive M2 macrophages. HKUOT-S2 increased the CD206 positive M1 macrophages. Figure 6G ) HKUOT-S2 increased the protein level of ARG-1, a marker of M2 macrophages, in M1 macrophages. Figure 6H ) HKUOT-S2 increased the anti-inflammatory gene AMPKα1 in M1 macrophages. Fig.6I ) HKUOT-S2 decreased CCL17, CCL22, CXCL16, GDF-15, OPN in M1 macrophage CM, but increased CD14 and CD54 cytokines. HKUOT-S2 also increased G-CSF in M2 macrophage CM, but decreased GDF-15 cytokine. Figures 6J-6K ) HKUOT-S2-treated M1 macrophage CM significantly increased osteoblast biomineralization. Figure 6J , 6L ) HKUOT-S2 treated M2 macrophage CM significantly increased osteoblast biomineralization. Statistical analysis performed using Prism 5 software was performed using one-way ANOVA followed by Turkey test post hoc comparisons for normal distribution and Kruskal-Wallis post hoc comparisons for nonparametric tests. Values ​​are shown as mean ± SEM, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 were considered significant. Diff med = differentiation medium, CM = conditioned medium.

[0021] Figures 7A-7O .HKUOT-S2 regulates the mTOR1 / 4E-BP1 / AKT1 axis to promote osteogenesis. Fig. 7A ) XL388-induced mTOR inhibition had no effect on ALP expression. Figures 7B-7F )HKUOT-S2 blocks XL388-induced mTOR inhibition to increase RUNX2, mTOR1, 4E-BP1, AKT1, and S6K1 expression, thereby enhancing osteogenesis. Figure 7G-7M) HKUOT-S2 blocked XL388-induced mTOR inhibition to increase the phosphorylation of mTOR1 and 4E-BP1 proteins. Figure 7N ) HKUOT-S2 treatment also increased total AKT1 protein levels. Fig.7O ) proposed a mechanism by which HKUOT-S2 regulates the mTOR1 / 4E-BP1 / AKT1 axis to promote osteogenesis. Values ​​are shown as mean ± SEM, X-125 = 125 nM XL388, (n = 4). *p < 0.05 was considered significant under one-way ANOVA.

[0022] Figures 8A-8H .HKUOT-S2 enhances neuronal differentiation and maturation in vivo. Fig. 8A , Figure 8C , Fig. 8E , Fig.8F , Figure 8H )HKUOT-S2 treatment significantly increased the expression of neuronal differentiation and maturation genes Ngn1, TH, Gap43, Map2 and Eno2. Figure 8B , Fig.8D , Figure 8G )HKUOT-S2 treatment had no significant effect on the expression of neuronal differentiation and maturation genes Ngn2, Neurod1 and Tuj1.

[0023] Figures 9A-9F HKUOT-S2 significantly enriched pathways and biological processes associated with the growth, development, and maturation of neurons, nerves, and the brain. Fig. 9A ) HKUOT-S2 treatment significantly enriched GO terms related to axon, cell body, and dendrite development. Fig. 9B ) Transcriptome data showed that HKUOT-S2 treatment significantly enriched GO terms related to axon development, regeneration, and injury repair in the bone defect model. Figures 9C-9D ) HKUOT-S2 treatment significantly enriched GO terms related to neuronal growth, development, differentiation, and function. Fig.9E ) HKUOT-S2 treatment significantly enriched GO terms related to neural morphogenesis and structural organization. Fig.9F ) HKUOT-S2 treatment significantly enriched GO terms related to brain development.

[0024] All statistical analyses performed using Prism 5 software were performed using one-way ANOVA followed by Turkey test post hoc comparisons for normal distribution and Kruskal-Wallis post hoc comparisons for nonparametric tests. Values ​​are shown as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered significant.

[0025] Figures 10A-10H .HKUOT-S2 enhances the expression and function of neuropeptide genes. Figures 10A-10D )qPCR results showed that HKUOT-S2 treatment significantly increased the expression of Cox2, Ptges, Ep4 and Calca in the bone defect model. Fig. 10E )qPCR results showed that HKUOT-S2 had no significant effect on the expression of calcitonin receptor-like receptor gene Crlr. Figures 10F-10G ) Transcriptome data showed that HKUOT-S2 treatment significantly enriched GO terms related to some neuropeptide genes in the bone defect model. Fig. 10H ) Transcriptome data showed that HKUOT-S2 treatment significantly enriched pathways and biological processes related to neuropeptide synthesis, release, and activity in the bone defect model.

[0026] Figures 11A-11E : HKUOT-S2 treatment promotes neuronal differentiation. Fig.11A , 11B )HKUOT-S2 treatment had no effect on the viability of Neuro2A cells. Figures 11C-11E )HKUOT-S2 treatment promotes Neuro2A-neuronal cell differentiation.

[0027] Figures 12A-12H HKUOT-S2 prevents the development of OVX-induced osteoporosis in vivo. Fig. 12A ) Transcriptome data showed that HKUOT-S2 treatment significantly enriched GO terms related to the regulation of estrogen receptor binding and signaling pathways. Fig. 12B )HKUOT-S2 treatment prevented bone loss in OVX mice. Fig. 12C )HKUOT-S2 treatment increased bone volume in OVX mice. Fig.12D ) HKUOT-S2 treatment reduced the bone surface-to-volume ratio in OVX mice. Fig.12E )HKUOT-S2 treatment increased bone surface density in OVX mice. Fig.12F ) HKUOT-S2 treatment increased trabecular bone thickness in OVX mice. Figure 12G ) HKUOT-S2 treatment increased the number of trabecular bone in OVX mice. Fig.12H ) HKUOT-S2 treatment reduced trabecular bone separation in OVX mice. All statistical analyses performed using Prism 5 software were performed using one-way ANOVA followed by Turkey test post hoc comparisons for normal distribution and Kruskal-Wallis post hoc comparisons for nonparametric tests. The values ​​are shown as mean ± SEM, n = 8, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 were considered significant

[0028] Figures 13A-13H .HKUOT-S2 inhibits the development of dexamethasone (Dex)-induced progressive osteoporosis. Fig.13A ) in wild-type mice, 1.09 and 2.18 mg kg -1 HKUOT-S2 was significantly enriched for biological processes and functions related to responses to glucocorticoid stimulation, including dexamethasone (Dex). Fig. 13B ) HKUOT-S2 inhibited bone loss in Dex-treated mice at week 4 of the experimental endpoint. Fig. 13C ) HKUOT-S2 treatment increased the bone volume in Dex-treated mice. Fig.13D ) HKUOT-S2 treatment reduced the bone surface-to-volume ratio in Dex-treated mice. Fig.13E ) HKUOT-S2 treatment increased bone surface density in Dex-treated mice. Fig.13F ) HKUOT-S2 treatment increased trabecular bone thickness in Dex-treated mice. Figure 13G ) HKUOT-S2 treatment increased the number of trabecular bone in Dex-treated mice. Fig.13H ) HKUOT-S2 treatment reduced trabecular bone separation in Dex-treated mice.

[0029] Figures 14A-14B HKUOT-S2 treatment enhanced glucose metabolism and insulin function in wild-type mice. Figures 14A-14B )1.09 and 2.18 mg kg -1 HKUOT-S2 treatment significantly enriched several signaling pathways, biological processes, and functions related to glucose metabolism and insulin function in wild-type male mice.

[0030] Figures 15A-15H HKUOT-S2 treatment increased functional pancreatic β-cell gene expression in vitro. Fig.15A )HKUOT-S2 treatment had no significant effect on INS-1E cell viability. Figures 15B-15H )HKUOT-S2 treatment significantly increased the expression of functional pancreatic β-cell genes in INS-1E cells.

[0031] Figures 16A-16B Effects of HKUOT-S2 treatment on reproduction and embryonic development in vivo. Fig.16A )1.09 and 2.18 mg / kg -1 HKUOT-S2 treatment significantly enriched biological processes and functions related to oogenesis, spermatogenesis, and reproductive processes in wild-type mice. Fig. 16B )1.09 and 2.18 mg kg -1HKUOT-S2 treatment significantly enriched biological processes and functions related to embryonic development in wild-type mice.

[0032] Figures 17A-17I .HKUOT-S2 isolation and characterization. Fig.17A ) Crude Dioscorea opposita protein extract. HKUOT-D3 protein isolated from crude Dioscorea opposita protein extract. HKUOT-P1 protein isolated from protein fraction HKUOT-D3. Fig. 17B ) HKUOT-S2 protein purified from HKUOT-P1. Fig. 17C ) Silver staining of HKUOT-S2 and HKUOT-P1 proteins in 15% SDS-PAGE. Fig.17D ) The molecular weight of HKUOT-S2 was determined by mass spectrometry. Figures 17E-17H ) HKUOT-S2 de novo peptide sequencing analysis on PEAKS Studio X-Pro. Fig.17I ) The molecular weight (MW) of HKUOT-S2 was mathematically predicted using a standard curve of calibrated Superdex 75 Increase 10 / 300GL column information and the Kav equation.

[0033] Figures 18A-18W .HKUOT-S2 has no toxic effect on mice. Fig.18A ) HKUOT-S2 did not induce hemolysis in mouse blood. Fig.18B , 18C ) HKUOT-S2 inhibits hypotonic (0.45% saline)-induced hemolysis. Fig.18D )Acute toxic effects of HKUOT-S2 body weight, liver and kidney histology and gene expression, hematocrit, and clinical biochemistry were evaluated. Fig.18E )HKUOT-S2 had no effect on body weight. Fig.18F , 18G ) HKUOT-S2 had no effect on liver and kidney histology. Figures 18H-18K ) HKUOT-S2 had no effect on liver gene expression. Figures 18L-18O ) HKUOT-S2 had no effect on renal gene expression. Figure 18P-18Q ) HKUOT-S2 treatment had no effect on hematocrit. Figure 18R-18W ) Clinical biochemistry of serum showed that HKUOT-S2 treatment had no effect on serum levels of ALP, ALT, AST, ALP / AST, creatinine and urea in vivo. One-way ANOVA was used as a statistical tool for analysis. Values ​​are shown as mean ± SEM (n = 9 per group). *p < 0.05 was considered significant. HTC = hematocrit.

[0034] Figures 19A-19F.Transcriptome analysis of HKUOT-S2-induced fracture repair. Fig.19A , 19B )1.09mg kg - 1 HKUOT-S2 significantly enriched KEGG pathways. Fig.19C , 19D )2.18mg kg -1 HKUOT-S2 significantly enriched KEGG pathways. Fig.19E )1.09 and 2.18 mg kg -1 Significantly enriched GO terms shared by both HKUOT-S2 treatments. Fig.19F )4.36mg kg - 1 Significantly enriched GO terms in HKUOT-S2. One-way ANOVA was used as the statistical tool for analysis. Values ​​are shown as mean ± SEM (n = 3). *p < 0.05 was considered significant.

[0035] Figures 20A-20N .Yam protein extract increases the expression of RUNX2 in differentiated osteoblasts. Figures 20A-20D ) Crude yam protein extracts HKUOT-D3, HKUOT-P1 and HKUOT-S2 had no effect on the proliferation and viability of hTMSCs and RAW264.7 cells. Figures 20E-20L ) Crude yam protein extract increased RUNX2 expression in hMSC- and MC3T3-1E cell-derived osteoblasts. Figure 20M , 20N )0.01 μg ml -1 The HKUOT-S2 de novo peptide sequence TKSSLPGQTK (SEQ ID NO: 83) promoted osteoblast differentiation by increasing ALP and COL-1A expression in vitro.

[0036] Figures 21A-21B .HKUOT-S2 regulates the mTOR1 / 4E-BP1 axis to promote osteogenesis. Fig.21A ) HKUOTS-2 promotes hMSC-osteoblast differentiation by increasing ALP expression. Fig. 21B ) HKUOT-S2 blocks XL388-induced mTOR inhibition to increase phosphorylation of mTOR1 and 4E-BP1 proteins and increase total AKT1 protein levels. Values ​​are shown as mean ± SEM, (n = 4). *p < 0.05 was considered significant under one-way ANOVA.

[0037] Figures 22A-22H .HKUOT-S2 and SEQ ID NO:83(TK) significantly inhibit the development of osteoporosis. Fig.22A) μCT scans of femurs in the sham control group, OVX control group, HKUOT-S2, and TK-treated groups. Figures 22B-22H ) μCT analysis results of BV / TV, BS / TV ratio, Tb.th, Tb.N, BS / BV and Tb.Sp of femur. Values ​​are shown as mean ± SEM, n = 3. HKUOT-S2 = 2.18 mg / kg, TK = 0.5 mg / kg, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0038] Figures 23A-23F .HKUOT-S2 and SEQ ID NO:83(TK) significantly promoted osteoblast differentiation. Fig.23A ) HKUOT-S2 and SEQ ID NO: 83 treatment increased ERα expression. Fig. 23B ) Treatment with HKUOT-S2 and SEQ ID NO:83 increased ALP expression. Figures 23C-23D ) Treatment with HKUOT-S2 and SEQ ID NO: 83 increased ALP activity in osteoblasts. Figures 23E-23F ) HKUOT-S2 and SEQ ID NO: 83 treatment increased osteoblast biomineralization. Values ​​are shown as mean ± SEM, n = 4. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0039] Figures 24A-24J .HKUOT-S2 promotes osteoblast differentiation by regulating estrogen receptors. Figures 24A-24C )HKUOT-S2 treatment increased the expression of ERα and GPR30, but had no effect on the expression of ERβ. Figures 24D-24F )HKUOT-S2 treatment increased the expression of ALP, COL1A1, and RUNX2 during osteoblast differentiation. Figures 24G-24H )HKUOT-S2 treatment increased ALP activity in osteoblasts. Figures 24I-24J ) HKUOT-S2 treatment increased osteoblast biomineralization. Values ​​are shown as mean ± SEM, n = 4. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0040] Figures 25A-25D HKUOT-S2 treatment upregulates estrogen receptor in OVX mice. Figures 25A-25D ) HKUOT-S2 treatment increased ERα, ERβ and GPR30 protein expression in bone tissue. Values ​​are shown as mean ± SEM, n = 4. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0041] Figures 26A-26G .HKUOT-S2 significantly inhibits the development of lumbar osteoporosis. Fig.26A ) μCT scans of L5 in the sham-operated control group, OVX control group, and HKUOT-S2-treated group. Figures 26B-26G )μCT analysis of BV / TV, BS / TV ratio, Tb.th, Tb.N, BS / BV and Tb.Sp of L5. Values ​​are shown as mean ± SEM, n = 8. HKUOT-S2 = 2.18 mg / kg, TK = 0.5 mg / kg treatment, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0042] Fig. 27 The molecular weight (MW) of S2 and S3 proteins was determined using the Kav equation. The column standard curve was calibrated with proteins of known MV.

[0043] A brief description of the sequence

[0044] SEQ ID NO: 1: Alp primer forward

[0045] SEQ ID NO:2: Alp primer reverse

[0046] SEQ ID NO:3: Col primer forward

[0047] SEQ ID NO:4: Col Primer Reverse

[0048] SEQ ID NO:5: Opn primer forward

[0049] SEQ ID NO:6: Opn primer reverse

[0050] SEQ ID NO:7: Runx 2 primer forward

[0051] SEQ ID NO:8: Runx 2 primer reverse

[0052] SEQ ID NO:9: Arg-1 Primer Forward

[0053] SEQ ID NO: 10: Arg-1 primer reverse

[0054] SEQ ID NO:11: Mgl-1 primer forward

[0055] SEQ ID NO: 12: Mgl-1 primer reverse

[0056] SEQ ID NO:13: Cd206 primer forward

[0057] SEQ ID NO: 14: Cd206 primer reverse

[0058] SEQ ID NO:15:Ym1 primer forward

[0059] SEQ ID NO: 16: Ym1 primer reverse

[0060] SEQ ID NO: 17: Socs3 primer forward

[0061] SEQ ID NO: 18: Socs3 primer reverse

[0062] SEQ ID NO: 19: Tnfα primer forward

[0063] SEQ ID NO:20: Tnfα primer reverse

[0064] SEQ ID NO:21: iNOS primer forward

[0065] SEQ ID NO:22: iNOS primer reverse

[0066] SEQ ID NO:23: IL-6 Primer Forward

[0067] SEQ ID NO:24: IL-6 Primer Reverse

[0068] SEQ ID NO:25: IL-1β Primer Forward

[0069] SEQ ID NO:26: IL-1β Primer Reverse

[0070] SEQ ID NO:27: Mcp-1 primer forward

[0071] SEQ ID NO:28: Mcp-1 primer reverse

[0072] SEQ ID NO:29: Prkaa1 primer forward

[0073] SEQ ID NO:30: Prkaa1 primer reverse

[0074] SEQ ID NO:31: Prkaa2 primer forward

[0075] SEQ ID NO:32: Prkaa2 primer reverse

[0076] SEQ ID NO:33: Prkab1 primer forward

[0077] SEQ ID NO:34: Prkab1 primer reverse

[0078] SEQ ID NO:35: Prkab2 primer forward

[0079] SEQ ID NO:36: Prkab2 primer reverse

[0080] SEQ ID NO:37: Prkag1 primer forward

[0081] SEQ ID NO:38: Prkag1 primer reverse

[0082] SEQ ID NO:39: Prkag2 primer forward

[0083] SEQ ID NO:40: Prkag2 primer reverse

[0084] SEQ ID NO:41: Prkaig3 primer forward

[0085] SEQ ID NO:42: Prkag3 primer reverse

[0086] SEQ ID NO:43: Gapdh Primer Forward

[0087] SEQ ID NO:44: Gadph primer reverse

[0088] SEQ ID NO:45: Bglap1 primer forward

[0089] SEQ ID NO:46: Bglap1 primer reverse

[0090] SEQ ID NO:47: Bglap2 primer forward

[0091] SEQ ID NO:48: Bglap2 primer reverse

[0092] SEQ ID NO:49: Got1 (Ast) primer forward

[0093] SEQ ID NO:50: Got1 (Ast) primer reverse

[0094] SEQ ID NO:51: Gpt1 (Ast) Primer Forward

[0095] SEQ ID NO:52: Gpt1 (Ast) primer reverse

[0096] SEQ ID NO:53: Ckb (creatine) primer forward

[0097] SEQ ID NO:54: Ckb (creatine) primer reverse

[0098] SEQ ID NO:55: mTOR primer forward

[0099] SEQ ID NO:56: mTOR primer reverse

[0100] SEQ ID NO:57: Bmp2 primer forward

[0101] SEQ ID NO:58: Bmp2 primer reverse

[0102] SEQ ID NO:59: Bmrp2 primer forward

[0103] SEQ ID NO:60: Bmpr2 primer reverse

[0104] SEQ ID NO:61: Bmp7 primer forward

[0105] SEQ ID NO:62: Bmp7 primer reverse

[0106] SEQ ID NO:63: Tgfbr2 primer forward

[0107] SEQ ID NO:64: Tgfbr2 primer reverse

[0108] SEQ ID NO:65: ALP primer forward

[0109] SEQ ID NO:66: ALP primer reverse

[0110] SEQ ID NO:67: COL1A primer forward

[0111] SEQ ID NO:68COL1A primer reverse

[0112] SEQ ID NO:69:OPN Primer Forward

[0113] SEQ ID NO:70: OPN primer reverse

[0114] SEQ ID NO:71:RUNX2 primer forward

[0115] SEQ ID NO:72:RUNX2 primer reverse

[0116] SEQ ID NO:73: GAPDH primer forward

[0117] SEQ ID NO:74: GAPDH primer reverse

[0118] SEQ ID NO:75:4 EBP1 primer forward

[0119] SEQ ID NO:76:4 EBP1 primer reverse

[0120] SEQ ID NO:77: AKT1 Primer Forward

[0121] SEQ ID NO:78: AKT1 Primer Reverse

[0122] SEQ ID NO:79: S6K1 Primer Forward

[0123] SEQ ID NO:80: S6K1 primer reverse

[0124] SEQ ID NO:81: HKUOT-S2 peptide

[0125] SEQ ID NO:82: HKUOT-S2 peptide

[0126] SEQ ID NO:83: HKUOT-S2 peptide

[0127] SEQ ID NO:84: HKUOT-S2 peptide

[0128] SEQ ID NO:85: HKUOT-S2 peptide

[0129] SEQ ID NO:86: HKUOT-S2 peptide

[0130] SEQ ID NO:87: HKUOT-S2 peptide

[0131] SEQ ID NO:88: HKUOT-S2 peptide

[0132] SEQ ID NO:89: HKUOT-S2 peptide

[0133] SEQ ID NO:90: ​​HKUOT-S2 peptide

[0134] SEQ ID NO:91: HKUOT-S2 peptide

[0135] SEQ ID NO:92: HKUOT-S2 peptide

[0136] SEQ ID NO:93: HKUOT-S2 peptide

[0137] SEQ ID NO:94: HKUOT-S2 peptide

[0138] SEQ ID NO:95: HKUOT-S2 peptide

[0139] SEQ ID NO:96: HKUOT-S2 peptide

[0140] SEQ ID NO:97: HKUOT-S2 peptide DETAILED DESCRIPTION OF THE INVENTION

[0142] Selected Definition

[0143] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, to the extent that the terms "including," "includes," "having," "has," or "with," or variations thereof, are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising." The transitional terms / phrases (and any grammatical variations thereof) "comprising," "comprises," or "comprises," "consisting essentially of," or "consists essentially of," and "consisting or "consists" may be used interchangeably.

[0144] The phrase "consisting essentially of or consists essentially of" indicates that the claim covers embodiments including the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claim.

[0145] The term "about" means within an acceptable error range of a particular value determined by a person of ordinary skill in the art, which depends in part on how the value is measured, i.e., the limitations of the measurement system. In the context of compositions containing a certain amount of ingredients using the term "about", these compositions contain the ingredients in that amount with a variation (error range) of 0%-10% (X ± 10%) around the value. In other contexts, the term "about" provides a variation (error range) of 0%-10% (X ± 10%) around a given value. Obviously, this variation represents a range of up to 10% above or below a given value, such as X ± 1%, X ± 2%, X ± 3%, X ± 4%, X ± 5%, X ± 6%, X ± 7%, X ± 8%, X ± 9%, or X ± 10%.

[0146] In the present disclosure, ranges are stated in shorthand to avoid having to elaborate and describe each and every value in the range. Where appropriate, any suitable value in the range can be selected as the upper limit, lower limit or end of the range. For example, the range of 0.1-1.0 represents terminal values ​​0.1 and 1.0, and intermediate values ​​0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges included in 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc. It is conceivable that there are values ​​with at least two significant figures in a range, for example, the range of 5-10 represents all values ​​between 5.0 and 10.0 and between 5.00 and 10.00, including the terminal value. When using ranges in this article, combinations and sub-combinations of ranges (e.g., sub-ranges within the disclosed ranges) and specific embodiments thereof are explicitly included.

[0147] As used herein, an "isolated" or "purified" compound is substantially free of other compounds. In certain embodiments, the purified compound is at least 60% by weight (dry weight) of the compound of interest. Preferably, the preparation is at least 75% by weight of the compound of interest, more preferably at least 90%, and most preferably at least 99%. For example, a purified compound is a compound that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or 100% (W / W) by weight of the desired compound. Purity is measured by any suitable standard method, such as by column chromatography, thin layer chromatography or high performance liquid chromatography (HPLC) analysis.

[0148] As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as sequences explicitly specified. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985) and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0149] By "reduced" is meant a negative change of at least 1%, 5%, 10%, 25%, 50%, 75% or 100%.

[0150] By "increase" is meant a positive change of at least 1%, 5%, 10%, 25%, 50%, 75% or 100%.

[0151] As used herein, the term "sample" refers to a sample comprising at least one protein, peptide or nucleic acid, including a blood sample. In one embodiment, the term "biological sample" as used herein refers to a sample obtained from a subject, wherein the sample comprises at least one protein, peptide or nucleic acid. Although not required or required, the term "biological sample" is intended to encompass samples that are processed prior to assay using the systems and methods described herein.

[0152] As used herein, the term "subject" refers to a plant or animal, particularly a person, from which a biological sample is obtained or derived. As used herein, the term "subject" covers both humans and non-human animals. The term "non-human animal" includes all vertebrates, such as mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and non-mammals such as chickens, amphibians, reptiles, etc. In one embodiment, the subject is a person. In another embodiment, the subject is an experimental animal or an animal substitute as a disease model. In some embodiments, the term "subject" refers to a mammal, including but not limited to mice, apes, humans, cats, dogs, horses, cattle, mammal farm animals, mammal sports animals, and mammal pets. In the context of the present invention, a preferred subject is a person. The subject can be of any age or developmental stage, including infants, toddlers, teenagers, adolescents, adults, or the elderly.

[0153] As used herein, the terms "therapeutically effective amount", "therapeutically effective dose", "effective amount" and "effective dose" are used to refer to an amount or dose of a protein, peptide or composition thereof that, when administered to a subject, is capable of treating or ameliorating a condition, disease or disorder of the subject, or is capable of providing an enhancement of the health or function of an organ, tissue or body system. In other words, the amount is "therapeutically effective" when administered to a subject. The actual amount will vary depending on many factors, including, but not limited to, the specific condition, disease or disorder being treated or improved; the severity of the condition; the specific organ, tissue or body system for which enhancement of health or function is desired; the patient's weight, height, age and health status; and the route of administration.

[0154] In some embodiments of the present invention, the method includes administering multiple doses of the composition of the present invention. The method may include administering a therapeutically effective dose of a composition three times a week, once a week or more frequently, the composition comprising a protein, peptide or its composition of the present invention as described herein. In some embodiments, the dosage is administered over a period of 1 week, 2 weeks or more than 3 weeks. In addition, treating a subject with a therapeutically effective amount of the composition of the present invention may include a single treatment or may include a series of treatments. It should also be understood that the effective dose of a protein, peptide or its composition for treatment may increase or decrease during a specific treatment. Changes in dosage may result in and make the results of diagnostic assays or imaging techniques known in the art for detecting or visualizing bone become apparent. In some embodiments of the present invention, the method includes administering the composition several times a day, including but not limited to 2 times a day, 3 times a day and 4 times a day.

[0155] As used herein, in the context of describing two or more polynucleotide sequences, the term "identical" or "percent identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides over a comparison region. For example, the homologous nucleotide sequence used in the method of the present invention has at least 80% sequence identity, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the reference sequence, when compared and aligned over a comparison window or over a specified region to obtain maximum correspondence, using a comparison algorithm or measured by manual alignment and visual inspection. With respect to polynucleotide sequences, this definition also refers to the complementary sequence of the test sequence.

[0156] As used herein, in the context of describing two or more amino acid sequences, the term "identical" or "percent identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acids over a comparison region. For example, the homologous nucleotide sequence used in the method of the present invention has at least 80% sequence identity, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the reference sequence, when compared and aligned over a comparison window or over a specified region to obtain maximum correspondence, using a comparison algorithm or measured by manual alignment and visual inspection. With respect to amino acid sequences, this definition also refers to the complementary sequence of the test sequence.

[0157] The recitation of a list of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of the listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0158] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0159] Other features and advantages of the present invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.All references cited herein are incorporated herein by reference.

[0160] Protein HKUOT-S2 and its composition

[0161] In certain embodiments, the composition of the present invention comprises a protein isolated from Chinese yam: HKUOT-S2. HKUOT-S2 protein can be separated from Chinese yam by continuous ion exchange, hydrophobic interaction and high-resolution size exclusion chromatography. As determined by mathematical modeling, silver staining and mass spectrometry, the molecular weight of HKUOT-S2 protein is 32kDa. HKUOT-S2 is further characterized using high-resolution liquid chromatography tandem mass spectrometry (LC-MS / MS), de novo peptides and N-terminal peptide sequencing. In certain embodiments, the composition comprises a peptide selected from the HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity with SEQ ID NOs: 81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity with SEQ ID NOs: 81-97.

[0162] In certain embodiments, HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity with SEQ ID NOs: 81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity with SEQ ID NOs: 81-97 can be added to the composition at a concentration of about 0.0001% to about 50% by weight (wt%), preferably about 0.01 wt% to about 10 wt%, and most preferably about 0.1 wt% to about 10 wt%. In another embodiment, the HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity with SEQ ID NOs: 81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity with SEQ ID NOs: 81-97 can be combined with an acceptable carrier and / or excipient, wherein the protein or peptide may be present in a concentration of about 0.0001% to about 50% (v / v), preferably about 0.01% to about 10% (v / v), more preferably about 0.1% to about 10% (v / v).

[0163] In one embodiment, the subject composition is formulated as an oral consumable product, such as food, capsule, pill or drinkable liquid. The medicine that can be delivered orally is any physiologically active substance delivered via the initial absorption in the gastrointestinal tract or oral mucosa. The subject composition can also be formulated into a solution, which can be used via, for example, injection, including intravenous, intraperitoneal, intramuscular, intrathecal, intracerebroventricular or subcutaneous. In other embodiments, the subject composition is formulated as via the skin by a patch or directly applied to the skin to obtain a local or systemic effect. The composition can be used sublingually, through the cheek, through the rectum or vaginally. In addition, the composition can be sprayed into the nose to absorb, atomize, inhale via the mouth or nose or use in the eyes or ears by the nasal membrane.

[0164] An orally consumable product according to the present invention is any preparation or composition suitable for consumption, for nutrition, for oral hygiene or for pleasure, and is intended to be introduced into the oral cavity of a human or animal, kept there for a certain period of time, and then swallowed (e.g., food or pills to be consumed) or taken out of the oral cavity again (e.g., chewing gum or oral hygiene products or medical mouthwashes). Although an orally deliverable drug can be formulated into an orally consumable product, and an orally consumable product can contain an orally deliverable drug, the two terms are not meant to be used interchangeably herein.

[0165] Orally consumable products include all substances or products intended to be ingested by humans or animals in a processed, semi-processed or unprocessed state. This also includes substances that are added to orally consumable products (especially foods and pharmaceuticals) during their production, handling or processing and are intended to be introduced into the oral cavity of humans or animals.

[0166] Orally consumable products may also include materials intended to be swallowed by humans or animals and then digested in an unmodified, prepared or processed state; therefore, orally consumable products according to the present invention also include a shell, coating or other packaging intended to be swallowed or expected to be swallowed with the product.

[0167] In one embodiment, the oral consumable product is a capsule, pill, syrup, emulsion or liquid suspension comprising a desired oral delivery material. In one embodiment, the oral consumable product may comprise an orally deliverable material in powder form that may be mixed with water or another liquid to produce a drinkable oral consumable product.

[0168] In some embodiments, the oral consumable product according to the present invention may include one or more preparations intended for nutrition or pleasure. These include baked products (e.g., bread, dry biscuits, cakes and other cakes), candies (e.g., chocolate, chocolate bar products, other bar products, fruit gums, coated tablets, hard caramels, toffees and caramels and chewing gum), alcoholic or non-alcoholic beverages (e.g., cocoa, coffee, green tea, black tea, black tea or green tea beverages rich in green tea or black tea extracts, Rooibos tea, other herbal teas, lemonade containing fruit, isotonic beverages, soft drinks, nectar, fruit and vegetable juices and fruit or vegetable juice preparations), instant beverages (e.g. instant cocoa drinks, instant tea drinks and instant coffee drinks), meat products (e.g. ham, fresh or raw sausage products and seasoned or cured fresh or cured meat products), eggs or egg products (e.g. dried whole eggs, egg whites and egg yolks), cereal products (e.g. breakfast cereals, granola bars and precooked ready-to-eat rice products), dairy products (e.g. whole or reduced-fat or skim milk drinks, rice pudding, yogurt, kefir, cream cheese, soft cheese, hard cheese, dried milk powder, whey, butter, cheese The invention relates to products of the present invention which are prepared from soy protein or other soy components (e.g., soy milk and products prepared therefrom, beverages containing isolated or enzyme-treated soy protein, beverages containing soy flour, preparations containing soy lecithin, fermented products such as tofu or its prepared tempeh products and mixtures with fruit preparations and optionally flavoring substances), fruit preparations (e.g., jams, fruit ice creams, fruit spreads and fruit fillings), vegetable preparations (e.g., ketchup, sauces, dried vegetables, deep-frozen vegetables, pre-cooked vegetables and boiled vegetables), snack products (e.g., baked or fried potato chips (crisps) or potato dough products and corn or peanut-based extrudates), products based on fats and oils or their emulsions (e.g., mayonnaise, mayonnaise and dressings), other ready-made meals and soups (e.g., dry soups, instant soups and pre-cooked soups), seasonings (e.g., sprinkleable seasonings), sweetener compositions (e.g., tablets, sachets and other preparations for sweetening or whitening beverages or other foods). The composition of the invention may also be used as a semi-finished product for the production of other compositions intended for nutrition or pleasure.

[0169] The subject composition may further comprise one or more pharmaceutically acceptable carriers and / or excipients, and may be formulated into a preparation, such as a solid, semisolid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

[0170] As used herein, the term "pharmaceutically acceptable" means compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.

[0171] Carriers and / or excipients according to the present invention may include any and all solvents, diluents, buffers (e.g., neutral buffered saline, phosphate buffered saline or optionally Tris-HCl, acetate or phosphate buffer), oil-in-water or water-in-oil emulsions, aqueous compositions with or without organic cosolvents suitable for, e.g., IV use, solubilizers (e.g., polysorbate 65, polysorbate 80), colloids, dispersion media, vehicles, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, fragrances, thickeners (e.g., carbomer, gelatin or sodium alginate), coatings, preservatives (e.g., thimerosal, benzyl alcohol, polyquaternium salts), antioxidants (e.g., ascorbic acid, sodium metabisulfite), tonicity control agents, absorption delaying agents, adjuvants, fillers (e.g., lactose, mannitol), and the like. The use of carriers and / or excipients in the pharmaceutical and supplement arts is well known. In addition to any conventional media or agent that is incompatible with the target health promoting substance or with the composition, a carrier or excipient is contemplated for use in the subject compositions.

[0172] In one embodiment, the composition of the present invention can be made into aerosol formulations, so that for example, it can be atomized or inhaled. Suitable pharmaceutical preparations used in aerosol or spray form are for example powders, particles, solutions, suspensions or emulsions. The preparations used for oral or nasal aerosols or inhalation can also be prepared with a carrier, and the carrier includes for example salt, polyethylene glycol or ethylene glycol, DPPC, methylcellulose, or mixed with a powder dispersant or a fluorocarbon. Aerosol formulations can be placed in pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, fluorocarbon and / or other solubilizing agents or dispersants known in the art. Illustratively, delivery can be by using any other in a disposable delivery device, a mist nebulizer, a breath-activated powder inhaler, an aerosol metered dose inhaler (MDI) or numerous nebulizer delivery devices available in this area. In addition, spray tents can also be used or directly applied by an endotracheal tube.

[0173] In one embodiment, the composition of the present invention can be formulated for administration via injection, for example as a solution or suspension. The solution or suspension may contain a suitable non-toxic, parenterally acceptable diluent or solvent, such as mannitol, 1,3-butylene glycol, water, Ringer's solution, or isotonic sodium chloride solution, or a suitable dispersant or wetting agent and suspending agent, such as a sterile, non-irritating fixed oil, including synthetic monoglycerides or diglycerides, and fatty acids, including oleic acid. An illustrative example of a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600 and the balance USP water for injection (WFI). Other exemplary carriers for intravenous use include 10% USP ethanol and USP WFI; 0.01%-0.1% triethanolamine in USP WFI; or 0.01%-0.2% dipalmitoyl diphosphatidylcholine in USP WFI; and 1%-10% squalene or parenteral plant oil-in-water emulsions. Water or saline solutions and aqueous glucose and glycerol solutions can be preferably used as carriers, particularly for injectable solutions. Illustrative examples of carriers for subcutaneous or intramuscular use include phosphate buffered saline (PBS) solutions, 5% dextran in WFI and 0.01-0.1% triethanolamine in 5% dextran or 0.9% sodium chloride in USP WFI, or a 1 to 2 or 1 to 4 mixture of 10% USP ethanol, 40% propylene glycol, the balance being an acceptable isotonic solution, such as 5% dextran or 0.9% sodium chloride; or 0.01%-0.2% dipalmitoyl diphosphatidylcholine and 1% to 10% squalene in USP WFI or parenteral vegetable oil in water emulsions.

[0174] In one embodiment, the composition of the present invention can be formulated for external application to the skin, for example, as an external composition, including flushing agents, sprays or drops, lotions, gels, ointments, creams, foams, powders, solids, sponges, tapes, steam, pastes, tinctures, or using transdermal patches. In addition to any pharmaceutically active carrier, the suitable formulation of external application can include, for example, emollients, such as carnauba wax, cetyl alcohol, cetyl ester wax, emulsifying wax, aqueous lanolin, lanolin, lanolin alcohol, microcrystalline wax, paraffin, vaseline, polyethylene glycol, stearic acid, stearyl alcohol, white beeswax or yellow beeswax. In addition, the composition can contain a wetting agent such as glycerol, propylene glycol, polyethylene glycol, sorbitol solution and 1,2,6 hexanetriol or a penetration enhancer such as ethanol, isopropyl alcohol or oleic acid.

[0175] Methods of using HKUOT-S2 and compositions thereof

[0176] In certain embodiments, HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97 can be administered to a subject. Any mode of administration that can allow a peptide or protein to contact cells in a subject is contemplated in the subject method, including, for example, oral, intravenous, intraperitoneal, intramuscular, intrathecal, or subcutaneous. In certain embodiments, a peptide or protein can contact cells of a subject, including, for example, macrophages, osteoblasts, chondrocytes, osteoclasts, and MSCs.

[0177] In certain embodiments, the proteins and / or peptides of the present invention can induce macrophage polarization and human MSC (hMSC)-derived osteoblast mineralization. In certain embodiments, the proteins and / or peptides of the present invention can modulate genes enriched in focal adhesion, mTOR, AMPK and BMP signaling pathways. The proteins and / or peptides of the present invention can also induce genes involved in bone cell development (Ptpn6, Fli1, Med1, Tnfsf11, Rabgap1l, Pip4k2a, Src, Sh2b3, Zfpm1, Ep300, Nbeal2) and genes involved in new bone formation (Tgfb1, Rsad2, Hif1a, Ltbp3, Alox5, Fam20c, Smad7, Ddr2, Isg15, Smad6, Suv39h1, S1pr1, Adrb2, Sgms2, Mapk14, Bcl2, Kl, Mapk3, Mapk1, Sox11, Zbtb16 and Adgrv1). In certain embodiments, HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97 can promote human mesenchymal stem cells (hMSCs) to differentiate into osteoblasts by increasing estrogen receptor α (ERα), estrogen receptor β (ERβ) and ALP mRNA expression. In certain embodiments, HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence with at least 90% sequence identity to SEQ ID NOs: 81-97 can upregulate osteogenic gene expression of ALP, COL1A1 and RUNX2.

[0178] In certain embodiments, the proteins and / or peptides of the present invention can enhance bone defect repair by effectively regulating cellular functions, such as macrophage polarization regulating pro-inflammatory and anti-inflammatory activity, bone resorption of osteoclasts, and new bone formation of osteoblasts. The proteins and / or peptides of the present invention can also enhance osteogenic molecular functions by differentially stimulating osteogenic gene expression that promotes biomineralization and increases BMD to promote bone defect repair. In certain embodiments, the proteins and / or peptides of the present invention induce multiple osteogenic signaling pathways, such as focal adhesion, AMPK, PI3K / Akt / mTOR, and BMP / TGF-β signaling pathways, as well as biological functions and processes, such as osteoblast and osteoclast differentiation, musculoskeletal morphogenesis, and development to produce desired new bone formation.

[0179] In certain embodiments, the proteins and / or peptides of the present invention are non-hemolytic and protect red blood cells (RBCs) from hypotonic-induced hemolysis (RBC rupture). In certain embodiments, the proteins and / or peptides of the present invention induce differentially expressed genes that regulate chondrocyte differentiation (Slc39a14, Col11a2, Ift80, Adamts12), osteoblast differentiation (Fbxo5, Gli3, Men1, Il6, Fam20c, Ddr2, Wwtr1, Ctnnbip1, Ltf, Cebpb, Sox11, Jund) and osteoclast differentiation (Tcirg1, Tgfb1, Gab2, Epha2, Tnfsf11, Tnf, Csf1r, Tnfrsf11a, Traf6, Tyrobp, Trf, Mitf, Sn x10, Junb, Mapk14, Fcer1g, Fos, Tnfsf11, Creb1, Car2, Klf10), endochondral ossification (Mmp14, Col1a1, Ift80, Mmp16, Mmp13, Col13a1) and intramembranous ossification (Col1a1, Mmp2), chondrogenesis (Col1a1, Ift80, Mmp13, Bmp1, Col11a2, Cfh, Col11a1, Bmp3, Bgn, Smad1, Mmp13), odontogenesis (Lamb1, Id3, Fst, Lrp4, Slc4a2, Pdgfra, Fgfr2, Phex ), neurogenesis (Fabp7, Btbd1, Lrp4, Tal1, Cit, Dbn1, Cdk5rap2, Nup133, Serpinf1, Mdk, Ngf, Ptprd, Mtor, Plxna4, Plxnd1, Aspm, Lif, Eef2k, Zfp335, Shtn1, Robo2, Kdr), neuronal development (Cfh, Ngf, Gba2, Fgfr2, Lif, Hs6st1, Robo2, Htra2, Nanos1) and axon development (Mtr, Mmp2, Camsap2), neuropeptide synthesis (Sema4f, Sema6a, Ptges2, Map2, Sema6c, Sema4b, Ptges3, Sema3c, Sema4a), as well as enhancing liver development (Foxm1, Vwf, Med1, Sec63, Xbp1, Stat5b, Ptcd2, Il6, Notch1, Sp3, Aurka, Notch2, Aacs, Ezh2, Atg7, Hmbs, Pkd2, Hlx, Icmt, Onecut1, Zmpste24, Taf10, Arf6, Onecut2, Jun, Klf1, Cebpb, Mpst, Atf7), kidney development (Bax, Bloc1s6,Qrich1, Tgfbr1, Ap1b1, Slc5a1, Odc1, Glis2, Sgpl1, Id2, Gli3, Ptcd2, Robo1, Adamts1, Bag6, Pdgfrb, Cat , Bcl2l11, Pds5a, Cux1, Nek1, Ctnnd1, Wfs1, Greb1l, Fmn1, Pou3f3, Pygo2, Agtr1a, Zbtb14, Foxc1, Bcl2, Z btb16, Mpst, Smo, Glis2, Ednrb, Prom1, Pou3f3, Cd24a, Ext1) and cardiomyocyte development (Tbx2, Id2, Zfpm2, Csrp3, Sin3b, Tbx18, Gjc1, Sgcg, Tbx20, Ccnb1, Mapk14, Mapk1, Tgfbr1, Pi16, G6pdx, Tgfbr2, Parp2, Rgs4, Gsk3a, G6pd2).

[0180] In certain embodiments, the proteins and / or peptides of the present invention can increase neuronal function regulatory genes, such as Ep4, Ptges, Cox2, Eno2 and Calca, to enhance fracture repair. In certain embodiments, the proteins and / or peptides of the present invention can mechanically activate the mTOR / 4E-BP1 axis to promote osteogenesis.

[0181] In certain embodiments, HKUOT-S2 protein, at least one peptide according to at least one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity thereto, or a protein comprising an amino acid sequence according to at least one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity thereto can be administered at about 0.01 mg kg -1 About 50 mg kg -1 , about 0.1mgkg -1 About 10 mg kg -1 , about 1.09mgkg -1 , about 2.18 mg kg -1 or about 4.36 mg kg -1 The dose is administered to the subject.

[0182] In certain embodiments, the subject compositions can be used in methods for treating osteoporosis.In certain embodiments, the proteins and / or peptides of the invention can inhibit bone loss by inhibiting the development of OVX-induced and drug (glucocorticoid)-induced osteoporosis.

[0183] In certain embodiments, the subject composition can be used in methods for osteogenesis and treating new bone formation problems. In certain embodiments, the protein and / or peptide of the present invention significantly enhances osteoblast differentiation and promotes the function of osteogenesis and new bone formation. The subject composition can activate the mTOR / 4E-BP1 / AKT1 axis to promote osteogenesis. uCT scanning can be used to measure the progressive enhancement of fracture repair in vivo. Enhanced fracture repair can be assessed by measuring the percentage of bone defect site, bone volume, bone mineral density, trabecular thickness, separation and quantity, and / or bone surface area to tissue volume (BS / TV) ratio. In certain embodiments, the method can provide a combination of faster repair or structurally more sound repair.

[0184] In certain embodiments, the subject compositions can be used in methods for treating delayed fracture repair. In certain embodiments, the proteins and / or peptides of the present invention significantly enhance fracture repair and can be used as interventions for delayed fracture repair. Delayed bone healing indicates that fractures take longer to heal than normal. In certain embodiments, fracture healing can be considered delayed when fracture healing requires at least about 3 months. The time frame for delayed healing may vary depending on different bone types.

[0185] In certain embodiments, the subject compositions can be used in methods for treating inflammatory diseases such as rheumatoid arthritis. In certain embodiments, the proteins and / or peptides of the present invention can modulate M1 and M2 macrophage polarization and maintain a steady-state balance between M1 and M2 macrophage functions to inhibit the development of inflammatory diseases such as osteoarthritis.

[0186] In certain embodiments, the subject compositions can be used in methods of treating spinal cord injury (SCI).In certain embodiments, the proteins and / or peptides of the invention promote neurogenesis and modulate macrophage polarization, which are essential components of SCI repair.

[0187] In certain embodiments, the subject compositions can be used in methods for treating liver and kidney diseases. In certain embodiments, the proteins and / or peptides of the present invention enhance the expression of Ast and Ckb genes in liver and kidney function. In certain embodiments, the proteins and / or peptides of the present invention induce differentially expressed genes, which are significantly enriched in the biological functions and processes regulating liver and kidney and heart development. In certain embodiments, the proteins and / or peptides of the present invention can enhance the expression of key liver genes (Rps6ka1, Il6, Cebpb, Notch2) and kidney genes (Bax, Bloc1s6, Qrich1, Tgfbr1, Ap1b1, Slc5a1, Odc1, Glis2, Sgpl1, Id2, Gli3, Ptcd2, Robo1, Adamts1, Ba g6, Pdgfrb, Cat, Bcl2l11, Pds5a, Cux1, Nek1, Ctnnd1, Wfs1, Greb1l, Fmn1, Pou3f3, Pygo2, Agtr1a, Zbtb14, Foxc1, Bcl2, Zbtb16, Mpst, Smo, Glis2, Ednrb, Prom1, Pou3f3, Cd24a, Ext1) gene expression, Hematopoiesis (Foxm1, Vwf, Med1, Sec63, Xbp1, Stat5b, Ptcd2, Il6, Notch1, Sp3, Aurka, Notch2, Aacs, Ezh2, Atg7, Hmbs, Pkd2, Hlx, Icmt, Onecut1, Zmpste24, Taf10, Arf6, Onecut2, Jun, Klf1, Cebpb, Mpst, Atf7), renal regeneration (Tgfb1, Aqp1, Dlg1, Smad7, Cat, Smad9, Kank2, Smad6, Greb1l, Pou3f3, Foxc1, Bcl2, Smo, Glis2, Gli3, Ednrb, Prom1, Pou3f3, Cd24a, Ext1) and macrophage polarization (Socs3, Tnfα, Arg-1, Mgl-1 Ampka1, Pparg, Lgals9, Havcr2, Gpr137b, Snca, Il1rl1, Cd1d1, Stap1, Atm, Lrrk2, Tlr4, Sphk1, Muc5b, Jund, Ulbp1) to inhibit liver-related diseases, such as hepatitis and cirrhosis, and kidney diseases, such as nephritis and nephrotic syndrome.

[0188] In certain embodiments, the subject compositions can be used in methods for treating cardiovascular-related problems. In certain embodiments, the proteins and / or peptides of the invention induce differentially expressed genes that regulate hematopoiesis (Ap2a2, Nfe2l2, Eif2ak2, Zbtb1, Sos2, Ankle1, Foxc1, Kit1, Cxcl1, Atxn1l), angiogenesis (Itgb2, Hk2, Aqp1, Notch4, Nfe2l2, Ptgis, Nr2e1, Xbp1, Hif1a, Sema5a, Cyp1b1, C3, Itgb8, Cxcr2, Jak1, Add1, Ctsh, Tgfbr2, Ccr3, Btg1, Hspb6, Lrg1, Serpinel, Pik3cd, Agtr1a, C5ar1, Sphk1, Chil1), angiogenesis (Ccm2, Fo The biological functions and processes of the genes involved in the genes encoding for mitochondria (Mdm2, Zfpm2, Robol, Smad7, Parva, Tbx20, Tgfbr2, Smad6, Fzdl, Zfpml, Trp53, and Soxl1) were significantly enriched. In certain embodiments, the proteins and / or peptides can treat, for example, hypertension and cardiomyopathy.

[0189] In certain embodiments, the subject compositions can be used in methods for treating diabetes. In certain embodiments, the proteins and / or peptides of the present invention induce differentially expressed genes that are significantly enriched in regulating the following biological functions and processes: positive regulation of insulin secretion in response to glucose stimulation of cells (Arrb1, Ppp3cb, Camk2n1, Gpr68), insulin receptor signaling pathway (Tsc2, Ankrd26, Rarres2, Pip4k2b, Rps6kb1, Mstn, Pip4k2a, Src, Zbtb7b, Ybx1, Sesn3, Ccnd3, Ncoa5, Pik3r1, Sorl1, Prkaa1, Socs3, Gsk3a), insulin signaling pathway (Hk2, Araf, Tsc2, Prkar2b, Gys1, Crk1, Ppp1cb, Crk, Mknk2, Shc2, Rps6kb1, Prkar1a, Pygl, Pdpk1, Hras, ​​Hk3, Pik3r3, Mknk1, Rheb, Add1, Prkab1, Kras, Pde3b, Pik3cb, Cbl, Sos2, Phkb, Pik3cd, Pik3r1, Mapk10, Prkaa1, Socs3, Irs4, Flot1, Mapk3, Mapk1), positive regulation of insulin secretion (Arrb1, Ppp3cb, Camk2n1, Gpr68, Gja1, Bglap2), regulation of glucose metabolism (Kat2b, Slc35b4, Grb10, Usp7, Mtor, Bckdk, Ogt, Foxk2) and catabolic processes (Hk1, Foxk2), cellular glucose homeostasis (Slc39a14, Pck1, Ogt, Hk1, Foxk2).

[0190] In certain embodiments, the subject composition can be used in a method for wound healing. In certain embodiments, the protein and / or peptide of the present invention induces differentially expressed genes, which are significantly enriched in positive regulation of wound (Cldn13, Dmtn, Enpp4, Vegfb, Mtor, Foxc2) and vascular wound (Vegfb, Foxc2) healing, angiogenesis (Ndnf, Kdr) involved in wound healing, and regulation of inflammatory response to wound (Git1, Alox5, Cd24a). In certain embodiments, the protein and / or peptide of the present invention enhances macrophage polarization, which also plays an important role in wound healing.

[0191] In certain embodiments, the subject composition can be used in the method for the treatment of postmenopausal syndrome. In certain embodiments, the protein and / or peptide of the present invention inhibits the osteoporosis induced by ovarian ablation (OVX), which is one of the models of postmenopausal syndrome. In certain embodiments, the protein and / or peptide induce differentially expressed genes, which are significantly enriched in the positive regulation of the intracellular estrogen receptor signaling pathway and the response to estrogen. In certain embodiments, the protein and / or peptide can treat some postmenopausal syndromes, such as osteoporosis.

[0192] In certain embodiments, the subject compositions can be used in methods for infertility treatment. In certain embodiments, the proteins and / or peptides induce differentially expressed genes that are significantly enriched in oocyte differentiation (Atm, Bcl2) and maturation (Fbxo5, Rps6ka2, Ereg, Ccnb1, Foxo3), spermatogenesis (Axl, Bax, Crkl, Sfmbt1, Bcl2l1, Herc4, Sgpl1, Ube2b, Apob, Tlk2, Vipas39, Mettl3, Bcl6, Bsg, Bag6, Prdx4, Prnd, Bcl2l11, S100a11, Celf3, Spaca1, Pum1, Ndc1, Inpp5b, Nek1, Mlh1, Wdr48, Ythdc1, Siah1a, Parp11, Mical2, Arid4b, Tdrd6, Slco4c1, Dmxl2, Katnal1, Setx, Cfap43, Zfp41, Catsperg2, Prss43, Cdyl, Hspa2, H3f3a, Cyp26b1, Nr6a1, Dazap1, Btbd18, Mfsd14a, Fancf, Syne1), sperm mitochondrial organization (Ddhd1, Vps13a, Nectin2, Mfsd14a), fertilization regulation (Cfap69), developmental processes involved in reproduction (Pparg, W dr77, Axl, Tgfb1, Bax, Stat5a, Ptpn6, Prlr, Celf1, Crkl, Sfmbt1, Tgfbr1, Bcl2l1, Arid1a, Map3k4, Ptgis, Med1, Lhx9, Ubb, Fbxo5, Rspo3, Kitl, H erc4, Sgpl1, Ube2b, Rps6kb1, Myocd, Apob, Tlk2, Stat5b, Vipas39, Hif1a, Zfp36l1, Ctsl, Mettl3, Bcl6, Dlg1, Tnk2, Bsg, Rps6ka2, Birc6, Epas1, M sh2, C3, Bag6, Prdx3, Prdx4, Ube3a, Plk4, Serpine2, Asb1, Lhx4, Nr5a1, Sp3, Prnd, Bcl2l11, Src, Ncoa3, Smad9, S100a11, Celf3, Spaca1, Pum1, Nd c1, Pithd1, Inpp5b, Ereg, Tmf1, Ddias, Tial1, Flna, Nek1, Irx5, Fem1b, Mlh1, Wdr48, Fanca, Irf2bpl, Atm, Hectd1, Ythdc1, Siah1a, Paqr7, Icam1,Serpine1, Pank2, Parp11, Mical2, Ncoa6, Taf4, Abhd2, Arid4b, Tdrd6, Slco4c1, Dmxl2, Katnal1, Ccnb1, Snai1, Serpinb6b, Greb1l, Setx, Adam6a, Cf ap43, Ermp1, Zfp41, Pygo2, Foxo3, Catsperg2, Tifab, Foxc1, Lin28a, Junb, Socs3, Mapk14, Sec23ip, Dach1, Cebpb, Bcl2, Bak1, Prss43, Rsl1, Cdyl, Hs pa2, H3f3a, Mapk3, Mapk1, Cyp26b1, Nr6a1, Dazap1, Etnk2, Ccnf, Fsip2, Syna, Xist, Btbd18, Mfsd14a, Fancf, Syne1), cellular processes involved in the reproduction of multicellular organisms (Wdr77, Tgfb1, Bax, Celf1, Tgfbr1, Bcl2l1, Zmiz1, Ubb, Fbxo5, Kitl, Ube2b, Rps6kb1, Top2a, Mettl3, Tnk2, Rps6ka2, Msh2, Src, Spaca1, Pithd1, Ereg, Tmf1, Etv6, Dd ias, Tial1, Ccnb2, Mlh1, Fanca, Atm, Siah1a, Paqr7, Pank2, Ttk, Abhd2, Tdrd6, Ccnb1, Cfap43, Spesp1, Pygo2, Foxo3, Foxc1, Lin28a, Sec23ip, Bcl2, Prss43, Cdyl, Hspa2, Prmt7, H3f3a, Cyp26b1, Zbtb16, Fsip2, Btbd18, Mfsd14a), blastocyst development regulation (Ttll4, Agbl4), inner cell mass cell proliferation (Setdb1, Prpf19, Gins4, Chek1, Nca pg2, Palb2), intrauterine embryonic development (Ccm2, Smo, Keap1, Dll3, Tgfbr1, Bcl2l1, Zmiz1, Cr1l, Med1, H13, Cops3, Ube2b, Apob, Gna13, Kat2a, Gli3, Dab2, Zfpm2, Msh2, Mib1, Pdgfrb, Zfand5, Sp3, Traf6, Bcl2l11, Dhx35, Mbnl1, Cpt2, Ybx1, Inpp5b, Add1, Grin2b, Atp11a, Ccnb2, Tgfbr2, Npat, Epn1, Ankrd11, Ythdc1, Gpi1,Wdtc1, Sec24d, Sec24c, Ccnb1, Maff, Ubr3, Palb2, Foxc1, Slc39a1, Junb, Klf2, Trp53, Zbtb18, Uty, Etnk2, Atf7), positive regulation of uterine smooth muscle contraction, response to progesterone (Tgfb1, Ube3a, Src, Wbp2, Abhd2, Mbp), progesterone receptor signaling pathway (Ube3a, Src, Klf9, Wbp2, Yap1), and Response to estrogen (Adcy3, Adcy7, Bcl2, Creb1, Creb3l1, Fos, Gnai1, Gp9, Hras, ​​Hspa2, Jun, Kcnj6, Kras, Krt14, Krt24, Krt26, Mapk1, Mapk3, Mmp2, Ncoa3, Oprm1, Pik3cb, Pik3cd, Pik3r1, Pik3r3, Plcb2, Shc1, Shc2, Sos1, Sos2, Src).

[0193] In certain embodiments, the subject compositions can be used in methods of treating hematopoietic disorders.In certain embodiments, the proteins and / or peptides induce differentially expressed genes that are significantly enriched in the regulation of hematopoietic or lymphoid organ development (Tgfb1, Stat5a, Cnn2, Crk1, Pknox1, Tgfbr1, Med1, Ikzf1, Kit1, Psen1, Sgpl1, Mknk2, Id2, Klf11, Stat5b, Hif1a, Tnfsf11, Epas1, Pdgfrb, Csf1r, Men1, Nfkb2, Slc40a1, Tnfrsf11a, Ptprc, Sp3, Bcl2l11, Slc46a2, Ddias, Ccnb2, Tgfbr2, Zbtb 1, Lyl1, Atm, Ttc7, Ccr7, Picalm, Cdk13, Lyn, Sh2b3, Onecut1, Zfp36l2, Wbp1l, Samd9l, Cxcr5, Selplg, Ccr2, Zfpm1, Bcl3, Bcl2, Trp53, Mapk3, Mapk1, Zbtb16, Ceacam1), hematopoietic progenitor cells (Ap2a2, Nfe2l2, Eif2ak2, Zbtb1, Sos2, Ankle1, Foxc1) and stem cell differentiation ((Ap2a2, Nfe2l2, Eif2ak2, Foxc1), hematopoietic stem cell homeostasis (Tcirg1, Glis2, F stl1, Arhgef5, Ubap2l, Emcn, Ext1), regulation of hematopoietic stem cell proliferation (Kitl, Eif2ak2, Cxcl1, Atxn1l), positive regulation of angiogenesis (Itgb2, Hk2, Aqp1, Notch4, Nfe2l2, Ptgis, Nr2e1, Xbp1, Hif1a, Sema5a, Cyp1b1, C3, Itgb8, Cxcr2, Jak1, Add1, Ctsh, Tgfbr2, Ccr3, Btg1, Hspb6, Lrg1, Serpine1, Pik3cd, Agtr1a, C5ar1, Sphk1, Chil1), involvement in sprouting blood vessels Positive regulation of proliferation of newly formed vascular endothelial cells (Hmox1, Vegfa, Ppp1r16b, Aplnr, Agtr1a), positive regulation of cell migration involved in sprouting angiogenesis (Hdac9, Hmox1, Akt3, Plk2, Vegfa, Anxa1, Srpx2, Foxc2, Kdr), angiogenesis (Ccm2, Foxm1, Smo, Tgfb1, Epha2, Zmiz1, Sgpl1, Myocd, Zfp36l1, Zfpm2, Itgb8, Spred1, Tbx20, Tgfbr2, Gjc1, Junb), angiogenesis involved in coronary vascular morphogenesis (Epor, Fgfr2).In certain embodiments, the proteins and / or peptides may also protect RBCs from hypotonicity-induced hemolysis. In certain embodiments, the proteins and / or peptides may therefore be therapeutic interventions for blood-related diseases such as hemolytic anemia.

[0194] Materials and methods

[0195] 5.1 Materials and Animals

[0196] 5.1.1.HKUOT-S2 Isolation Equipment, Materials and Reagents

[0197] A high-speed refrigerated centrifuge (hicmac CR21G II) from HITACHI and an AKTAexplorer purifier from GE Healthcare were provided by the Department of Surgery and the School of Chinese Medicine, University of Hong Kong (HKU), respectively. HKUOT-S2 separation columns, namely Hiprep DEAE FF 16 / 10 (Catalog No. 28-9365-41), Hiprep Phenyl FF (high Sub) 16 / 10 (Catalog No. 28-9365-45) and Superdex 75 Increase, 10 / 300 GL (Catalog No. 29-1487-21) were purchased from GE Healthcare, USA. The following reagents were also purchased for HKUOT-S2 extraction and purification (catalog number and supplier in parentheses): glacial acetic acid (catalog number 3839-2.5L, DUKSAN), β-mercaptoethanol (catalog number A2008-250ML, Biomatik), 99%, ultrapure ammonium sulfate (catalog number J11254-A1, Thermo Scientific), snake skin dialysis tubing (catalog number 68700, Thermo Scientific), ultrapure sodium chloride (catalog number J21618-A1, Thermo Scientific), Tris (catalog number 15504-020, Invitrogen), Na2HPO4 (catalog number 3153 / 500G, Tocris), ovalbumin (catalog number A5253, Sigma) and BSA (catalog number A-420-250, GoldBio), and aprotinin (catalog number 4139 / 10, Tocris)

[0198] 5.1.2. Surgical equipment and reagents

[0199] Vicryl suture 6-0 (catalog number W9981) and Mersilk suture 5-0 (W500) were purchased from Ethicon. Heparin sodium injection (catalog number HK-28227) was obtained from B. Braun Melsungen, and betadine was provided by the Department of Orthopaedics and Traumatology, HKU.

[0200] 5.1.3. Animal Permits and Ethical Approval

[0201] The animal license for the use of mice for this experiment [reference number: (20-1000) in DH / HT&A / 8 / 2 / 3Pt.14] was granted by the Licensing Office, Department of Health, Hong Kong Government. Ethical approval for the handling of animals in this study was obtained from the HKU Ethics Committee, Committee on the Use of Live Animals in Teaching and Research (CULATR) (CULATR 5502-20).

[0202] 5.1.4. Acute toxicity study equipment and reagents

[0203] Microhematocrit centrifuge (Catalog No. SKU: VQ5578(a)) and glass capillaries (Catalog No. 87002-161) were purchased from Iris Sample Processing and VITREX, respectively. Clinical chemistry analyzer (BS-230) was purchased from Mindray by the School of Chinese Medicine, HKU. Cuvettes for BS-230 (Catalog No. HTM 115-037543-00), ALP (Catalog No. HTM 105-000816-00a), ALT (Catalog No. HTM 105-000814-00a), AST (Catalog No. HTM 105-000815-00a), CREA-S (Catalog No. HTM 105-004614-00), urea (Catalog No. HTM 105-000824-00a), multi-serum calibrator (Catalog No. HTM 105-001144-00a), and CD80 detergent (Catalog No. HTM 105-000748-00) were purchased from Health Pro Technology Co. Ltd.

[0204] Cell lines

[0205] Mouse M0 macrophage RAW264.7 cell line (Catalog No. TIB-74), mouse preosteoblast MC3T3-1E subclone 4 (Catalog No. CRL-2593), and human premonocyte U937 cell line (Catalog No. CRL-1593.2) were purchased from ATCC.

[72] Harvest human turbinate mesenchymal stromal cell line (hMSC).

[0206] 5.1.6. In vitro cell culture reagents and media

[0207] DMEM low glucose medium (catalog number 11885076), DMEM high glucose medium (catalog number 12100-046), RMPI medium 1640 (1X) (catalog number a10491-01), fetal bovine serum (FBS) (catalog number 10270-106, Gibco), penicillin-streptomycin (P / S) (catalog number 15140122), L-glutamine (catalog number 21051-024), 0.25 μg / ml amphotericin B (catalog number 15290-018), 0.25% trypsin-EDTA (catalog number 25200-056) and 10X phosphate buffered saline (PBS) (catalog number 70011-044) were purchased from Gibco, Thermo Scientific. NaHCO3 (S5761-1KG), HEPES (H4034-1KG) and MTT (M5655-100MG) were purchased from Sigma.

[0208] 5.1.7. Cell polarization and differentiation compounds and chemicals

[0209] Lipopolysaccharide (LPS) (Catalog No. L4391-1MG), dexamethasone (Dex) (Catalog No. D4902-25MG), L-ascorbic acid (Asc) (Catalog No. A4544-100G), β-glycerophosphate disodium salt hydrate (β-Gly) (Catalog No. G9422-50G), phorbol 12-myristate 13-acetate (PMA) (Catalog No. 79346-1MG) were purchased from Sigma. Recombinant mouse IL-4 (Catalog No. 404ML) and recombinant human IL-4 (Catalog No. 204-IL) were purchased from R&D Systems.

[0210] 5.2. Extraction and separation of HKUOT-S2 protein

[0211] Dioscorea opposita tubers were purchased from a wet market in Hong Kong.

[38] ] Extraction and separation of HKUOT-S2 were performed. In short, weighed (g) tubers were peeled. 2 volumes (ml) of cold extraction buffer consisting of 5% acetic acid and 0.1% β-mercaptoethanol were added to 1 weight portion (g) of tubers and mixed in a hood. The resulting homogenized mixture was magnetically stirred for 3 hours at 4°C. The homogenate was supersaturated with 80% (NH4)2SO4 and stirred overnight at 4°C. The cold homogenate was centrifuged at 4°C and 14000rpm for 1 hour using a high-speed refrigerated centrifuge to precipitate the crude protein mixture. The crude protein mixture was resuspended in cold Milli-Q water, distributed in snake skin dialysis tubing (7000MWCO), dialyzed overnight in Milli-Q water at 4°C, and centrifuged at high speed refrigerated at 14000rpm for 2 hours. The collected dialyzed crude protein mixture was processed for downstream continuous fast protein liquid chromatography (FPLC) purification. To isolate the novel HKUOT-S2 protein, the dialyzed crude protein mixture was sequentially subjected to different column purifications. First, the dialyzed crude protein was lyophilized, dissolved in 100 mM tris buffer, and subjected to ion exchange chromatography using a HiPrep 16 / 10 DEAE FF column (GE Healthcare, Sweden), buffer A (100 mM Tris) and buffer B (1 M NaCl + 100 mM Tris, gradient (0-45%). The osteogenic fraction D3 (HKUOT-D3) obtained from the crude protein extract was then dialyzed and lyophilized. The concentrated solution of HKUOT-D3 in buffer A (Milli-Q water) was added to buffer B (10 mM Na2HPO4 + 1 M (NH4)2SO4) in a 1:1 ratio. The reconstituted HKUOT-D3 solution was subjected to hydrophobic interaction chromatography using a HiPrep Phenyl FF (high sub) 16 / 10 column (GE Healthcare, Sweden), buffer A and gradient buffer B (30-0%). The osteogenic fraction D3 obtained from HKUOT-D3 and dissolved in buffer A (50 mM The dialyzed and lyophilized osteogenic peak P1 (HKUOT-P1) in Na2HPO4+150mM NaCl) was subjected to size exclusion chromatography (SEC) using a Superdex 75Increase 10 / 300GL column (GE Healthcare, Sweden) and buffer A, and the column was calibrated with known molecular weight markers [aprotinin (6.5kDa), ovalbumin (44.287kDa) and BSA (66kDa)]. SEC produced pure fraction S2 (HKUOT-S2 protein). All columns were installed on an AKTA purifier (GE Healthcare, Sweden) FPLC system and operated according to the recommendations of the specific manufacturer. The dialyzed and lyophilized osteogenic HKUOT-S2 was stored at -80°C for downstream applications.

[0212] Characterization of HKUOT-S2 protein

[0213] 5.3.1. Prediction of molecular weight of HKUOT-S2 using mathematical model

[0214] The molecular weight (MW) of HKUOT-S2 was mathematically predicted using a standard curve of calibrated Superdex 75 Increase 10 / 300GL column information and the Kav equation.

[0215] According to the Kav equation formula,

[0216]

[0217] Where Ve = elution volume (11.232 ml), V0 = void volume (8 ml) and Vc = column volume (24 ml)

[0218]

[0219] The standard curve of the calibrated Superdex 75Increase 10 / 300GL column is shown in Fig. 27 middle.

[0220] From the standard curve, we can see that Kav=y and Log Mw=X.

[0221] From the linear equation of the standard curve, we know that Kav = -0.4268 (Log MW) + 2.1401

[0222]

[0223] Mw(HKUOT-S2)=104.5410=34753Da=34.8kDa

[0224] From the column calibration standard curve of proteins with known molecular weight, it can be seen that the Kav value is inversely proportional to the protein molecular weight.

[0225]

[0226]

[0227] Where K = constant = Kav(known protein)*MW(known protein)

[0228]

[0229] Ovalbumin with a Kav of 0.15 and a Mw of 44287 Da is used as a reference protein, and Kav(HKUOT-S2) is 0.202.

[0230]

[0231] Therefore, the MW (HKUOT-S2) ranged from 32.9 to 34.8 kDa.

[0232] 5.3.2. Determination of HKUOT-S2 MW by silver staining

[0233] HKUOT-P1 and HKUOT-S2 proteins were electrophoresed using 15% SDS-PAGE according to the manufacturer's protocol and analyzed using Pierce TM Silver staining was performed using a silver staining kit (Cat. No. 24612, Thermo Scientific). Silver staining showed a single band near 32.9 kDa for HKUOT-S2 and a double band between 32 kDA and 35 kDA for HKUOT-P1.

[0234] 5.3.3. HKUOT-S2 molecular mass determination and mass spectrometry identification

[0235] HKUOT-S2 protein bands were cut for sequential MALDI-MS and high-resolution LC-MS / MS analysis at the Proteomics and Metabolomics Core Facility (PMcore) of the Pan-omics Research Center (CPOS) of the LKS School of Medicine, University of Hong Kong. MALDI-MS determined that the molecular weight of HKUOT-S2 was approximately 32 kDa, consistent with the molecular weight determined by mathematical modeling and silver staining techniques. For LC-MS / MS analysis, in-gel protein trypsin digestion was performed to digest the HKUOT-S2 protein into peptides. The resulting peptides were extracted from the gel in sequence using 50% ACN / 5% FA and 100% ACN. The combined peptides were quickly vacuum dried, desalted and processed using C18 StageTips for LC-MS / MS analysis. The eluted peptides were analyzed using a Dionex Ultimate3000 nanoRSLC system coupled to an Orbitrap Fusion Tribid Lumos mass spectrometer (Thermo Fisher) and separated using a commercial C18 column coupled to a NanoTrap column (Thermo Fisher). The full mass spectrometer (MS) survey scan resolution was set to 120 000 with an automatic gain control (AGC) target value of 4 × 105, a maximum ion injection time (IT) of 30 ms, and a scan range of 400–1500 m / z. Spectra were acquired at 30 000 MS2 resolution, an AGC target of 5 × 104, a maximum IT of 80 ms, an isolation width of 1.6 m / z, and a normalized collision energy of 30. Raw MS data were processed using MaxQuant 1.6.14.0 and searched against the Dioscorea UniProt FASTA database (June 2020) containing 18,477 entries. Confident proteins were identified using a target-decoy approach with a reverse database, with a stringent false discovery rate of 1% at the peptide and peptide spectrum match (PSM) level.

[0236] 5.3.4. HKUOT-S2 de novo sequencing

[0237] Targeted LC-MS / MS de novo sequencing was performed to identify HKUOT-S2. Peptides obtained by LC-MS / MS were matched to the NCBInr Dioscorea protein database. The NCBI Protein Basic Local Alignment Search Tool (BlastP) result E-value <0.001 was considered significant and used as an inclusion criterion for high-quality alignment. Individually identified de novo peptides of the HKUOT-S2 protein had no significant matches to the NCBInr Dioscorea protein database. BlastP against the HKUOT-S2 de novo peptide sequence from Dioscorea also produced no significant peptide sequence matches. HKUOT-S2 may be a new candidate protein with a unique de novo peptide sequence. PEAKS Studio X-Pro analysis of these unique de novo sequences revealed peptide intensities and m / z ratios.

[0238] 5.3.5. HKUOT-S2 N-terminal peptide sequencing

[0239] The N-terminal sequencing of HKUOT-S2 using Edman degradation was completed by Creative Proteomics Company, USA. The N-terminal sequencing of HKUOT-S2 was performed on a polyvinylidene fluoride (PVDF) membrane. Analyzed on ABI Procise 494HT (Thermo Fisher). The N-terminal amino acid residues of HKUOT-S2 were cut off one at a time and identified by chromatography. Edman degradation chemistry involves first coupling the PITC reagent to the N-terminal amino group under alkaline conditions, and then cutting the N-terminal residue under acidic conditions. The PITC coupling residue was transferred to a flask, then converted into a PTH-residue and identified by HPLC chromatography. Then the next cycle was started to identify the next N-terminal residue. N-terminal sequencing of HKUOT-S2 by Edman degradation chemistry revealed a unique N-terminal sequence.

[0240] In vivo studies

[0241] A total of 96 C57BL / 6 male mice were used in these in vivo studies. Mice aged 8 to 10 weeks, weighing between 22 and 25 g, were obtained from the HKU Laboratory Animal Department (LAU) and provided with good temperature, clean shelter, food and water, good ventilation, and humane handling. Mice were housed in standard open-top cages in the housing department of the HKU Center for Comparative Medicine Research (CCMR). Before the start of the experiment, the mice were allowed to adapt to their new environment for one week after transportation.

[0242] 5.4.1. Effect of HKUOT-S2 treatment on hemolysis

[0243] Hemolysis test on mouse red blood cells (RBC) was performed to determine the anti-hemolytic effect of HKUOT-S2 protein

[47] Briefly, 1 mL of fresh heparinized whole blood samples were collected from euthanized C57BL / 6 male mice by cardiac puncture. Blood samples were centrifuged at 1,500 × g for 5 min, and the resulting RBC pellet was washed four times with 1 ml of 1X PBS. After the last centrifugation, the RBC pellet was washed with 1X PBS (negative control), 1X triton-X-100 (positive control), 3.13-200 μg mL -1 HKUOT-S2, diclofenac, melittin and venom treatment 200 ul in 0.5 mL Eppendorf tube 6.0 x10 7 RBC suspension. Incubate the blocked and parafilm-sealed tubes at 37°C for 1 hour and then centrifuge at 1,500 rpm for 5 min. Carefully transfer 50 ul of sample supernatant in triplicate to a 96-well plate. Measure the optical density of the samples at 540 nm (OD540) using a microplate reader.

[0244]

[0245] 5.4.2. Effect of HKUOT-S2 treatment on hypotonicity-induced hemolysis

[0246] According to previously published protocols

[73] A hypotonic solution-induced hemolysis assay was performed to investigate the hemolysis inhibitory effect of HKUOT-S2. Briefly, 10 μl of stock RBC suspension was added to a solution containing 0–2000 μg ml -1 HKUOT-S2 protein, BSA, diclofenac sodium and 1 ml hypotonic solution (0.45% NaCl, pH 7.4) of chloranthate. 10 ul RBC suspension in 1 ml isotonic (0.9% NaCl, pH 7.4) and hypotonic (0.45% NaCl, pH 7.4) solution without any drug was used as negative and positive controls, respectively. The mixture was incubated at room temperature for 1 hr and centrifuged at 1000 rpm for 10 min. 50 ul sample supernatant was carefully transferred to a 96-well plate in triplicate. The hemoglobin content in the supernatant was measured spectrophotometrically at 560 nm (OD560). The percentage of inhibition of hypotonic-induced hemolysis was calculated as:

[0247]

[0248] Wherein, ODα = OD560 of negative control (0.9% NaCl)

[0249] ODβ = OD560 of sample treated in 0.45% saline solution

[0250] ODγ = OD560 of positive control (0.45% NaCl)

[0251] 5.4.3. Acute toxicity study of HKUOT-S2 treatment in vivo

[0252] The acute toxicity of HKUOT-S2 treatment was evaluated in mice. The initial body weight and physical appearance of mice were recorded before HKUOT-S2 treatment. Mice were treated with 1.09-4.36 mg kg -1 HKUOT-S2 treatment was performed three times per week, while sham-operated controls were treated with 1XPBS (pH 7.4) only. On the first day of HKUOT-S2 treatment, mice were observed for any signs of toxicity in the first 30 min after drug administration, then observed every 4 hr, and then observed for signs of toxicity every day until the end of the experiment (week 4). During the entire study, changes in mouse body weight (g) were also measured weekly. 100 mg kg -1 Mice were euthanized by pentobarbital. Blood samples were collected via cardiac puncture for biochemical analysis, and liver and kidneys were surgically harvested and frozen for RNA extraction and histopathological analysis by qPCR. Since HKUOT-S2 treatment had no toxic effects in non-operated mice, the same experimental procedures and evaluations of the acute toxicity study of HKUOT-S2 treatment were also repeated in the bone defect mouse model.

[0253] 5.4.4. Effect of HKUOT-S2 treatment on hematocrit in mice

[0254] Manual microhematocrit test was performed to evaluate the effect of HKUOT-S2 treatment on RBC levels in vivo. About 9 μl of venous blood was collected from the saphenous vein of sham-operated control group and HKUOT-S2-treated mice into heparinized microhematocrit capillary tubes. The test tubes were sealed with model clay and centrifuged at 3000 g for 2 minutes using a StatSpin veterinary centrifuge. The hematocrit was manually determined using a microhematocrit capillary reader (StatSpin). The measurements were double-confirmed with a 30 cm ruler.

[0255] 5.4.5. Effects of HKUOT-S2 treatment on serum biochemistry

[0256] Clinical biochemical analysis is carried out to assess the impact of HKUOT-S2 treatment on liver and kidney function in vivo.Non-heparinized whole blood samples were collected from sham-operated control and HKUOT-S2-treated mice by cardiac puncture in 1.5ml Eppendorf tubes.The collected blood samples were kept at 4 ° C for 30 minutes to allow blood coagulation.The coagulated samples were centrifuged at 1000rpm for 10min.Serum was collected gently and aliquoted into new 0.2ml Eppendorf tubes.Using automated clinical chemistry analyzer (Mindray Bs-230), triplicate 70ul serum in colorimetric dish was subjected to biochemical analysis to determine serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), blood urea nitrogen (urea) and creatinine (CRE) levels.

[0257] Effects of HKUOT-S2 treatment on bone defect healing in vivo

[0258] All animal surgical procedures and protocols were performed in strict accordance with the methods approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong. Two days before and 5 days after surgery, mice were given a 4-mg / kg -1 ) in drinking water. Before surgery, 100 mg kg -1 Ketamine hydrochloride and 4mg kg -1 The mice were anesthetized with xylazine. The hair on the dorsal skin of the right femur was shaved. The mice were placed on a heating pad and given a subcutaneous injection of 1 ml of warm saline solution. The shaved skin was cleaned alternately with betadine and 70% ethanol 4 times. A sharp sterile razor blade was used to cut the superficial and subcutaneous skin and muscle layers along the femur to expose the femur. A circular bone defect was created 2 mm above the epiphyseal plate (diaphysis) of the distal femur on the right side. To create a bone defect, a sterile 21G needle was held in the hand, minimal but sufficient force was applied, and the surgically exposed bone was carefully and vertically pierced from the anterior surface down to the opposite posterior surface to drill a circular hole with a diameter of 0.9 mm. The bone defect site was rinsed with 1 ml of warm 1X PBS to flush out any bone fragments therein. The wound was closed by suturing the muscle with an absorbable Vicryl suture 6-0 system and suturing the subcutaneous and superficial skin layers with a Mersilk suture system. Mice were given an immediate subcutaneous injection of the antibiotic enrofloxacin (5 mg kg -1 ) and the analgesic buprenorphine (0.05 mg kg -1 ). Thereafter, the analgesic buprenorphine was administered twice daily for three days after surgery. The mice were randomly assigned to four experimental groups, namely, a sham control group, 1.09, 2.36, and 4.36 mg kg -1HKUOT-S2 treated group. Each experimental group contained 8 mice (n=8). Mice were given subcutaneous injections above the bone defect site immediately after surgery, 200 μl HKUOT-S2 solution in the HKUOT-S2 treated group and 200 μL 1X PBS in the sham control group, three times a week for 4 weeks. At the end of the experiment, blood samples were collected from sham control and HKUOT-S2 treated mice by cardiac puncture. Blood samples were processed for downstream applications such as hematocrit testing, clinical biochemistry, ELISA, and western blot analysis. Surgical and non-surgical femurs, livers, and kidneys were also harvested and processed for other applications such as qPCR, immunostaining, histology, and RNA-seq analysis.

[0259] 5.4.7. Microcomputed tomography (μCT) scanning analysis of bone defects.

[0260] μCT scans of the bone defect sites of anesthetized mice were performed using Skyscan (Catalog No. 1076, Bruker) immediately after surgery and then weekly for 1 month to monitor HKUOT-S2-induced bone defect repair in vivo. X-ray images obtained from bone scans were reconstructed into 2D images using software (NRecon) provided by Skyscan. 3-D images of sham-operated controls and HKUOT-S2-treated femurs were reconstructed using CTvox software. To determine the mineral density of bone samples, samples of known density (0.25 and 0.75 gcm) were scanned using the same μCT settings used for bone tissue scanning. - 3) Two phantoms of calcium hydroxyapatite (CaHA) rods (2 mm in diameter). 0.25 and 0.75 g / cm - The measured attenuation coefficients (AC) of the three phantoms are 0.02616 and 0.05773 mm respectively. -1 The BMD and TMD of μCT scanned bones were calibrated with the known density of CaHA and the corresponding AC using CTAn software. The effects of HKUOT-S2 treatment on bone parameters such as BV / TV, BMD, TMD, Tb.th, Tb.N, Tb.Sp, BS / BV, and BS / TV during bone defect repair were then evaluated using CTAn software.

[0261] 5.4.8. Histological processing of undecalcified bone tissue

[0262] Femurs from mice treated with sham-operated controls and HKUOT-S2 were harvested at the experimental endpoint. The harvested bone tissue was immediately transferred to 10% neutral buffered formalin (10% NBF) and fixed for 24 hr. Afterwards, the bone was dehydrated in increasing grades of ethanol (70%-100%), transparentized in xylene, and embedded in methyl methacrylate 1 (MMAI), methyl methacrylate II (MMAI+20g dibenzoyl peroxide) and methyl methacrylate III (MMA I+20g dibenzoyl peroxide+250ml dibutyl phthalate) in sequence. The embedded sample was cut into 200 μm, and further ground or polished into thinner slices between 50-70 μm. Selected slices were subjected to Giemsa staining.

[0263] 5.4.9. Histological processing of decalcified bone tissue

[0264] 10% NBF-fixed femurs harvested from sham-operated controls and HKUOT-S2-treated mice were decalcified in 0.5M ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich) for 48 hours. The decalcified bones were washed under running water for 30 minutes, dehydrated in increasing grades of ethanol (70%-100%), and then cleared in xylene. The samples were then processed and embedded in paraffin for downstream staining and analysis.

[0265] 5.4.10. Fluorescent dye labeling

[0266] Fluorescent dye labeling was used to evaluate new bone formation at the bone defect site during HKUOT-S2 treatment. Briefly, 10 mg kg -1 Calcein green (Catalog No. C0875-5G, Sigma-Aldrich) and 90 mg kg -1 Xylenol orange (Catalog No. X-0127, Sigma-Aldrich). Calcein green was administered intraperitoneally postoperatively (day 19). Xylenol orange was also administered intraperitoneally three days before euthanasia. Harvested undecalcified bone tissue was embedded in MMA and processed for inverted confocal fluorescence microscopy. An inverted confocal fluorescence microscope Carl Zeiss (LSM900) equipped with ZEISS (ZEN 3.4, blue version) software was used to acquire and quantify the in vivo fluorescent dye intensity.

[0267] 5.4.11. Hematoxylin and eosin (H&E) staining of decalcified bone

[0268] Paraffin-embedded femurs were sliced ​​at 4 μm using a rotary microtome. Paraffin sections were placed on adhesive tissue slides for histological staining. For H&E staining, bone tissue on slides was dewaxed with xylene and rehydrated in decreasing grades of ethanol (100%-70%) and water. The rehydrated bone tissue was stained with Harris hematoxylin modified solution (HHS32-1L, Sigma-Aldrich) for 2 minutes, washed in water, differentiated in 70% ethanol containing 1% HCl, bluing in 0.1% NH4OH solution, and counterstained with eosin (E03210-7G, UNI-Chem). The tissue was continuously dehydrated in 70%-100% ethanol, transparentized in xylene, dried, and covered with a coverslip using the mounting medium Pertex (Catalog No. 41-40011-00, Medite).

[0269] 5.4.12. Giemsa staining of MMA-embedded bone sections

[0270] 50-70 μm sections of MMA embedded bone tissue were slightly deplasticized in 2% HNO3 (aqueous solution) and immersed in xylene for 2 minutes. Bone tissue was rehydrated in ethanol (100%-70%) and ddH2O in descending order. The tissue was immersed in 10X Giemsa stain for 3-4 minutes, washed in ddH2O for 2 minutes, air-dried and covered with a cover glass using a mounting medium.

[0271] 5.4.13. Trichrome staining of paraffin-embedded bone sections

[0272] 4 μm sections of paraffin-embedded femur tissue were dewaxed, rehydrated, stained with prepared Weigert iron hematoxylin, washed with ddH2O and 1% acetic acid, and incubated in azo red solution (catalog number 100485 / 1, Sigma-Aldrich) for 10 minutes, then washed in 1% acetic acid for 30 seconds. The tissue was immersed in tungstophosphate orange G solution (catalog number 100485 / 2, Sigma-Aldrich) for 1 minute, and in light green SF solution (catalog number 100485 / 3, Sigma-Aldrich), washed with 1% acetic acid in between. Bone tissue was dehydrated in increasing order of ethanol, transparentized in xylene, air-dried, and fixed with coverslip using mounting medium. All histologically stained bone tissues were observed under an Eclipse 80i compound fluorescence microscope (Nikon) equipped with NIS-Elements software. Bright field images were taken at 10 times and 20 times magnification.

[0273] 5.4.14. Double staining of paraffin-embedded bone sections with TRACP and ALP

[0274] TRACP and ALP dual staining was performed on 4 μm bone sections using the TRACP&TRAP dual staining kit (catalog number mK300, Takara) following the manufacturer's guidelines. Briefly, 500 ul of acid phosphatase (ACP) substrate was added to the bone tissue and incubated at 37°C for 45 min, followed by washing in ddH2O. Then 500 ul of alkaline phosphatase (ALP) substrate was added to the tissue sections and incubated at 37°C for 45 min. The washed bone tissue was counterstained with a nuclear staining reagent for 5 min at room temperature. The bone tissue was washed, air-dried, mounted with a coverslip using mounting medium, and observed under an Eclipse 80i compound fluorescence microscope. TRAP at the bone defect site and growth plate was quantified using Image J software + and ALP + The number of cells.

[0275] 5.4.15. Transmission electron microscopy (TEM)

[0276] Bone tissue harvested from the sham control group and HKUOT-S2 treated group was decalcified. The 2 mm bone tissue including the bone defect site was subjected to TEM analysis in the HKU Electron Microscope Unit. Philips CM100 TEM was used to obtain detailed images of bone tissue sections at different magnifications (1200-5200X) for qualitative cellular and intracellular ultrastructural analysis.

[0277] 5.4.16. Enzyme-linked immunosorbent assay (ELISA)

[0278] HKUOT-S2-induced ALP and OCN levels during bone defect repair were assessed by mouse BALP (Catalog No. CSB-E11914m, CUSABIO) and mouse OC / GPG (Catalog No. NBP2-68151, NOVUSBIOLOGICAL) ELISA kits according to the manufacturer's instructions. Briefly, 100 μl of freshly prepared standards, serum, and bone lysate samples from the sham control group and HKUOT-S2 treatment group were added to the micro-ELISA plate in duplicate and incubated at 37°C for 90 or 120 minutes. The standard and sample solutions were replaced with freshly prepared 100 μl of biotin-antibody solution and incubated at 37°C for 60 minutes with gentle shaking. The micro-ELISA plate was washed 4 times with 200 μl of wash buffer. 100 μl of freshly prepared 1X HRP-conjugated working solution or 1X HRP-avidin was added to each well and incubated at 37°C for 30 or 60 minutes, followed by 4 washes. A sealed micro-ELISA plate containing 90 μl of substrate reagent or TMB substrate was incubated at 37 °C in the dark for 15 min. Then 50 μl of stop solution was added to the micro-ELISA plate and tapped gently to ensure adequate mixing. The OD value of each well was determined spectrophotometrically at 450 nm. The levels of BALP and OCN in serum and bone lysates of the sham control group and HKUOT-S2-treated group were determined using the standard curve linear equation. The measured total protein was used to normalize the levels of BALP and OCN in the bone lysate samples.

[0279] 5.4.17. RNA sequencing of rRNA depletion

[0280] Femurs harvested from the sham and HKUOT-S2 treated groups were wrapped in aluminum foil and ground in liquid nitrogen using a mortar and pestle to prevent RNA degradation. RNAiso plus reagent (Cat. No. 9109, Takara) and PureLink TM High-quality total RNA was extracted from bone tissue using RNA mini kit (Cat. No. 12183018A, Thermo Scientific). The OD260 / OD280 of the resulting RNA solution was also between 1.98 and 2.00, and the RNA integrity number (RIN) was >8. -1 (n=3), 2.18 mg kg -1 (n=3) and 4.36 mg kg -1(n=3) RNA samples from HKU OT-S2 treatment group were used for RNA sequencing (RNA-seq). Library preparation, Illumina sequencing (151 bp paired-end sequencing) and transcriptome bioinformatics were performed at the Genomics Core Facility of the Center for Pan-omics Science (CPOS) of HKU LKS School of Medicine. Before the RNA-seq process, the RNA samples were purified using FastSelect TM Multi RNA Depletion Kit (Human Complete rRNA & Globin mRNA, Catalog No. THS-201Z-24) depletes the bulk of rRNA in 0.5ug RNA samples to ensure RNA-seq quality captures the most informative portion of transcriptome data

[74] During next-generation sequencing (NGS) library preparation, FastSelect TM The reagent depletes cytoplasmic and mitochondrial rRNA. The cDNA library was prepared using the KAPA mRNA HyperPrep Kit (KR1352-v.116). Double-stranded (ds) cDNA was 3' adenylated and indexed by dual-indexed UMI aptamers. The aptamer-ligated library was enriched by 12 cycles of polymerase chain reaction (PCR), denatured and diluted to the optimal concentration. Paired-end 151bp sequencing was performed using Illumina NovaSeq 6000. RNA-seq reads were assigned to each experimental sample using Illumina (bcl2fastq) software. The average throughput per sample was 12.1Gb and the total throughput was 144.6Gb. In terms of sequence quality, an average of 92% of the bases reached a quality score of Q30, where Q30 indicates an accuracy of 99.9% for base identification. Transcriptome pairwise bioinformatics analysis was used to generate HKUOT-S2-induced differentially expressed genes and transcripts in defective bone. KEGG pathways and GO entries for HKUOT-S2-induced gene enrichment were evaluated using Partek genomic suite (PGS) software from HKU CPOS. The transcripts per million (TPM) values ​​from RNA-seq were used for further analysis. Unsupervised hierarchical clustering was plotted with gplots using the z-scores calculated for each gene in the sample.

[75] The most variable 1000 genes were included in the analysis.

[0281] 5.4.18. Validation of differentially expressed genes in HKUOT-S2-induced repair bone

[0282] qPCR analysis was performed on RNA from bones of sham control and HKUOT-S2 treated groups to validate HKUOT-S2 induced differentially expressed genes related to BMP, TGF-β, AMPK, and mTOR signaling pathways that promote bone defect repair in vivo. AMPK and mTOR related genes (Prkaa1, Prkaa2, Prkab1, Prkab2, Prkag1, Prkag2, Prkag3, and Mtor), BMP and TGF-β related genes (Bmp2, Bmp7, Bmpr2, Tgfβr2, and Bmpr7) induced by HKUOT-S2 were validated by qPCR.

[0283] In vitro studies

[0284] In vitro cell culture

[0285] hMSCs and human monocytes, U-937 cell lines were cultured and maintained in T75 flasks containing 10 ml of DMEM low glucose medium and in cell culture dishes containing 10 ml of RMPI medium 1640 (1X), respectively. Both cell culture media were supplemented with 10% heat-activated FBS, 100 U ml -1 P / S. RAW264.7 and MC3T3-1E cell lines were cultured and maintained in cell culture dishes and T75 flasks, respectively, supplemented with 3.72 mg / ml NaHCO3, 5.685 mg ml -1 , HEPES, 10% FBS, 2 mM L-glutamine), 0.25 μg mL -1 Amphotericin B, 100 U ml -1 10ml DMEM high glucose medium of P / S. hMSC and MC3T3-1E cell lines were cultured and maintained for 4-5 days, and the cell culture medium was changed every 3 days. Cells were passaged to 80% confluence by washing twice with warm 1X PBS, adding 1ml 0.25% trypsin-EDTA to each flask and incubating at 37°C for 3 minutes to detach the cells, adding 5ml normal cell culture medium to quench the cell trypsin action, centrifuging at 1000rpm for 5 minutes to collect the cell pellet, resuspending the cell pellet with 1ml culture medium, and finally, adding 100ul of cell suspension to 10ml culture medium in a new T75 flask. U-937 and RAW264.7 cells were passaged every 3 days, gently washing the cells twice with 5ml warm 1X PBS, removing loosely attached cells with 3ml fresh warm cell culture medium, and adding 100ul of cell suspension to 10ml culture medium in a new cell culture dish. All cells were maintained in a humidified incubator at 37 °C and 5% CO2.

[0286] Effects of HKUOT-S2 treatment on the viability of hMSCs and RAW264.7 cells

[0287] hMSCs and RAW264.7 cells were cultured at 1.5 × 10 3 cells / well and 2×10 4 Cells / well were seeded in triplicate into 96-well plates. Cells were incubated under cell culture conditions for 1, 2, and 3 days. At each time point, cells were washed with FBS-free medium. Then, 50 μl of 0.45 mg ml - 1 MTT-free FBS medium was added and incubated in the dark at 37°C and 5% CO2 for 4 hrs, after which the medium was carefully discarded. 100 μl of DMSO was added to each well and incubated for 15 minutes to dissolve the formazan crystals. The optical density of triplicate wells was measured at absorbance (OD) 570nm (OD570) and 640nm (OD640) reference. Cell viability % was calculated as:

[0288]

[0289] 5.5.3. Effect of HKUOT-S2 on hMSC differentiation into osteoblasts

[0290] hMSC and MC3T3-1E cell lines were cultured at 2.5×10 4 cells / well and 2.5×10 3 Cells / well were seeded into 6-well plates containing 2 ml of normal cell culture medium. The cell culture medium was then replaced with osteogenic culture medium containing 100 nM Dex, 10 mM β-Gly and 0.28 mM Asc, with or without HKUOT-S2 protein. The cells were maintained in osteogenic culture medium for 14 days, with the culture medium replaced every 3 days. Undifferentiated cells maintained in normal cell culture medium were used as controls. The cell pellets were then collected and processed for osteogenic gene expression analysis by qPCR at the end of the experiment.

[0291] 5.5.4. Effect of HKUOT-S2 on macrophage polarization

[0292] RAW264.7 cells were cultured at 2 × 10 5 Cells / well were seeded into 6-well plates and cultured for 24 hours. -1 LPS and 20 ng ml -1 Mouse IL-4 24 hr, in the presence or absence of 0.01-1.0 μg ml -1HKUOT-S2 protein was used to polarize cells into M1 and M2 macrophages. U937 cells were cultured at 2×10 4 Cells / well were seeded into 6-well plates and cultured for 24 hr. U937 cell culture medium was supplemented with 20 ng ml -1 The cells were incubated for 48 hr to activate the suspended pro-monocytes to attach to the bottom of the wells and differentiate into mature M0 macrophages. The attached cells were washed twice with 1X PBS and treated with 100 ng ml -1 LPS and 20 ng ml -1 Human IL-4 24 hr, in the presence or absence of 0.01-1.0 μg ml -1 M1 and M2 macrophage polarization was performed under HKUOT-S2 protein. Non-polarized cells were used as controls. RAW264.7 and U937-derived M1 and M2 macrophages were scraped and the collected pellets were processed for qPCR, flow cytometry, and western blot analysis. Conditioned medium (CM) from U937-derived M1 and M2 macrophages was collected for hMSC-osteoblast differentiation and cytokine array analysis.

[0293] 5.5.5. Effects of HKUOT-S2- and HKUOT-P-induced M1 and M2 macrophage CM on biomineralization of hMSC-derived osteoblasts

[0294] 1×10 4hMSCs seeded into 24-well plates were cultured for 24 hrs with cells / well. In the continuous presence or absence of HKUOT-S2 and HKUOT-P proteins, the cell culture medium was replaced with a 1:1 ratio of culture medium containing osteogenic medium and U937 cell-derived macrophage CM. The cells were maintained in osteogenic macrophage CM culture medium for 10 days, with the culture medium replaced every 3 days. At the end of the experiment, the cells were gently washed 4 times with warm 1X PBS and fixed with 4% paraformaldehyde (4% PFA) at room temperature for 15 minutes. The fixed cells were gently washed 4 times with ddH2O and then stained with 0.5ml 40mM Alizarin Red S (ARS) in the dark for 45 minutes at room temperature. The stained cells were gently washed 6 times with ddH2O. The plate was air-dried overnight in the hood, and then 0.2ml 10% acetic acid was added to each well and gently shaken for 45 minutes. The Th cells scraped in acetic acid were transferred to a 1.5ml Eppendorf tube and vortexed for 30 seconds. The sealed tubes were placed in a heating block at 85°C for 10 minutes. The tubes were then cooled on ice for 5 minutes and then centrifuged at 20000g for 15 minutes. 200ul supernatant was transferred to a clean 0.5ml Eppendorf tube, and 75ul 10% NH4OH was added to each tube to quench the effect of acid. ARS standards were prepared as 40mM, 20mM, 10mM, 5mM, 0.25mM, 0.125mM, 0.0625mM and 0mM by serial dilution. 50 μl standards and samples were transferred to 96-well plates in triplicate, and absorbance was read at 405nM using a microplate reader. The amount of biomineralization in each treatment group was determined using the linear equation generated from the standard curve.

[0295] 5.5.6. HKUOT-S2 treatment inhibits XL388-induced mTOR inhibition to promote osteoblast differentiation

[0296] In the presence / absence of 125-500 XL388 or 0.1 μg ml -1 hMSCs differentiated into osteoblasts when HKUOT-S2 was treated for 7 days and the culture medium was replenished every 3 days. The collected cell pellets were processed for qPCR and western blot analysis to establish the mechanism by which HKUOT-S2 promotes osteogenesis and new bone formation. A list of all genes and corresponding primer pairs and primary and secondary antibodies used for qPCR and western blot analysis is listed in Tables 6, 7, and 8.

[0297] Table 6: Mouse genes and primer pairs used for qPCR

[0298]

[0299]

[0300]

[0301]

[0302]

[0303] Table 7: Human genes and primer pairs used for qPCR

[0304]

[0305]

[0306] Table 8: Primary and secondary antibodies used for Western blotting

[0307]

[0308] 5.6 Experimental Results Analysis and Evaluation

[0309] Quantitative real-time PCR (qPCR) analysis

[0310] According to the manufacturer's protocol, high-quality total RNA with an OD260 / OD280 between 1.98 and 2.00 was extracted from cell lines for in vitro experiments and tissues harvested from in vivo experiments using RNAiso plus reagent (Cat. No. 9109). The quality and quantity of the extracted RNA were determined using a NanoDrop One (C) UV-VIS microspectrophotometer (Thermo Scientific). Equal amounts of RNA in the experimental groups were reverse transcribed into complementary DNA (cDNA) using a PrimeScriptTM RT reagent kit (Cat. No. RR037A, Takara) and a Peltier thermal cycler (Cat. No. PTC-100, MJ Research) according to the manufacturer's instructions. Specific primer pairs, equal amounts of cDNA from different experimental groups, and PowerUp TM SYBR TM Green premix (Cat. No. A25742, Thermo Scientific) was used to prepare the qPCR reaction mixture. Using 10 μl of qPCR reaction mixture of control and HKUOT-S2-treated groups loaded into 96-well plates (0.2 ml), HKUOT-S2-induced gene expression in cell lines from in vitro and harvested tissues from in vivo experiments was evaluated using the QuantStudio 5 Real-Time PCR System (ThermoScientific). After normalization of housekeeping genes, the expression of HKUOT-S2-induced genes was evaluated by 2 -ΔΔCt The fold changes of differential gene expression induced by HKUOT-S2 were calculated by qPCR. A list of all genes and corresponding primer pairs used for qPCR is listed in Table 3.

[0311] Table 3: De novo peptide sequences of HKUOT-S2 protein

[0312]

[0313]

[0314] 5.6.2. Flow cytometric analysis of primary M0-derived macrophages treated with HKUOT-S2

[0315] Primary bone marrow macrophages were harvested from 8-week-old mice according to published protocols.

[76] In brief, pentobarbital (100 mg kg -1 )Euthanize the mice. Harvest the femurs into cell culture dishes under sterile conditions. In a sterile cell culture hood, add 10 ml of 70% ethanol to the femur for 1 minute. Transfer the femur, which has been washed 3 times with warm 1XPBS, to another 100 mm cell culture dish containing 10 ml of mouse macrophage (RAW264.7 cell) culture medium. Use a pair of scissors to cut both ends of the femur. Use sterile forceps to fix the femur vertically. Use a 26G×1 / 2” needle (filled with cell culture medium) attached to a 10 ml syringe to rinse the bone marrow cells several times into a 50 ml test tube. Pass the cell suspension through a 70 μm cell strainer into another 50 ml test tube and then centrifuge at 1000 rpm for 5 minutes. Resuspend the cell pellet in 4% paraformaldehyde supplemented with 10 ng ml -1 100 mm cell culture dishes were added with 10 ml of cell culture medium supplemented with murine recombinant M-CSF. The cells were incubated under cell culture conditions for one week, with the medium changed every 3 days. On day 7, the cells were passaged with cell culture medium not supplemented with M-CSF. Primary M0 macrophages were cultured at 2 × 10 5 Cells / well were seeded in 6-well plates and cultured under appropriate cell culture conditions for 24 h. -1 LPS and 20 ng ml -1 Mouse IL-4 was added for 24 h, and the cells were polarized into M1 and M2 macrophages in the presence or absence of HKUOT-S2 or HKUOT-P1 protein treatment, respectively. 0.5 ml of the treatment was 2.0 × 10 7 The cells were suspended in a single cell suspension at 100 cells / ml and co-stained with fluorescently labeled CD206 and MGL-1 antibodies for flow cytometry analysis using an Agilent NovoCyte Quanteon analyzer.

[0316] 5.6.3. Cytokine array analysis of HKUOT-S2 polarized M1 and M2 macrophage CM

[0317] According to the manufacturer's scheme, the cytokine array analysis of M1 and M2 macrophage CM induced by HKUOT-S2 was performed using Mouse XL cytokine array kit (catalog number ARY028, R&D Systems). In short, 4 membranes placed in the hole 1-4 of a 4-well multi-well culture dish containing 2ml blocking buffer were incubated on an oscillator for 1 hour. The blocking buffer was replaced with 1.5ml of each sample solution so that hole 1-4 contained M2 (M2 macrophage positive control), M1 (M1 macrophage positive control), M1+HKUOT-S2 and M1+HKUOT-S2 CM respectively. The membrane was incubated overnight at 4 ° C on an oscillator, and washed several times with 1X washing buffer. The membrane in 1.5mL 1X array buffer 4 / 6 containing 30 μl of detection antibody mixture was incubated in oscillation at room temperature, and then washed with 1X washing buffer. Each membrane was incubated in 2.0 mL 1X Streptavidin-HRP at room temperature for 30 minutes, washed, and treated with 1 ml Chemi reagent mix for 60 seconds. The detected cytokine array intensity was visualized using GE Amersham Imager AI680 and quantified by ImageJ software.

[0318] Western blot analysis

[0319] 2×10 5 RAW264.7 cells were seeded at 10 cells / well and cultured for 24 h. - 1 HKUOT-S2 or 5 μg ml -1 In the case of HKUOT-P1, the cells were polarized into M1 and M2 macrophages. The collected cell pellets were washed with 5% paraformaldehyde supplemented with 25X cOmplete TM The cells were lysed with 60 μl of RIPA buffer (Cat. No. 89900, Thermo Scientific) and EDTA-free protease inhibitor cocktail (Cat. No. 1187358001, Roche Diagnostics). The proteins were analyzed using the Pierce Coomassie Plus (Bradford) Protein Assay Kit (Cat. No. 23236, Thermo Scientific) and Pierce TM Total protein concentration was measured spectrophotometrically using bovine serum albumin standard (Catalog No. 23209). 30 μg of protein lysate (12.5 ul reaction mixture) separated by 8% SDS-PAGE in running buffer and 10 μl of PageRuler TMPlus prestained protein ladder (Catalog No. 26619, Thermo Scientific) was transferred to the PVDF membrane in cold transfer buffer for 2 hours. The PVDF membrane was blocked with 10 ml of PBST (0.5 ml Tween 20 in 1 L PBS) containing 5% BSA (Catalog No. A-420-250, GoldBio) for 30 minutes. The PVDF membrane was then incubated in a 5% PBS containing 10 ul of arginase-1 (D4E3M TM ) Rabbit mAb (1:1000 dilution) (Cat. No. 93668, Cell Signaling Technology) in 10 ml blocking buffer (PBST + 5% BSA) was incubated at 4°C with gentle shaking overnight. The washed membrane was incubated with ECL TM Anti-rabbit IgG-HRP secondary antibody (Catalog No. NA934, Amersham) was incubated on a shaker at room temperature for 2 h. The membrane was washed with 1X PBST and incubated with Pierce TM ECL Western Blotting Substrate (Catalog No. 32209, Thermo Scientific) was incubated for 1 minute. ARG-1 protein band intensity was visualized using GE Amersham Imager AI680 and quantified using ImageJ software. Table 4 lists all primary and secondary antibodies used for Western blotting.

[0320] Table 4: Number of differentially expressed genes and transcripts

[0321]

[0322] All patents, patent applications, provisional applications, and publications mentioned or cited herein, including all figures and tables, are incorporated by reference in their entirety to the extent they are not inconsistent with the explicit teachings of this specification.

[0323] The following are examples illustrating procedures for practicing the present invention. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight and all solvent mixture ratios are by volume.

[0324] Example 1 - Isolation of a new HKUOT-S2 protein from Dioscorea opposita

[0325] To isolate the novel osteogenic HKUOT-S2 protein from Dioscorea species, crude protein ( Fig.17A The novel HKUOT-S2 protein was isolated from the crude protein extract using ion exchange, hydrophobic interaction, and high-resolution size exclusion chromatography techniques to sequentially separate the osteogenic fractions D3 (HKUOT-D3), P1 (HKUOT-P1), and the novel S2 (HKUOT-S2) proteins, respectively ( Figures 17A-17B ).

[0326] Example 2 - Phytochemical Screening of Crude Protein Extracts and Fractions

[0327] Dioscorea plants are not only carbohydrate-rich tubers, but also contain other essential components such as water, inulin, tannins, organic acids, phenols, proteins and antioxidants. [39,40] Qualitative phytochemical screening of the crude protein extract of D. opposita and its serially purified derivatives for the presence or absence of carbohydrates, saponins, phytosterols, phenolics, flavonoids, amino acids, and proteins

[41] . As expected, the results showed that the crude protein extracts HKUOT-D3 and HKUOT-P1 did not contain carbohydrates (Table 1). However, the crude protein extracts contained saponins, phytosterol-triterpenoids, and flavonoids, which were not present in HKUOT-D3 and HKUOT-P1. Both HKUOT-D3 and HKUOT-P1 contained amino acids and proteins (Table 1). Therefore, it can be inferred from the phytochemical results that HKUOT-S2 derived from HKUOT-P1 is a protein molecule.

[0328] Table 1: Phytochemical analysis of yam protein extracts and isolates

[0329]

[0330] Example 3 - Characterization of HKUOT-S2 protein

[0331] The downstream applications of the new HKUOT-S2 were characterized by different techniques, such as molecular weight determination using mathematical modeling, silver staining, mass spectrometry, de novo peptide sequencing, and N-terminal sequencing.

[0332] The molecular weight of HKUOT-S2 was predicted by mathematical model

[0333] High-resolution size exclusion chromatography (SEC) functions to separate molecules in solution according to their size or molecular weight.

[42] To determine the molecular weight of HKUOT-S2 protein, the column used in SEC was calibrated with proteins of known molecular weight. After protein elution, a standard curve of known proteins was plotted using the column and elution information. The linear equation of the standard curve (y = mx + c) and the Kav equation were used. [35,42] The molecular weight of HKUOT-S2 was predicted to be in the range of 32.9-34.8 kDa (Table 2).

[0334] Table 2: Prediction of HKUOT-S2 molecular weight by mathematical model

[0335]

[0336] Determination of the molecular mass of HKUOT-S2 by silver staining and mass spectrometry

[0337] To further confirm the molecular weight of HKUOT-S2, HKUOT-P1 (P1) and HKUOT-S2 (S2) proteins were run in 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then silver stained to visualize the protein bands for molecular markers therein ( Fig. 17C ). Silver staining results showed a single band of HKUOT-S2 with a molecular weight of about 32 kDa ( Fig. 17C HKUOT-P1 has two close bands (lower band and upper band). The lower band of HKUOT-P1 has a similar molecular weight to that of HKUOT-S2 ( Fig. 17C ). Next, the HKUOT-S2 protein band in 15% SDS-PAGE was excised and subjected to mass spectrometry analysis to accurately determine the molecular weight. Mass spectrometry determined the molecular weight of HKUOT-S2 to be 32.22 kDa ( Fig.17D ).

[0338] HKUOT-S2 is a novel protein

[0339] Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) analysis was performed to identify the HKUOT-S2 protein. The HKUOT-S2 peptide was not mapped to any reference protein database. Therefore, high-resolution liquid chromatography tandem mass spectrometry (LC-MS / MS) analysis was performed on the HKUOT-S2 protein for de novo peptide sequencing. The results showed that the de novo derived peptides had high scores, which showed correctly sequenced strings. Two analysis methods were used to identify the target HKUOT-S2: 1) the HKUOT-S2 protein sequence was matched with the entire plant kingdom protein sequence downloaded from Uniprot using a conventional protein database analysis method, but there was no significant peptide match, 2) a targeted de novo sequence method was used to confirm the novelty of the HKUOT-S2 protein. The NCBInr Dioscorea protein database was used for matching. The NCBI Protein Basic Local Alignment Search Tool (BlastP) results with an E value <0.001 were considered significant and used as inclusion criteria for high-quality alignments. From this analysis, several candidate de novo peptides were identified. However, BlastP of the de novo peptide sequence validation method for HKUOT-S2 from Dioscorea also resulted in no significant protein matches. Therefore, it was concluded that HKUOT-S2 might be a novel candidate protein. LC-MS / MS results also revealed that HKUOT-S2 had unique de novo peptide sequences, such as KTVSLPR (SEQ ID NO: 81), KGNLLECDGGNTAQMMAR (SEQ ID NO: 82), TKSSLPGQTK (SEQ ID NO: 83), and KEVSLPR (SEQ ID NO: 84) ( Figures 17E-17H , Table 3). Analysis of these de novo sequences on PEAKS StudioX-Pro also revealed peptide intensities and m / z ratios ( Figures 17E-17H , Table 3).

[0340] N-terminal sequencing of HKUOT-S2 by Edman degradation chemistry

[0341] Finally, N-terminal sequencing analysis of HKUOT-S2 was performed to further identify its N-terminal peptide sequence. Here, the HKUOT-S2 protein run in 15% SDS-PAGE was transferred to a polyvinylidene fluoride (PVDF) membrane. The amino acid residues on the PVDF membrane were cut off one at a time and identified by chromatography. The N-terminal sequence of HKUOT-S2 obtained is IKITTYRQ (SEQID NO: 86) (Mw = 1022.55Da), corresponding to the three-letter sequence Ile-Lys-Ile-Thr-Thr-Tyr-Arg-Gln (SEQID NO: 86) (Mw = 1022.55Da).

[0342] Example 4 - In vivo studies

[0343] Before the HKUOT-S2 protein was used in vivo for bone defect repair, a comprehensive acute toxicity study was performed on HKUOT-S2 to ensure the safe application of this new protein in vivo. It was determined that the therapeutic dose of HKUOT-S2 used in this study had no toxic effects on mice ( Figures 18A-18W ).

[0344] Example 5 - HKUOT-S2 significantly enhances bone defect repair in vivo

[0345] Dioscorea plant proteins such as diosgenin have been shown to stimulate osteoblast differentiation in vitro

[35] . The role of Dioscorea proteins in fracture healing in vivo has not been fully explored. Here, a bone defect model was used to investigate the bone defect repair potential of HKUOT-S2. A circular hole was drilled in the femur of mice and then HKUOT-S2 was treated for 4 weeks. Weekly micro-computed tomography (μCT) scan analysis revealed that HKUOT-S2 protein promoted bone defect healing in vivo ( Figure 1A-1B Quantification of the CT scan images using CTAn software revealed that 2.18 mg kg -1 and / or 4.36 mg kg -1 HKUOT-S2 treatment gradually and significantly increased bone volume (BV / TV), bone mineral density (BMD), tissue mineral density (TMD), trabecular thickness (Tb.th), trabecular number (Tb.N), and the ratio of bone surface area to tissue volume (BS / TV), but reduced trabecular separation (Tb.Sp) and had no significant effect on the ratio of bone surface area to bone volume (BS / BV) ( Figure 1C-1J ). These results demonstrate that HKUOT-S2 treatment significantly enhances bone defect repair in vivo.

[0346] Example 6-Fluorescent dye labeling reveals HKUOT-2-induced bone defect repair

[0347] To monitor HKUOT-S2-induced new bone formation during bone defect repair, sham-operated control and HKUOT-S2-treated mice were injected with calcein green (calcein G) and xylenol orange (xylenol O) on days 19 and 26 after surgery, respectively. The mice were killed on day 29 after surgery. The harvested femurs with bone defects were fixed, processed and embedded in methyl methacrylate (MMA) plastic blocks. MMA-embedded bone tissue sections were processed for confocal fluorescence microscopy. Fluorescence microscopy analysis showed that the fluorescent pigment intensity of calcein G at the bone defect site in the HKUOT-S2-treated group was significantly higher than that in the sham-operated control group ( Figure 2A-2B ). The results showed that HKUOT-S2 treatment promoted new bone formation.

[0348] Example 7 - Histological analysis shows that HKUOT-2 promotes bone defect repair

[0349] To evaluate the effect of HKUOT-S2 on bone defect repair, harvested bones were fixed, paraffin-embedded, processed for hematoxylin and eosin (H&E), and stained with Masson-Goldner trichrome. MMA-embedded bone tissue was processed for Giemsa staining. Observation of H&E-stained samples under a light microscope revealed that HKUOT-S2 treatment repaired femoral fractures (yellow oval scars) within 4 weeks after surgery compared with the sham control group ( Figure 2C Bone tissue and collagen fibers appear light pink under Giemsa staining ( Figure 2D In the HKUOT-S2-treated group, the collagen fibers at the bone defect site were well organized, indicating that the bone defect healing was almost complete ( Figure 2D One of the main characteristics of Masson-Goldner trichrome staining is that immature (unmineralized) and mature (mineralized) bone tissue stain green and red, respectively.

[43] Under Masson-Goldner trichrome staining, the bone defect sites in the HKUOT-S2-treated group stained redder (mature bone matrix) than those in the sham-operated control group, indicating that HKUOT-S2 treatment induced bone mineralization ( Figure 2E ). Since bone mineralization is the function of osteoblasts, it can be inferred that HKUOT-S2 treatment enhances the activity of osteoblasts to promote bone defect repair.

[0350] Example 8 - HKUOT-2 enhances bone defect repair via modulation of osteoclast and osteoblast activity.

[0351] Bone remodeling involves sequential regulation of osteoclast-induced bone resorption and osteoblast-stimulated bone formation

[18] Osteoblast activity usually exceeds osteoclast activity to favor new bone formation. Therefore, it was hypothesized that HKUOT-S2 treatment could reduce osteoclast activity but increase osteoblast function to enhance new bone formation. To test this hypothesis, 4 μm sections of paraffin-embedded bone tissue were double immunostained for TRAP and ALP. The results revealed that compared with sham controls and 1.09 mg kg - 1 Compared with the HKUOT-S2 treated group, 2.18 mg kg -1 and 4.36 mg kg -1 HKUOT-S2 treatment significantly reduced the number of TRAP+ cells (red arrows, osteoclasts) in the bone defect site ( Figure 2F-2G As expected, ALP+ cells (osteoblasts) were more concentrated in the growth plate ( Figure 2H). Quantification of osteoclasts (red arrows) and osteoblasts (black arrows) in growth plates ( Figure 2H-2I ). This is consistent with the observations at the bone defect site, 2.18 mg kg -1 and 4.36 mg kg -1 HKUOT-S2 treatment significantly decreased the number of osteoclasts at the growth plate, but increased the number of osteoblasts ( Fig.2I Immunohistochemistry (IHC) staining results confirmed that HKUOT-S2 treatment regulated osteoclast and osteoblast activities to enhance bone defect repair.

[0352] Example 9 - Transmission electron microscopy (TEM) analysis of osteoclast and osteoblast cytology in HKUOT-2 treated bone tissue

[0353] IHC results showed that HKUOT-S2 treatment reduced and increased the number of osteoclasts and osteoblasts, respectively, to enhance new bone formation. It can be assumed that HKUOT-S2 treatment impaired the structural integrity and ultrastructure of osteoclasts to reduce their number, thereby favoring new bone formation. To resolve this hypothetical debate, fixed and decalcified bone defect areas from the sham control group and HKUOT-S2-treated groups were used for TEM. TEM analysis at low magnification showed normal cytology and cellular architecture in all experimental groups. Consistent with the IHC results ( Figure 2F-2I ), compared with the sham control group and 1.09 mg kg -1 Compared with the HKUOT-S2 treated group, -1 and 4.36 mg kg -1 A decrease in the number of multinucleated osteoclast-like cells, but an increase in the number of osteoblast-like cells was observed in the HKUOT-S2-treated group ( Figure 2J TEM analysis of osteoblasts and osteoclasts at high magnification revealed normal cellular ultrastructures such as cell membrane, endoplasmic reticulum (ER), mitochondria, and nuclei in all treatment groups ( Figure 2K-2L ). However, TEM analysis of osteoblast and osteoclast nuclei revealed that osteoblast nuclei in the HKUOT-S2 treated group were generally lighter in color (more euchromatic in nature) compared to those in the sham control group ( Figure 2K ), whereas osteoclast nuclei are typically darker (more heterochromatic in nature) ( Figure 2L ). Euchromatic nuclei are usually rich in gene transcriptional activity, while heterochromatic nuclei are less active in gene transcription. Therefore, HKUOT-S2 treatment can modulate the differential gene transcriptional potential of osteoblasts and osteoclasts to promote new bone formation and defect healing in vivo at the experimental endpoint.

[0354] Example 10 - HKUOT-S2-induced osteogenic gene expression in bone defects

[0355] The TEM results led to the hypothesis that HKUOT-S2 could increase osteogenic gene expression to stimulate bone defect healing. To test this hypothesis, total RNA extracted from the operated femurs was processed for osteogenic gene expression analysis. qPCR results showed that HKUOT-S2 treatment significantly increased the expression of Alp, Bglap1, Bglap2, and Runx2 in defective bones ( Figures 3A-3D ). The results determined that HKUOT-S2 treatment generally increased osteogenic gene expression to enhance bone defect repair.

[0356] Example 11-HKUOT-S2 treatment significantly increased BALP and OCN levels in vivo

[0357] In vitro and in vivo data consistently demonstrated that HKUOT-S2 treatment induced osteogenesis by increasing Alp expression. In addition, HKUOT-S2 treatment induced Bglap1 and Bglap2 gene expression in surgical bone tissue. Both Bglap1 and Bglap2 encode osteocalcin (OCN) protein. Therefore, it is expected that bone and / or serum ALP and OCN levels will also increase after HKUOT-S2 treatment. To evaluate the ALP and OCN levels elevated by HKUOT-S2, serum and bone lysates from the sham control group and HKUOT-S2-treated group were processed for enzyme-linked immunosorbent assay (ELISA) analysis using bone-specific ALP (BALP) and osteocalcin (OCN) ELISA kits. The results demonstrated that HKUOT-S2 treatment significantly increased BALP and OCN levels in serum and bone lysates, indicating that HKUOT-S2 treatment induced osteoblast activity to enhance new bone formation and defect repair in vivo ( Figure 3E-3H ).

[0358] Example 12 - Transcriptomic analysis of HKUOT-S2-induced bone defect healing

[0359] All the current data lead to the hypothesis that HKUOT-S2 treatment can induce differentially expressed genes to promote bone defect repair. To test this hypothesis, high-quality RNA extracted from defect bones of sham control and HKUOT-S2 treated groups was processed for RNA-seq with rRNA depletion. Transcriptome data analysis showed that HKUOT-S2 treatment induced differentially expressed genes and transcripts in defect bones (Tables 4 and 5). Filtering for differentially expressed genes and transcripts with FDR < 0.05 revealed that 1.09 mg / kg -1 , 2.18mg kg -1 and 4.36 mg kg -1HKUOT-S2 treatment induced 6038, 6158, and 1064 genes, 9477, 9974, and 2871 transcripts, respectively (Table 4). Among the differentially expressed genes, 792 genes were common to all three doses, and 3490 genes were only expressed at 1.09 mg kg -1 and 2.18 mg kg -1 The dose was 2.18 mg kg, with 111 genes only -1 and 4.36 mg kg -1 The dose was found to be only 1.09 mg kg for 50 genes -1 and 4.36 mg kg -1 The total number of HKUOT-S2 treatments was ( Figure 4A (Left)). 54% of the differentially expressed genes were up-regulated, while 46% were down-regulated ( Figure 4B , Table 5). In addition, among the differentially expressed genes, 440 up-regulated and 351 down-regulated genes were found to be common to all three doses of HKUOT-S2 treatment ( Figure 4A (center and right). Hierarchical clustering of the top 1000 most differentially expressed genes using heatmaps also illustrates the transcriptional distance between sham-operated controls and HKUOT-S2-treated groups ( Figure 4C ). The heat map results revealed that the sham control group was transcriptionally separated from the HKUOT-S2 treated group ( Figure 4C ). The HKUOT-S2 treated groups shared some common transcriptional patterns. -1 and 2.18 mg kg -1 The HKUOT-S2 treated group had a close transcriptional distance ( Figure 4C ). Therefore, the experimental groups were ranked in descending order according to their transcriptional separation as sham control, 4.36 mg kg -1 , 2.18mg kg -1 and 1.09 mg kg -1 HKUOT-S2 treated group. Then, the differentially expressed genes with FDR < 0.05 were analyzed using Partek Genomics Suite (PGS) software. HKUOT-S2 induced differentially expressed genes were significantly enriched in the maintenance, proliferation, development, differentiation, and function of neutrophils, monocytes, macrophages, osteoclasts, stem cells, and osteoblasts ( Figure 4D-4I ). Pathway enrichment analysis showed that HKUOT-S2 treatment significantly enriched genes in many Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways ( Figure 4J , 4K ; Figures 19A-19DHKUOT-S2 treatment significantly enriched genes in many KEGG pathways, such as AMPK signaling, focal adhesion, foxO signaling, osteoclast differentiation, PI3K-AKT signaling, MAPK signaling, VEGF signaling, TNF signaling, mTOR, and TGF-β signaling pathways ( Figure 4J , 4K ; Figures 19A-19D Functional analysis further revealed that HKUOT-S2 treatment significantly enriched genes in gene ontology (GO) terms such as regulation of anatomical structure morphogenesis, osteoblast, osteoclast and odontoblast differentiation, stem cell development and differentiation, intramembranous and endochondral bone formation, trabecular bone development, bone remodeling, bone and tooth mineralization, odontogenesis, musculoskeletal development and motility, chondrogenesis, and regulation of BMP signaling pathways ( Figure 4L ; 19E-19F). HKUOT-S2-induced differentially expressed genes were also significantly enriched in the mTOR protein complex and signal transduction pathway ( Figure 4M ).

[0360] Table 5: Percentage of differentially expressed genes

[0361]

[0362]

[0363] Example 13 - Verification of genes related to BMP, AMPK and mTOR signaling pathways

[0364] To verify some differentially expressed genes related to BMP, TGF-β, AMPK and mTOR signaling pathways, qPCR analysis was performed on RNA from defective bones. The results showed that HKUOT-S2 treatment significantly upregulated AMPK and mTOR-related genes in repaired bone tissue, such as Prkaa1, Prkaa2, Prkab1, Prkab2, Prkag1, Prkag2, Prkag3 and Mtor ( Figures 5A-5H HKUOT-S2 treatment also significantly increased the expression of Bmp2, Bmp7, and Bmpr2 in repaired bone tissue, but did not increase the expression of Tgfβr2 ( Figure 5I-5L ). Validation of these HKUOT-S2-induced differentially expressed genes suggested that HKUOT-S2 might regulate AMPK, mTOR, and BMP signaling pathways to enhance bone defect repair in vivo.

[0365] Example 14 - HKUOT-S2 Enhanced hMSC-Osteoblast Differentiation

[0366] Studies have shown that extracts from the rhizome and bark of Dioscorea batatas exhibit osteogenic activity by promoting osteoblast differentiation and function

[35] In this study, 5–20 μg ml -1 Crude protein extracts of HKUOT-D3 and HKUOT-P1) and 0.01–1 μg ml -1 HKUOT-S2 had no significant effect on the viability of hMSCs ( Fig. 20A , 20D ). It also showed that 5 μg ml -1 The crude yam protein extract exhibited osteogenic activity by increasing RUNX2 expression in hMSC- and MC3T3-E1 cell-derived osteoblasts ( Fig. 20H , 20L ). Transcriptome data from in vivo studies also showed that HKUOT-S2-induced differentially expressed genes were significantly enriched in the maintenance, proliferation, development, differentiation, and function of stem cells and osteoblasts ( Figure 4H , 4I To test and confirm the osteogenic activity of HKUOT-S2 protein, hMSCs were differentiated into osteoblasts in the presence or absence of HKUOT-S2. Quantitative polymerase chain reaction (qPCR) results showed that 0.1 μg ml -1 HKUOT-S2 enhances hMSC-osteoblast differentiation by significantly increasing ALP expression ( Fig. 6A The in vitro results support the osteogenic activity of HKUOT-S2 in enhancing bone defect repair in vivo.

[0367] Example 15 - HKUOT-S2 enhances polarization of M1 and M2 macrophages

[0368] Dioscorea villosa extract reportedly induces anti-inflammatory activity in mice

[44] Dioscorin from Dioscorea was also shown to trigger immunomodulatory activity in RAW264.7 cells and mice

[36] In this study, it was determined that 5-20 μg mL -1 Crude protein extracts, HKUOT-D3 and HKUOT-P1) and 0.01-1 μg mL -1 HKUOT-S2 had no significant effect on the viability of RAW264.7 cells ( Fig. 20B , 20C This study also revealed that HKUOT-S2-induced differentially expressed genes were significantly enriched in the regulatory functions of monocytes and macrophages in promoting bone defect healing in vivo ( Figure 4E). Therefore, RAW264.7 cells were polarized to M1 and M2 macrophages in the presence or absence of HKUOT-S2. The results showed that HKUOT-S2 significantly increased the M1 macrophage markers Socs3 and Tnfα, as well as the M2 macrophage markers Arg-1 and Mgl-1 ( Figure 6B-6E ). In addition, mouse primary M0 (monocyte) macrophages were subjected to M1 and M2 macrophage polarization with or without HKUOT-S2 treatment. The polarized macrophages were co-stained with fluorescently labeled CD206 and MGL-1 antibodies, and then subjected to flow cytometry analysis. The results showed that the number of CD206 and MGL-1 double-stained M1 macrophages polarized by LPS+HKUOT-S2 (70.5% CD206+ and MGL-1+ cells) was reduced compared with the positive control (LPS group; 81.9% CD206+ and MGL-1+ cells). However, the number of CD206+ cells induced by HKUOT-S2 in polarized M1 macrophages (18.4% CD206+ cells) was increased compared with the positive control (LPS group; 12.4% CD206+ cells) ( Fig. 6F In addition, IL4+HKUOT-S2 slightly increased CD206 and MGL-1 double-stained M2 macrophages (92.1% CD206+ and MGL-1+ cells) compared with the positive control (IL4 group; 87.6% CD206+ and MGL-1+ cells) ( Fig. 6F In addition, total protein lysates from HKUOT-S2 polarized M1 and M2 macrophages were subjected to Western blot analysis using ARG-1 antibody (M2 macrophage marker). Surprisingly, LPS+HKUOT-S2 increased ARG-1 protein levels in the M1 macrophage phenotype ( Figure 6G HKUOT-S2 treatment also increased the anti-inflammatory gene Ampkα1 in the polarized M1 macrophage phenotype ( Figure 6H ).

[0369] Example 16-HKUOT-S2 treatment significantly induced changes in the cytokine array of M1 and M2 macrophage conditioned medium (CM)

[0370] HKUOT-S2 has the potential to regulate M1 and M2 macrophage polarization ( Figure 6B-6E). Therefore, it was hypothesized that HKUOT-S2 might induce polarized macrophages to secrete some soluble factors for bone immunomodulatory activity. To test this hypothesis, cytokine array analysis was performed on HKUOT-S2-induced M1 and M2 macrophage CM. The results showed that HKUOT-S2 treatment reduced CCL17, CCL22, CXCL16, GDF-15, OPN in M1 macrophage CM, but increased CD14 and CD54 cytokines. HKUOT-S2 treatment also increased G-CSF in M2 macrophage CM, but reduced GDF-15 cytokine ( Fig.6I These results support reports that increases and decreases in these cytokines are beneficial for osteogenesis.

[45] .

[0371] Example 17 - HKUOT-S2-induced macrophage CM enhances osteoblast biomineralization

[0372] Next, U937 cells (a human monocytic cell line) were polarized into M1 and M2 macrophages in the presence or absence of HKUOT-S2. The collected macrophage CM was used to differentiate hMSCs into osteoblasts for 10 days. Alizarin Red S staining results showed that HKUOT-S2-treated M1 macrophage CM significantly increased osteoblast biomineralization ( Figure 6J , 6K), which is consistent with Lu et al.

[23] and Huang et al.

[46] HKUOT-S2 treated M2 macrophage CM also increased osteoblast biomineralization ( Figure 6J , 6L ).

[0373] Example 18 - HKUOT-S2 regulates the MTOR1 / 4E-BP1 / AKT1 axis to promote osteogenesis

[0374] The evolutionarily conserved mammalian target of rapamycin (mTOR) signaling pathway plays a key role in regulating osteogenesis and bone integrity [47,48] Ablation of mTOR1 signaling reportedly impairs skeletal development and growth in mice

[48] . Transcriptome data revealed that HKUOT-S2-induced differentially expressed genes were significantly enriched in the mTOR protein complex and signal transduction pathway ( Figure 4M ). Therefore, we hypothesized that HKUOT-S2 treatment could mechanistically regulate mTOR to enhance osteogenesis. To test this hypothesis, we first determined the -1 hKUOT-S2 can enhance hMSC-osteoblast differentiation in vitro ( Fig.21AhMSCs were then differentiated into osteoblasts in the presence / absence of mTOR inhibitor (XL388) or HKUOT-S2, followed by qPCR and western blot analysis. qPCR results showed that 125nM XL88 treatment significantly inhibited the expression of RUNX2, MTOR1, 4E-BP1, AKT1, and S6K1 ( Figures 7A-7F ). However, 0.1 μg ml -1 HKUOT-S2 treatment blocked the inhibitory effect of XL388 on increasing the expression of RUNX2, MTOR1, 4E-BP1, AKT1, and S6K1 during osteoblast differentiation ( Figures 7A-7F Western blot analysis further confirmed that 250 nM XL88 treatment prevented the phosphorylation of MTOR1 and its downstream target 4E-BP1. HKUOT-S2 treatment blocked the XL388 inhibitory effect and increased the phosphorylation of MTOR1 and 4EBP1 proteins ( Figure 7G , 7H , 7J). HKUOT-S2 treatment also increased total AKT1 protein levels, another downstream target of MTOR ( Figure 7N ). Normalize protein with β-actin or total protein if necessary ( Figure 7G-7N ). Therefore, in this study, it was proposed that HKUOT-S2 treatment can activate the MTOR1 / 4E-BP1 / AKT1 signaling axis to promote osteogenesis ( Fig.7O ).

[0375] Example 19 - HKUOT-S2 treatment induces neuromodulation to enhance bone defect repair

[0376] A circular hole was drilled in the femur of mice, followed by HKUOT-S2 treatment for 4 weeks. Weekly micro-computed tomography (μCT) scan analysis showed that HKUOT-S2 protein promoted bone defect healing in vivo. HKUOT-S2 treatment also induced neuromodulatory effects to promote bone defect repair in vivo ( Figures 8A-8H , 9A-9F, 10A-10H).

[0377] Example 20-HKUOT-S2 treatment promotes neuronal differentiation

[0378] The effect of HKUOT-S2 protein on the viability of neuroblastoma cell line Neuro2A was studied in vitro. The results showed that HKUOT-S2 had no significant effect on the cell viability of Neuro2A cells ( Fig.11A , 11B). In addition, the effect of HKUOT-S2 protein on the differentiation of Neuro2A cells into neurons was studied in vitro. When (1) the axon length > 2X the cell body diameter and (2) the differentiated cells (neurons) had both axons and dendrites, the number of differentiated cells in each field of view of the three groups was counted. The results showed that HKUOT-S2 protein significantly promoted the differentiation of Neuro2A cells into neurons in vitro ( Figures 11C-11E ).

[0379] Example 21 - HKUOT-S2 prevents ovariectomy (OVX)-induced osteoporosis

[0380] Estrogen is one of the important body hormones that promotes and maintains good bone health and integrity. The ovaries are the main estrogen manufacturing factories in women. Aging in women leads to a gradual decline in ovarian function. At the same time, estrogen levels also decline and reach their lowest levels during the menopausal stage. The decline in estrogen levels is associated with bone porosity and the subsequent development of osteoporosis. FDA-approved estrogen replacement is one of the commonly used therapeutic interventions to prevent osteoporosis. However, it has been reported that the continuous use of estrogen replacement therapy for osteoporosis has disadvantages, such as blood clotting problems and the development of breast cancer. Finding appropriate osteogenic interventions with no or minimal side effects can help maintain good bone health, integrity and function during the age-related decline in estrogen and help prevent the development of osteoporosis. It has been demonstrated in our laboratory that the new HKUOT-S2 protein can promote osteogenesis and the formation of new bone to promote the repair of bone defects in vivo. The safety of therapeutic doses of HKUOT-S2 in in vivo applications has been confirmed. Transcriptome data from in vivo studies also revealed that HKUOT-S2 treatment significantly enriched biological processes and functions associated with responses to the estrogen receptor signaling pathway in wild-type mice ( Fig. 12A ). Therefore, it was hypothesized that HKUOT-S2 protein could modulate the estrogen receptor signaling pathway to prevent the development of osteoporosis in vivo. The aim of this study was to investigate the clinical application of HKUOT-S2 protein in preventing the development of menopause-associated osteoporosis in ovariectomized (OVX) mice.

[0381] It has been demonstrated that OVX induces significant bone loss in C57BL / 6J mice that responds to estrogen treatment. A total of 32 female C57BL / 6J mice aged 8-12 weeks and weighing between 22-25 g were used in this study. All mice underwent dorsal and ventral surgery under anesthesia. Sham-operated controls underwent surgery without ovariectomy. The ovaries in the OVX group were completely removed from the anterior horn of the uterus. There were four groups, namely sham-operated control group (n=8), OVX control group (n=8), OVX+30 μg kg -1 Estrogen (E2, n = 8) and OVX + 2.18 mg kg -1HKUOT-S2 (n=8) treatment group. Sham control and OVX control mice were given 200 μl 1XBP subcutaneously. OVX+E2 and OVX+HKUOT-S2 groups were given 200 μl E2 and HKUOT-S2 via subcutaneous injection and IP injection, respectively. All treatments were started 4 hours after surgery, three times a week, and continued for 4 weeks after surgery. MicroCT (μCT) scans were performed on day 0 and every two weeks after surgery. At the end of the experiment, all mice in each group were killed under overdose anesthesia, and bone tissue was harvested and processed for bone tissue morphometry and immunohistochemistry analysis.

[0382] Our preliminary results revealed that 1.09 mg kg -1 and 2.18 mg kg -1 HKUOT-S2 treatment significantly enriched the biological processes and functions related to the estrogen receptor signaling pathway in wild-type mice ( Fig. 12A ). μCT scan results showed that 2.18 mg kg -1 HKUOT-S2 treatment prevented OVX-induced bone loss and osteoporosis by increasing bone volume (BV / TV (%)), bone surface density (BS / TV), trabecular thickness (Tb.th), and trabecular number (Tb.N), but decreasing bone surface / volume ratio (BS / BV) and trabecular separation (Tb.Sp). Figures 12B-12H ).

[0383] Example 22 - HKUOT-S2 inhibits progressive glucocorticoid-induced osteoporosis

[0384] Glucocorticoids such as cortisone, dexamethasone, and prednisolone exert anti-inflammatory and immunosuppressive activities. They are often used as medical interventions to treat pathological conditions caused by an overactive immune system. Therefore, glucocorticoids are used to treat autoimmune diseases, asthma, and severe allergies. In the early days of the COVID-19 pandemic, a dexamethasone and hydroxychloroquine mixture was even considered as a short-term treatment for COVID-19 patients with severe symptoms. Many side effects of long-term use of glucocorticoid drugs are well documented, as they are usually administered in high doses to induce the desired therapeutic effect. It is well documented that long-term use of glucocorticoids such as dexamethasone is detrimental to bone health. Glucocorticoid treatment has been reported to increase bone resorption by promoting osteoclastogenesis and inhibit new bone formation by reducing osteoblastogenesis in patients. Therefore, patients receiving glucocorticoid therapy are at very high risk of osteoporosis. It is recommended that all patients receiving glucocorticoid therapy take bisphosphonates, vitamin D, and calcium supplements to prevent progressive bone loss due to drug-induced osteoporosis. However, long-term use of bisphosphonates (anti-osteoporosis therapy) also has well-documented disadvantages, such as the development of osteonecrosis, esophageal inflammation, and severe muscle and joint pain. Therefore, there is a high demand for medical interventions against glucocorticoid-induced osteoporosis with minimal or no side effects. Our group discovered a new osteogenic HKUOT-S2 protein with fewer safety issues for clinical use. It was further demonstrated that the HKUOT-S2 protein can enhance inflammatory M1 and anti-inflammatory M2 macrophage polarization, osteogenesis, and new bone formation to promote bone defect repair in vivo. Transcriptome data from in vivo studies also revealed that HKUOT-S2 treatment significantly enriched biological processes and functions related to the response of wild-type mice to glucocorticoid stimulation ( Figure 1A ). This study hypothesized that HKUOT-S2 protein could enhance new bone formation by increasing osteoblastogenesis to inhibit the development of progressive glucocorticoid-induced osteoporosis in vivo. The purpose of this study was to investigate and establish the possible mechanism by which HKUOT-S2 protein inhibits the development of progressive glucocorticoid-induced osteoporosis.

[0385] Dexamethasone treatment has been shown to induce osteoporosis in animal models. In this study, dexamethasone (Dex) will be used as a drug to induce osteoporosis in mice. A total of 32 male C57BL / 6J mice, 8-10 weeks old and weighing between 22-25 g, will be used in this study. To test this research hypothesis, mice will undergo dexamethasone (Dex)-induced osteoporosis. There are four groups, namely a sham control group (n=8), 50 mg kg -1 Dex control group (n = 8), Dex + 10 mg kg -1 Risedronate (Ris) (positive control, n = 8) and Dex + 2.18 mg kg-1 HKUOT-S2 (n = 8) treatment group. Sham control and Dex control mice were given an intraperitoneal (IP) injection of 200 μl 1XBP. Dex + Ris and Dex + HKUOT-S2 groups will be given 200 μl Ris and HKUOT-S2 via IP injection. All treatments will be applied three times a week for 4 weeks after surgery. Micro-CT (μCT) scans will be performed on day 0 and every two weeks after surgery. At the end of the experiment, all mice in each group were killed under overdose anesthesia, and bone tissue was harvested and processed for bone tissue morphometry and immunohistochemical analysis.

[0386] Preliminary results showed that 1.09 mg kg -1 and 2.18 mg kg -1 HKUOT-S2 was significantly enriched in biological processes and functions related to responses to glucocorticoid stimulation, including dexamethasone (Dex), in wild-type mice ( Fig.13A In the Dex-induced osteoporosis model, preliminary results showed that, like the positive control, 2.18 mg kg -1 HKUOT-S2 can increase bone volume percentage (BV / TV), bone surface density (BS / TV), Tb.th and trabecular number (Tb.N) ( Fig. 13B , 13C , 13E-13G) but reduced bone surface / volume ratio (BS / BV) and trabecular separation (Tb.Sp) ( Fig.13D , 13H ), to inhibit the development of Dex-induced osteoporosis.

[0387] Example 23 - Study on the Anti-diabetogenesis and Anti-diabetes of HKUOT-S2

[0388] Diabetes is a multifactorial, chronic metabolic disorder that is often characterized by hyperglycemia. The World Health Organization (WHO) estimates that there are 422 million people with diabetes worldwide, making it the ninth leading cause of death worldwide in 2019. Approximately 1.5 million people with diabetes die each year. The main form of diabetes is type 2 diabetes, which is caused by insulin resistance. Type 1 diabetes is an autoimmune disease that results in insufficient insulin production by pancreatic beta cells. Gestational diabetes leads to elevated blood glucose levels in pregnant women. Impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) mediate between normal and hyperglycemic conditions. Pharmacological treatments for diabetes target glucose metabolism and / or insulin production and act to bias hyperglycemic conditions toward normoglycemia. Dioscorea extracts have been reported to have antidiabetic properties. However, the antidiabetic components and underlying mechanisms of the bioactive Dioscorea extracts have not been fully explored. In the ongoing exploration of the clinical application of the novel HKUOT-S2 protein, it was found that HKUOT-S2 treatment significantly enriched several signaling pathways, biological processes, and functions related to glucose metabolism and insulin function in wild-type male mice ( Fig.14A , 14B ). Therefore, it was hypothesized that HKUOT-S2 could regulate glucose metabolism and insulin function to inhibit the onset of diabetes development in mice.

[0389] Type 1 diabetes mellitus (T1DM) animal model

[0390] Multiple low-dose treatments of STZ

[0391] Multiple low-dose STZ treatments induce inflammatory effects, which partially destroy pancreatic islets and β cells, impair insulin production and cause hyperglycemia, which is a pathologically similar phenotype of T1DM. In this model, C57BL / 6 male mice will be given 40 mg / kg streptozotocin (STZ) in citrate buffer for 5 days. Four groups should be set up, namely the sham control group (citrate buffer, n=15), STZ CTL group (n=15), STZ+insulin group (n=15) and STZ+HKUOT-S2 group (n=15). All injections should be performed via the IP route. For the first five days, mice will be provided with normal food and 10% sucrose water. Thereafter, sucrose water will be replaced with normal water. On days 7, 14, 21 and 28 of the experiment, 2g kg -1 Glucose will be given to mice fasted for 6 hours. Blood glucose levels will be measured using a glucometer before and after fasting. Blood glucose levels will also be measured 15, 30, 60 and 120 minutes after the glucose load. Blood insulin levels will also be measured. Histological, immunohistochemical and electron microscopic techniques as well as downstream mechanistic studies will be used to determine the anti-diabetic and anti-diabetogenic properties of the new HKUOT-S2 protein.

[0392] Single high-dose STZ treatment

[0393] A single high-dose STZ treatment destroys pancreatic beta cells, leading to a T1DM phenotype. In this model, C57BL / 6 male mice will be given 200 mg / kg streptozotocin (STZ) in citrate buffer. The experimental groups, number of mice per group, and evaluation procedures will be the same as for multiple low-dose STZ treatment experiments.

[0394] Type 2 diabetes mellitus (T2DM) animal model

[0395] Insulin deficiency model

[0396] Nicotinamide (NA) and SZT co-administration protect mice from STZ-induced diabetes by partially protecting pancreatic β-cells. Under the influence of STZ and NA treatment, insufficient insulin produced by pancreatic β cells leads to T2DM. In this model, C57BL / 6 male mice fasted for 6-8 hours before STZ and NA treatment. Mice will first be given an IP injection of 210mg / kg NA. 15 minutes after NA injection, mice will be given 180mg / kg STZ via IP injection. The experimental group, the number of mice in each group and the evaluation procedure will be the same as the T1DM experiment.

[0397] Insulin resistance model

[0398] It has been reported that feeding mice with a high-fat diet followed by treatment with moderate doses of STZ results in hyperglycemia, hyperinsulinemia, and insulin resistance. In this model, C57BL / 6 male mice will be fed a high-fat diet for 21 days. On day 22, the mice will be fasted for 6-8 hours and then injected with 40mg / kg STZ IP. Ten days after STZ treatment, insulin resistance will be assessed in the mice. The experimental groups, number of mice per group, and assessment procedures will be the same as for the insulin deficiency model. Ethical approval and animal license will be obtained to initiate this project.

[0399] The results showed that HKUOT-S2 treatment significantly enriched the related glucose metabolism and insulin function signaling pathways, biological processes and functions in wild-type male mice ( Fig.14A , 14B In vitro results showed that HKUOT-S2 treatment had no effect on the viability of INS-1E cells (insulinoma cells) ( Fig.15A However, HKUOT-S2 treatment significantly increased pancreatic β-cell functional genes such as Ins-1, Mafa, Pdx-1, and HNF-1α in INS-1E cells ( Figures 15B-15H ).

[0400] Example 24 - Study of HKUOT-S2 protein in male and female reproduction and embryonic development.

[0401] Transcriptome data generated from RNA sequencing of rRNA-depleted bone tissue of HKUOT-S2-treated mice were highly reliable in all of our research directions. Transcriptome data from in vivo studies also revealed that HKUOT-S2 treatment significantly enriched biological processes and functions related to oogenesis, spermatogenesis, reproductive processes, and embryonic development in wild-type mice ( Fig.16A , 16B ). HKUOT-S2 protein promotes reproductive and embryonic development in vivo. In an animal model of menopausal syndrome, HKUOT-S2 treatment significantly modulated estrogen receptor signaling and inhibited the progression of OVX-induced osteoporosis. HKUOT-S2 may be a potential intervention for some fertility problems.

[0402] Transcriptome data from in vivo studies revealed that HKUOT-S2 treatment significantly enriched biological processes and functions related to oogenesis, spermatogenesis, reproductive processes, and embryonic development in wild-type mice ( Fig.16A , 16B ).

[0403] Example 25 - HKUOT-S2-derived peptides robustly inhibit the development of osteoporosis

[0404] HKUOT-S2 or SEQ ID NO: 83 HKUOT-S2 peptide: TKSSLPGQTK (referred to herein as TK) has been shown to prevent the development of osteoporosis in the femur of ovariectomized (OVX) mice. Micro-computed tomography (μCT) scanning analysis showed that HKUOT-S2 protein or its derivative SEQ ID NO 83 treatment inhibited the progression of osteoporosis by significantly increasing bone volume (BV / TV), bone surface area to tissue volume (BS / TV) ratio, trabecular thickness (Tb.th), trabecular number (Tb.N) but reducing bone surface area to bone volume ratio (BS / BV) and trabecular separation (Tb.Sp). Figures 22A-22G ). It should be noted that 2.18mg / Kg HKUOT-S2 and 0.5mg / Kg SEQ ID NO:83 treatment is the best dose to prevent the development of osteoporosis. In contrast, 0.5mg / Kg SEQ ID NO:83 treatment has a stronger osteoporosis inhibitory effect than the 2.18mg / Kg HKUOT-S2 treatment group. In addition, in vitro studies have shown that 0.1μg / ml HKUOT-S2 and 0.01μg / ml SEQ ID NO:83 treatment promotes the differentiation of human mesenchymal stem cells (hMSC) into osteoblasts by increasing the expression of estrogen receptor α (ERα) and ALP mRNA ( Figures 23A-23BThe results also showed that 0.1 μg / ml HKUOT-S2 and 0.01 μg / ml SEQ ID NO: 83 treatment significantly increased ALP activity and osteoblast biomineralization in vitro ( Figures 23C-23F ). SEQ ID NO: 83 has a robust osteogenic effect compared to HKUOT-S2 treatment. The osteogenic effects of other HKUOT-S2 derived peptides such as SEQ ID NO: 82 and 85 are under investigation.

[0405] Example 26 - HKUOT-S2 regulates estrogen receptors to promote osteogenesis and inhibit osteoporosis

[0406] In an in vitro study, hMSCs were differentiated into osteoblasts in the presence or absence of estrogen (E2, positive control) or HKUOT-S2. The results showed that HKUOT-S2 treatment promoted osteoblast differentiation by increasing the expression of estrogen receptors ERα and GPR30, thereby upregulating the osteogenic gene expression of ALP, COL1A1, and RUNX2 ( Figures 24A-24F HKUOT-S2 treatment also significantly enhanced ALP activity and osteoblast biomineralization ( Figures 24G-24J ). In the osteoporosis model, OVX mice were treated with E2 or HKUOT-S2 protein. Western blot analysis showed that HKUOT-S2 treatment significantly increased ERα, ERβ, and GPR30 protein levels in bone tissue when compared with the sham control group, OVX control group, or E2-treated group ( Figures 25A-25D ). Microcomputed tomography (μCT) scan analysis of the lumbar vertebrae (L5) showed that HKUOT-S2 protein treatment inhibited osteoporosis by significantly increasing bone volume (BV / TV), bone surface area to tissue volume (BS / TV) ratio, trabecular thickness (Tb.th), and trabecular number (Tb.N) but decreasing the bone surface area to bone volume ratio (BS / BV) and having no effect on trabecular separation (Tb.Sp) ( Figures 26A-26G ). Generally, there was no significant difference between the E2 and HKUOT-S2 protein-treated groups in inhibiting the development of osteoporosis.

[0407] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that those skilled in the art will propose various modifications or changes thereto, and that these modifications or changes will be included in the spirit and scope of the present application and in the scope of the appended claims. In addition, any element or limitation of any invention or embodiment thereof disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or embodiment thereof, and all such combinations are considered within the scope of the present invention without limitation thereto.

[0408] sequence

[0409] SEQ ID NO: 1: Alp primer forward: 5'-CCAGCAGGTTTCTCTCTTGG-3'

[0410] SEQ ID NO: 2: Alp primer reverse: 5'-GGGATGGAGGAGAGAAGGTC-3'

[0411] SEQ ID NO:3: Col Primer Forward: 5'-GAGCGGAGAGTACTGGATCG-3'

[0412] SEQ ID NO:4: Col Primer Reverse: 5'-GTTCGGGCTGATGTACCAGT-3'

[0413] SEQ ID NO:5: Opn primer forward: 5'-TCTGATGAGACCGTCACTGC-3'

[0414] SEQ ID NO:6: Opn primer reverse: 5'-AGGTCCTCATCTGTGGCATC-3'

[0415] SEQ ID NO:7: Runx 2 primer forward: 5'-CCCAGCCACCTTTACCTACA-3'

[0416] SEQ ID NO:8: Runx 2 primer reverse: 5'-TATGGAGTGCTGCTGGTCTG-3'

[0417] SEQ ID NO:9: Arg-1 Primer forward: 5'-CTCCAAGCCAAAGTCCTTAGAG-3'

[0418] SEQ ID NO: 10: Arg-1 primer reverse: 5'-AGGAGCTGTCATTAGGGACATC-3'

[0419] SEQ ID NO: 11: Mgl-1 Primer forward: 5'-TGAGAAAGGCTTTAAGAACTGGG-3'

[0420] SEQ ID NO: 12: Mgl-1 primer reverse: 5'-GACCACCTGTAGTGATGTGGG-3'

[0421] SEQ ID NO: 13: Cd206 Primer forward: 5'-TTCCATCGAGACTGCTGCT-3'

[0422] SEQ ID NO: 14: Cd206 primer reverse: 5'-CCAGAGGGATCGCCTGTTT-3'

[0423] SEQ ID NO: 15: Ym1 primer forward: 5'-CATGAGCAAGACTTGCGTGAC-3'

[0424] SEQ ID NO: 16: Ym1 primer reverse: 5'-GGTCCAAACTTCCATCCTCCA-3'

[0425] SEQ ID NO: 17: Socs3 primer forward: 5'-GATTTCGCTTCGGGACTAG-3'

[0426] SEQ ID NO: 18: Socs3 reverse primer: 5'-CGGCGGCGGGAAACTTGCTG-3'

[0427] SEQ ID NO: 19: Tnfα Primer forward: 5'-TCTCAGCCTCTTCTCATTCCTGC-3'

[0428] SEQ ID NO: 20: Tnfα reverse primer: 5'-AGAACTGATGAGAGGGAGGCCAT-3'

[0429] SEQ ID NO:21: iNOS primer forward: 5'-AATCTTGGAGCGAGTTGTGG-3'

[0430] SEQ ID NO: 22: iNOS reverse primer: 5'-CAGGAAGTAGGTGAGGGCTTG-3'

[0431] SEQ ID NO: 23: IL-6 Primer forward: 5'-ACAAAGCCAGAGTCCTTCAGAGAG-3'

[0432] SEQ ID NO: 24: IL-6 reverse primer: 5'-TTGGATGGTCTTGGTCCTTAGCCA-3'

[0433] SEQ ID NO: 25: IL-1β Primer forward: 5'-AGAGCTTCAGGCAGGCAGTA-3'

[0434] SEQ ID NO: 26: IL-1β reverse primer: 5'-AGGTGCTCATGTCCTCATCC-3'

[0435] SEQ ID NO: 27: Mcp-1 primer forward: 5'-CCAGCAAGATGATCCCAATG-3'

[0436] SEQ ID NO: 28: Mcp-1 reverse primer: 5'-TTCTTGGGGTCAGCACAGAC-3'

[0437] SEQ ID NO:29: Prkaa1 Primer forward: 5'-GGTGTACGGAAGGCAAAATGGC-3'

[0438] SEQ ID NO:30: Prkaa1 reverse primer: 5'-CAGGATTCTTCCTTCGTACACGC-3'

[0439] SEQ ID NO:31: Prkaa2 Primer forward: 5'-CTGAAGCCAGAGAATGTGCTGC-3'

[0440] SEQ ID NO:32: Prkaa2 reverse primer: 5'-GAGATGACCTCAGGTGCTGCAT-3'

[0441] SEQ ID NO:33: Prkab1 Primer forward: 5'-CCAAAAGTGCTCCGATGTGTCTG-3'

[0442] SEQ ID NO:34: Prkab1 reverse primer: 5'-GGGCTTTGAACCTCTCTTCTGG-3'

[0443] SEQ ID NO:35: Prkab2 Primer Forward: 5'-GACTTCGTTGCCATCCTGGATC-3'

[0444] SEQ ID NO:36: Prkab2 reverse primer: 5'-CCAAGCTGACTGGTAACCACAG-3'

[0445] SEQ ID NO:37: Prkag1 Primer forward: 5'-TCTCCGCCTTACCTGTAGTGGA-3'

[0446] SEQ ID NO:38: Prkag1 reverse primer: 5'-GCAGGGCTTTTGTCACAGACAC-3'

[0447] SEQ ID NO:39: Prkag2 primer forward: 5'-CTCCTCATCCAAAGAGTCTTCGC-3'

[0448] SEQ ID NO:40: Prkag2 reverse primer: 5'-TGGGTGTTGACGGAGAAGAGGA-3'

[0449] SEQ ID NO:41: Prkaig3 Primer forward: 5'-AAGCGGCTACTCAAGTTCCTGC-3'

[0450] SEQ ID NO:42: Prkag3 reverse primer: 5'-CCAGAACTACAGCCAAATCTCGG-3'

[0451] SEQ ID NO:43: Gapdh Primer Forward: 5'-CATCACTGCCACCCAGAAGACTG-3'

[0452] SEQ ID NO:44: Gadph primer reverse: 5'-ATGCCAGTGAGCTTCCCGTTCAG-3'

[0453] SEQ ID NO:45: Bglap1 Primer forward: 5'-GCAATAAGGTAGTGAACAGACTCC-3'

[0454] SEQ ID NO:46: Bglap1 reverse primer: 5'-CCATAGATGCGTTTGTAGGCGG-3'

[0455] SEQ ID NO:47: Bglap2 Primer forward: 5'-GCAATAAGGTAGTGAACAGACTCC-3'

[0456] SEQ ID NO:48: Bglap2 reverse primer: 5'-GCGTTTGTAGGCGGTCTTCAAG-3'

[0457] SEQ ID NO:49: Got1 (Ast) Primer forward: 5'-TGCTACTGGGATGCGGAGAAGA-3'

[0458] SEQ ID NO:50: Got1 (Ast) Primer Reverse: 5'-TGCATGACAGCAGCGATCTGCT-3'

[0459] SEQ ID NO:51: Gpt1 (Ast) Primer forward: 5'-CCACTCAGTCTCTAAGGGCTAC-3'

[0460] SEQ ID NO:52: Gpt1 (Ast) Primer Reverse: 5'-ACACAACCGCACGCTCATCAGT-3'

[0461] SEQ ID NO:53: Ckb (creatine) Primer forward: 5'-GCTCATTGACGACCACTTCCTC-3'

[0462] SEQ ID NO:54: Ckb (creatine) primer reverse: 5'-CCTCCTCGTTAATCCACACCAG-3'

[0463] SEQ ID NO:55: mTOR Primer forward: 5'-AGAAGGGTCTCCAAGGACGACT-3'

[0464] SEQ ID NO:56: mTOR primer reverse: 5'-GCAGGACACAAAGGCAGCATTG-3'

[0465] SEQ ID NO:57: Bmp2 primer forward: 5'-AACACCGTGCGCAGCTTCCATC-3'

[0466] SEQ ID NO:58: Bmp2 primer reverse: 5'-CGGAAGATCTGGAGTTCTGCAG-3'

[0467] SEQ ID NO:59: Bmrp2 Primer forward: 5'-AGAGACCCAAGTTCCCAGAAGC-3'

[0468] SEQ ID NO:60: Bmpr2 primer reverse: 5'-TCTCCTCAGCACACTGTGCAGT-3'

[0469] SEQ ID NO:61: Bmp7 primer forward: 5'-GGAGCGATTTGACAACGAGACC-3'

[0470] SEQ ID NO:62: Bmp7 reverse primer: 5'-AGTGGTTGCTGGTGGCTGTGAT-3'

[0471] SEQ ID NO:63: Tgfbr2 Primer forward: 5'-CCTACTCTGTCTGTGGATGACC-3'

[0472] SEQ ID NO:64: Tgfbr2 reverse primer: 5'-GACATCCGTCGTCTTGAACGAC-3'

[0473] SEQ ID NO:65: ALP primer forward: 5'-ATGGGATGGGTGTCTCCACA-3'

[0474] SEQ ID NO:66: ALP primer reverse: 5'-CCACGAAGGGGAACTTGTC-3'

[0475] SEQ ID NO:67: COL1A Primer forward: 5'-ATGACTATGAGTGGGAAGCA-3'

[0476] SEQ ID NO:68 COL1A reverse primer: 5'-TGGGTCCCTCTGTTACACTTT-3'

[0477] SEQ ID NO:69:OPN Primer Forward: 5'-CTCAGGCCAGTTGCAGCC-3'

[0478] SEQ ID NO:70:OPN primer reverse: 5'-CAAAAGCAAATCACTGCAATTCTC-3'

[0479] SEQ ID NO:71:RUNX2 Primer forward: 5'-CCTGAACTCTGCACCAAGTC-3'

[0480] SEQ ID NO:72:RUNX2 reverse primer: 5'-GAGGTGGCAGTGTCATCATC-3'

[0481] SEQ ID NO:73: GAPDH primer forward: 5'-GGCATCCACTGTGGTCATGAG-3'

[0482] SEQ ID NO:74: GAPDH reverse primer: 5'-TGCACCACCAACTGCTTAGC-3'

[0483] SEQ ID NO:75:4 EBP1 Primer forward: 5'-CACCAGCCCTTCCAGTGATGAG-3'

[0484] SEQ ID NO:76:4 EBP1 reverse primer: 5'-CCTTGGTAGTGCTCCACACGAT-3'

[0485] SEQ ID NO:77: AKT1 Primer forward: 5'-TGGACTACCTGCACTCGGAGAA-3'

[0486] SEQ ID NO:78: AKT1 reverse primer: 5'-GTGCGGCAAAAGGTCTTCATGG-3'

[0487] SEQ ID NO:79: S6K1 Primer forward: 5'-TATTGGCAGCCCACGAACACCT-3'

[0488] SEQ ID NO:80: S6K1 reverse primer: 5'-GTCACATCCATCTGCTCTATGCC-3'

[0489] SEQ ID NO:81:HKUOT-S2 peptide:KTVSLPR

[0490] SEQ ID NO:82:HKUOT-S2 peptide:KGNLLECDGGNTAQMMAR

[0491] SEQ ID NO:83:HKUOT-S2 peptide:TKSSLPGQTK

[0492] SEQ ID NO:84:HKUOT-S2 peptide:KEVSLPR

[0493] SEQ ID NO:85:HKUOT-S2 peptide:IKITTYRQ

[0494] SEQ ID NO:86:HKUOT-S2 peptide:AASECEEAGFSVCVEVNGR

[0495] SEQ ID NO:87:HKUOT-S2 peptide:APSTYGGGLSVSSSR

[0496] SEQ ID NO:88:HKUOT-S2 peptide:DDSITPTEDSIKR

[0497] SEQ ID NO:89:HKUOT-S2 peptide:DTANLFPQTSLSLFMKPDTAGTFDVECLTT-DHYTGGMKQK

[0498] SEQ ID NO:90:HKUOT-S2 peptide:FKLLNYCIPK

[0499] SEQ ID NO:91:HKUOT-S2 peptide:HAETSSGGQAASSHEQAR

[0500] SEQ ID NO:92:HKUOT-S2 peptide:QADLILTAGTVTMKMAPSLVRLYEQMAEPK

[0501] SEQ ID NO:93:HKUOT-S2 peptide:RSKLGSHHIDR

[0502] SEQ ID NO:94:HKUOT-S2 peptide:SISISVAR

[0503] SEQ ID NO:95:HKUOT-S2 peptide:SLVNLGGSK

[0504] SEQ ID NO:96:HKUOT-S2 peptide:TEDGSDPPSGDFLTEGGGVR

[0505] SEQ ID NO:97:HKUOT-S2 peptide:VATVSLPR

[0506] Implementation

[0507] Embodiment 1. Comprising the amino acid sequence according to SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97 or any combination thereof. Implementation .

[0508] Embodiment 2. Comprising the protein of claim 1 Implementation .

[0509] Embodiment 3. The method of claim 2 Implementation , which further comprises at least one carrier or excipient.

[0510] Embodiment 4. The embodiment of claim 2, wherein the protein is HKUOT-S2.

[0511] Embodiment 5. The embodiment of claim 4, wherein HKUOT-S2 is 32 kDA.

[0512] Embodiment 6. An osteogenesis embodiment, comprising administering to a subject an effective amount of a composition comprising a protein comprising an amino acid sequence according to any one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 81-97, or a peptide according to any one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 81-97.

[0513] Embodiment 7. The embodiment of claim 6, further comprising increasing the expression of Ep4, Ptges, Cox2, Eno2, Calca, or any combination thereof in the subject.

[0514] Embodiment 8. The embodiment of claim 6, further comprising activating mTOR / 4E-BP1, AMPK, and BMP signaling pathways in the subject.

[0515] Embodiment 9. The embodiment of claim 8, further comprising increasing the expression of Prkaa1, Prkaa2, Prkab1, Prkab2, Prkag1, Prkag2, Prkag3 and Mtor or any combination thereof in the subject.

[0516] Embodiment 10. The embodiment of claim 6, further comprising inhibiting ovariectomy (OVX)-induced and glucocorticoid-induced osteoporosis.

[0517] Embodiment 11. The method of claim 6 Implementation wherein the protein or the peptide is present in the composition at about 0.1 mg kg -1 About 10 mg kg -1 A dose of is administered to the subject.

[0518] Embodiment 12. The embodiment of claim 11, wherein the dose is about 2.18 mg kg -1 .

[0519] Embodiment 13. The embodiment of claim 6, wherein the composition further comprises at least one carrier or excipient.

[0520] Embodiment 14. An embodiment for treating inflammatory diseases, spinal cord injury, liver and kidney diseases, cardiovascular diseases, diabetes, postmenopausal syndrome, infertility or hematopoietic diseases, or enhancing wound healing, comprising: administering to a subject an effective amount of a composition comprising a protein comprising an amino acid sequence according to any one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 81-97, or a peptide according to any one of SEQ ID NOs: 81-97 or a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 81-97.

[0521] Embodiment 15. The embodiment of claim 14, further comprising increasing the expression of Ep4, Ptges, Cox2, Eno2, Calca, or any combination thereof in the subject.

[0522] Embodiment 16. The embodiment of claim 14, further comprising activating the mTOR / 4E-BP1, AMPK and / or BMP signaling pathway in the subject.

[0523] Embodiment 17. The embodiment of claim 16, further comprising increasing the expression of Prkaa1, Prkaa2, Prkab1, Prkab2, Prkag1, Prkag2, Prkag3 and Mtor or any combination thereof in the subject.

[0524] Embodiment 18. The embodiment of claim 14, further comprising inhibiting ovariectomy (OVX)-induced and glucocorticoid-induced osteoporosis.

[0525] Embodiment 19. The method of claim 14 Implementation wherein the protein or the peptide is present in the composition at about 0.1 mg kg -1 About 10 mg kg -1 A dose of is administered to the subject.

[0526] Embodiment 20. The embodiment of claim 11, wherein the dose is about 2.18 mg kg -1 .

[0527] Embodiment 21. The embodiment of claim 11, wherein the liver or kidney disease is hepatitis, cirrhosis, or nephropathy; the cardiovascular disease is hypertension or cardiomyopathy; the inflammatory disease is osteoarthritis; the hematopoietic disease is hemolytic anemia; and the postmenopausal syndrome is osteoporosis.

[0528] References

[0529] [1]G.B.D.F.Collaborators,Global,regional,and national burden of bonefractures in 204countries and territories,1990-2019:a systematic analysisfrom the Global Burden of Disease Study 2019,Lancet Healthy Longev 2(9)(2021)e580-e592.

[0530] [2]C.M.Court-Brown,B.Caesar,Epidemiology of adult fractures:A review,Injury 37(8)(2006)691-7.

[0531] [3]S.Polinder,J.Haagsma,M.Panneman,A.Scholten,M.Brugmans,E.Van Beeck,The economic burden of injury:Health care and productivity costs of injuriesin the Netherlands,Accid Anal Prev 93(2016)92-100.

[0532] [4]C.I.Hsieh,K.Zheng,C.Lin,L.Mei,L.Lu,W.Li,F.P.Chen,Y.Wang,X.Zhou,F.Wang,G.Xie,J.Xiao,S.Miao,C.F.Kuo,Automated bone mineral density predictionand fracture risk assessment using plain radiographs via deep learning,NatCommun 12(1)(2021)5472.

[0533] [5]O.T.G.f.F.o.O.G.f.C.M.o.P.O.i.H.Kong,T.P.Ip,S.K.Cheung,T.C.Cheung,T.C.Choi,S.L.Chow,Y.Y.Ho,S.Y.Kan,W.C.Kung,K.K.Lee,K.L.Leung,Y.Y.Leung,S.T.Lo,C.T.Sy,Y.W.Wong,K.Osteoporosis Society of Hong,The Osteoporosis Society ofHong Kong(OSHK):2013OSHKguideline for clinical management of postmenopausalosteoporosis in Hong Kong,Hong Kong Med J 19 Suppl 2(2013)1-40.

[0534] [6]J.R.Perez,D.Kouroupis,D.J.Li,T.M.Best,L.Kaplan,D.Correa,TissueEngineering and Cell-Based Therapies for Fractures and Bone Defects,FrontBioeng Biotechnol 6(2018)105.

[0535] [7]M.T.Corvol,K.Tahiri,A.Montembault,A.Daumard,J.F.Savouret,F.Rannou,[Cell therapy in cartilage repair:cellular and molecular bases],J Soc Biol202(4)(2008)313-21.

[0536] [8]A.Laurent,A.Porcello,P.G.Fernandez,A.Jeannerat,C.Peneveyre,P.Abdel-Sayed,C.Scaletta,N.Hirt-Burri,M.Michetti,A.de Buys Roessingh,W.Raffoul,E.Allemann,O.Jordan,L.A.Applegate,Combination of Hyaluronan andLyophilized Progenitor Cell Derivatives:Stabilization of Functional HydrogelProducts for Therapeutic Management of Tendinous Tissue Disorders,Pharmaceutics 13(12)(2021).

[0537] [9]L.Lafuente-Gracia,E.Borgiani,G.Nasello,L.Geris,Towards in silicoModels of the Inflammatory Response in Bone Fracture Healing,Front BioengBiotechnol 9(2021)703725.

[0538]

[10] R.E.Tomlinson,M.J.Silva,Skeletal Blood Flow in Bone Repair andMaintenance,Bone Res 1(4)(2013)311-22.

[0539]

[11] C.A.Loynes,J.A.Lee,A.L.Robertson,M.J.Steel,F.Ellett,Y.Feng,B.D.Levy,M.K.B.Whyte,S.A.Renshaw,PGE2 production at sites of tissue injurypromotes an anti-inflammatory neutrophil phenotype and determines the outcomeof inflammation resolution in vivo,Sci Adv 4(9)(2018)eaar8320.

[0540]

[12] T.A.Einhorn,L.C.Gerstenfeld,Fracture healing:mechanisms andinterventions,Nat Rev Rheumatol 11(1)(2015)45-54.

[0541]

[13] M.Phimphilai,Z.Zhao,H.Boules,H.Roca,R.T.Franceschi,BMP signalingis required for RUNX2-dependent induction of the osteoblast phenotype,J BoneMiner Res 21(4)(2006)637-46.

[0542]

[14] W.Huang,S.Yang,J.Shao,Y.P.Li,Signaling and transcriptionalregulation in osteoblast commitment and differentiation,Front Biosci 12(2007)3068-92.

[0543]

[15] S.Maeda,M.Hayashi,S.Komiya,T.Imamura,K.Miyazono,Endogenous TGF-beta signaling suppresses maturation of osteoblastic mesenchymal cells,EMBO J23(3)(2004)552-63.

[0544]

[16] C.Y.Tang,M.Wu,D.Zhao,D.Edwards,A.McVicar,Y.Luo,G.Zhu,Y.Wang,H.D.Zhou,W.Chen,Y.P.Li,Runx1 is a central regulator of osteogenesis for bonehomeostasis by orchestrating BMP and WNT signaling pathways,PLoS Genet 17(1)(2021)e1009233.

[0545]

[17] L.Claes,S.Recknagel,A.Ignatius,Fracture healing under healthy andinflammatory conditions,Nat Rev Rheumatol 8(3)(2012)133-43.

[0546]

[18] R.V.M.Groven,J.van Koll,M.Poeze,T.J.Blokhuis,M.van Griensven,miRNAs Related to Different Processes of Fracture Healing:An IntegrativeOverview,Front Surg 8(2021)786564.

[0547]

[19] S.Ansari,K.Ito,S.Hofmann,Cell Sources for Human In vitro BoneModels,Curr Osteoporos Rep 19(1)(2021)88-100.

[0548]

[20] R.L.Shin,C.W.Lee,O.Y.Shen,H.Xu,O.K.Lee,The Crosstalk betweenMesenchymal Stem Cells and Macrophages in Bone Regeneration:A SystematicReview,Stem Cells Int 2021(2021)8835156.

[0549]

[21] L.Gong,Y.Zhao,Y.Zhang,Z.Ruan,The Macrophage PolarizationRegulates MSC Osteoblast Differentiation in vitro,Ann Clin Lab Sci 46(1)(2016)65-71.

[0550]

[22] D.I.Cho,M.R.Kim,H.Y.Jeong,H.C.Jeong,M.H.Jeong,S.H.Yoon,Y.S.Kim,Y.Ahn,Mesenchymal stem cells reciprocally regulate the M1 / M2 balance in mousebone marrow-derived macrophages,Exp Mol Med 46(2014)e70.

[0551]

[23] L.Y.Lu,F.Loi,K.Nathan,T.H.Lin,J.Pajarinen,E.Gibon,A.Nabeshima,L.Cordova,E.Jamsen,Z.Yao,S.B.Goodman,Pro-inflammatory M1 macrophages promoteOsteogenesis by mesenchymal stem cells via the COX-2-prostaglandin E2pathway,J Orthop Res 35(11)(2017)2378-2385.

[0552]

[24] N.Zhang,C.W.Lo,T.Utsunomiya,M.Maruyama,E.Huang,C.Rhee,Q.Gao,Z.Yao,S.B.Goodman,PDGF-BB and IL-4 co-overexpression is a potential strategyto enhance mesenchymal stem cell-based bone regeneration,Stem Cell Res Ther12(1)(2021)40.

[0553]

[25] F.Moreno Sancho,Y.Leira,M.Orlandi,J.Buti,W.V.Giannobile,F.D’Aiuto,Cell-Based Therapies for Alveolar Bone and Periodontal Regeneration:Concise Review,Stem Cells Transl Med 8(12)(2019)1286-1295.

[0554]

[26] C.Dou,N.Ding,C.Zhao,T.Hou,F.Kang,Z.Cao,C.Liu,Y.Bai,Q.Dai,Q.Ma,F.Luo,J.Xu,S.Dong,Estrogen Deficiency-Mediated M2 MacrophageOsteoclastogenesis Contributes to M1 / M2 Ratio Alteration in OvariectomizedOsteoporotic Mice,J Bone Miner Res 33(5)(2018)899-908.

[0555]

[27] Y.Saxena,S.Routh,A.Mukhopadhaya,Immunoporosis:Role of InnateImmune Cells in Osteoporosis,Front Immunol 12(2021)687037.

[0556]

[28] D.H.Yang,M.Y.Yang,The Role of Macrophage in the Pathogenesis ofOsteoporosis,Int J Mol Sci 20(9)(2019).

[0557]

[29] P.Aveline,A.Cesaro,M.Mazor,M.B.T,E.Lespessailles,H.Toumi,Cumulative Effects of StrontiumRanelate and Impact Exercise on Bone Mass inOvariectomized Rats,Int J Mol Sci 22(6)(2021).

[0558]

[30] L.Gani,N.Anthony,L.Dacay,P.Tan,L.R.Chong,T.F.King,Incidence ofAtypical Femoral Fracture and Its Mortality in a Single Center in Singapore,JBMR Plus 5(8)(2021)e10515.

[0559]

[31] H.Hagino,T.Sugimoto,S.Tanaka,K.Sasaki,T.Sone,T.Nakamura,S.Soen,S.Mori,A randomized,controlled trial of once-weekly teriparatide injectionversus alendronate in patients at high risk of osteoporotic fracture:primaryresults of the Japanese Osteoporosis Intervention Trial-05,Osteoporos Int 32(11)(2021)2301-2311.

[0560]

[32] A.Corrado,E.R.Sanpaolo,S.Di Bello,F.P.Cantatore,Osteoblast as atarget of anti-osteoporotic treatment,Postgrad Med 129(8)(2017)858-865.

[0561]

[33] C.Tohda,X.Yang,M.Matsui,Y.Inada,E.Kadomoto,S.Nakada,H.Watari,N.Shibahara,Diosgenin-Rich Yam Extract Enhances Cognitive Function:A Placebo-Controlled,Randomized,Double-Blind,Crossover Study of Healthy Adults,Nutrients 9(10)(2017).

[0562]

[34] B.Padhan,D.Panda,Potential of Neglected and Underutilized Yams(Dioscorea spp.)for Improving Nutritional Security and Health Benefits,FrontPharmacol 11(2020)496.

[0563]

[35] S.Kim,M.Y.Shin,K.H.Son,H.Y.Sohn,J.H.Lim,J.H.Lee,I.S.Kwun,Yam(Dioscorea batatas)Root and Bark Extracts Stimulate Osteoblast Mineralizationby Increasing Ca and P Accumulation and Alkaline Phosphatase Activity,PrevNutr Food Sci 19(3)(2014)194-203.

[0564]

[36] Y.W.Liu,J.C.Liu,C.Y.Huang,C.K.Wang,H.F.Shang,W.C.Hou,Effects oforal administration of yam tuber storage protein,dioscorin,to BALB / c mice for21-days on immune responses,J Agric Food Chem 57(19)(2009)9274-9.

[0565]

[37] K.Y.Peng,L.Y.Horng,H.C.Sung,H.C.Huang,R.T.Wu,AntiosteoporoticActivity of Dioscorea alata L.cv.Phyto through Driving Mesenchymal Stem CellsDifferentiation for Bone Formation,Evid Based Complement Alternat Med 2011(2011)712892.

[0566]

[38] K.L.Wong,Y.M.Lai,K.W.Li,K.F.Lee,T.B.Ng,H.P.Cheung,Y.B.Zhang,L.Lao,R.N.Wong,P.C.Shaw,J.H.Wong,Z.J.Zhang,J.K.Lam,W.C.Ye,S.C.Sze,A Novel,Stable,Estradiol-Stimulating,Osteogenic Yam Protein with Potential for theTreatment of Menopausal Syndrome,Sci Rep 5(2015)10179.

[0567]

[39] N.Setyawan,J.S.Maninang,S.Suzuki,Y.Fujii,Variation in thePhysical and Functional Properties ofYam(Dioscorea spp.)Flour Produced byDifferent Processing Techniques,Foods 10(6)(2021).

[0568]

[40] S.Kumar,G.Das,H.S.Shin,J.K.Patra,Dioscorea spp.(A Wild EdibleTuber):A Study on Its Ethnopharmacological Potential and Traditional Use bythe Local People of Similipal Biosphere Reserve,India,Front Pharmacol 8(2017)52.

[0569]

[41] P.Tiwwari,B.Kumar,M.Kaur,G.Kaur,H.Kaur,Phytochemical screeningand Extraction:A Review INTERNATIONALE PHARMACEUTICA SCIENCIA 1(98-106)(2011).

[0570]

[42] P.Hong,S.Koza,E.S.Bouvier,Size-Exclusion Chromatography for theAnalysis of Protein Biotherapeutics and their Aggregates,J Liq ChromatogrRelat Technol 35(20)(2012)2923-2950.

[0571]

[43] M.Widbiller,C.Rothmaier,D.Saliter,M.Wolflick,A.Rosendahl,W.Buchalla,G.Schmalz,T.Spruss,K.M.Galler,Histology of human teeth:Standardand specific staining methods revisited,Arch Oral Biol 127(2021)105136.

[0572]

[44] C.M.Lima,A.K.Lima,M.G.Melo,M.R.Serafini,D.L.Oliveira,E.B.deAlmeida,R.S.Barreto,P.C.Nogueira,V.R.Moraes,E.R.Oliveira,R.L.de Albuquerque,Jr.,L.J.Quintans-Junior,A.A.Araujo,Bioassay-guided evaluation of Dioscoreavillosa-an acute and subchronic toxicity,antinociceptive and anti-inflammatory approach,BMC Complement Altern Med 13(2013)195.

[0573]

[45] J.Loffler,F.A.Sass,S.Filter,A.Rose,A.Ellinghaus,G.N.Duda,A.Dienelt,Compromised Bone Healing in Aged Rats Is Associated With ImpairedM2 Macrophage Function,Front Immunol 10(2019)2443.

[0574]

[46] R.Huang,X.Wang,Y.Zhou,Y.Xiao,RANKL-induced M1 macrophages areinvolved in bone formation,Bone Res 5(2017)17019.

[0575]

[47] J.Chen,F.Long,mTORC1 Signaling Promotes OsteoblastDifferentiation fromPreosteoblasts,PLoS One 10(6)(2015)e0130627.

[0576]

[48] S.Fitter,M.P.Matthews,S.K.Martin,J.Xie,S.S.Ooi,C.R.Walkley,J.D.Codrington,M.A.Ruegg,M.N.Hall,C.G.Proud,S.Gronthos,A.C.W.Zannettino,mTORC1 Plays an Important Role in Skeletal Development by ControllingPreosteoblast Differentiation,Mol Cell Biol 37(7)(2017).

[0577]

[49] B.Padhan,J.K.Nayak,D.Panda,Natural antioxidant potential ofselected underutilized wild yams(Dioscorea spp.)for health benefit,J Food SciTechnol 57(6)(2020)2370-2376.

[0578]

[50] R.S.Conlan,L.A.Griffiths,J.A.Napier,P.R.Shewry,S.Mantell,C.Ainsworth,Isolation and characterisation of cDNA clones representing thegenes encoding the major tuber storage protein(dioscorin)of yam(Dioscoreacayenensis Lam.),Plant Mol Biol 28(3)(1995)369-80.

[0579]

[51] J.P.Bonjour,Protein intake and bone health,Int J Vitam Nutr Res81(2-3)(2011)134-42.

[0580]

[52] E.Tsiridis,P.V.Giannoudis,Transcriptomics and proteomics:advancing the understanding of genetic basis of fracture healing,Injury 37Suppl 1(2006)S13-9.

[0581]

[53] A.Salhotra,H.N.Shah,B.Levi,M.T.Longaker,Mechanisms of bonedevelopment and repair,Nat Rev Mol Cell Biol 21(11)(2020)696-711.

[0582]

[54] J.Hu,H.Liao,Z.Ma,H.Chen,Z.Huang,Y.Zhang,M.Yu,Y.Chen,J.Xu,FocalAdhesion Kinase Signaling Mediated the Enhancement of Osteogenesis of HumanMesenchymal Stem Cells Induced by Extracorporeal Shockwave,Sci Rep 6(2016)20875.

[0583]

[55] Y.G.Wang,X.H.Qu,Y.Yang,X.G.Han,L.Wang,H.Qiao,Q.M.Fan,T.T.Tang,K.R.Dai,AMPK promotes osteogenesis and inhibits adipogenesis through AMPK-Gfi1-OPN axis,Cell Signal 28(9)(2016)1270-82.

[0584]

[56] H.B.Noor,N.A.Mou,L.Salem,M.F.A.Shimul,S.Biswas,R.Akther,S.Khan,S.Raihan,M.M.Mohib,M.A.T.Sagor,Anti-inflammatory Property of AMP-activatedProtein Kinase,Antiinflamm Antiallergy Agents Med Chem 19(1)(2020)2-41.

[0585]

[57] Y.Cheng,L.Huang,Y.Wang,Q.Huo,Y.Shao,H.Bao,Z.Li,Y.Liu,X.Li,Strontium promotes osteogenic differentiation by activating autophagy via thethe AMPK / mTOR signaling pathway in MC3T3E1 cells,Int J Mol Med 44(2)(2019)652-660.

[0586]

[58] H.Liu,X.Li,J.Lin,M.Lin,Morroniside promotes the osteogenesis byactivating PI3K / Akt / mTOR signaling,Biosci Biotechnol Biochem 85(2)(2021)332-339.

[0587]

[59] P.N.Tasli,S.Aydin,M.E.Yalvac,F.Sahin,Bmp 2 and bmp 7 induceodonto-and osteogenesis of human tooth germ stem cells,Appl BiochemBiotechnol 172(6)(2014)3016-25.

[0588]

[60] A.Bandyopadhyay,K.Tsuji,K.Cox,B.D.Harfe,V.Rosen,C.J.Tabin,Geneticanalysis of the roles of BMP2,BMP4,and BMP7 in limb patterning andskeletogenesis,PLoS Genet 2(12)(2006)e216.

[0589]

[61] E.H.Alcantara,M.Y.Shin,H.Y.Sohn,Y.M.Park,T.Kim,J.H.Lim,H.J.Jeong,S.T.Kwon,I.S.Kwun,Diosgenin stimulates osteogenic activity by increasing bonematrix protein synthesis and bone-specific transcription factor Runx2 inosteoblastic MC3T3-E1 cells,J Nutr Biochem 22(11)(2011)1055-63.

[0590]

[62] Y.W.Liu,H.F.Shang,C.K.Wang,F.L.Hsu,W.C.Hou,Immunomodulatoryactivity of dioscorin,the storage protein of yam(Dioscorea alata cv.TainongNo.1)tuber,Food ChemToxicol 45(11)(2007)2312-8.

[0591]

[63] T.Li,Z.L.Liu,M.Xiao,Z.Z.Yang,M.Z.Peng,C.D.Li,X.J.Zhou,J.W.Wang,Impact of bone marrow mesenchymal stem cell immunomodulation on theosteogenic effects of laponite,Stem Cell Res Ther 9(1)(2018)100.

[0592]

[64] X.Qu,Z.Zhai,X.Liu,H.Li,Z.Ouyang,C.Wu,G.Liu,Q.Fan,T.Tang,A.Qin,K.Dai,Dioscin inhibits osteoclast differentiation and bone resorption thoughdown-regulating the Akt signaling cascades,BiochemBiophys Res Commun 443(2)(2014)658-65.

[0593]

[65] C.Zhu,N.Bao,S.Chen,J.Zhao,Dioscin enhances osteoblastic celldifferentiation and proliferation by inhibiting cell autophagy via the ASPP2 / NF-kappabeta pathway,Mol Med Rep 16(4)(2017)4922-4926.

[0594]

[66] X.Chen,W.Chen,Z.M.Aung,W.Han,Y.Zhang,G.Chai,LY3023414 inhibitsboth osteogenesis and osteoclastogenesis through the PI3K / Akt / GSK3 signallingpathway,Bone Joint Res 10(4)(2021)237-249.

[0595]

[67] R.A.Saxton,D.M.Sabatini,mTOR Signaling in Growth,Metabolism,andDisease,Cell 168(6)(2017)960-976.

[0596]

[68] Iksen,S.Pothongsrisit,V.Pongrakhananon,Targeting the PI3K / AKT / mTOR Signaling Pathway in Lung Cancer:An Update Regarding Potential Drugs andNatural Products,Molecules 26(13)(2021).

[0597]

[69] M.Jhanwar-Uniyal,J.V.Wainwright,A.L.Mohan,M.E.Tobias,R.Murali,C.D.Gandhi,M.H.Schmidt,Diverse signaling mechanisms of mTOR complexes:mTORC1and mTORC2 in forming a formidable relationship,Adv Biol Regul 72(2019)51-62.

[0598]

[70] J.Chen,F.Long,mTORC1 signaling controls mammalian skeletal growththrough stimulation of protein synthesis,Development 141(14)(2014)2848-54.

[0599]

[71] J.Chen,N.Holguin,Y.Shi,M.J.Silva,F.Long,mTORC2 signaling promotesskeletal growth and bone formation in mice,J Bone Miner Res 30(2)(2015)369-78.

[0600]

[72] J.S.Kwon,S.W.Kim,D.Y.Kwon,S.H.Park,A.R.Son,J.H.Kim,M.S.Kim,invivo osteogenic differentiation of human turbinate mesenchymal stem cells inan injectable in situ-forming hydrogel,Biomaterials 35(20)(2014)5337-5346.

[0601]

[73] C.A.Anosike,O.Obidoa,L.U.Ezeanyika,Membrane stabilization as amechanism of the anti-inflammatory activity of methanol extract of garden egg(Solanum aethiopicum),Daru 20(1)(2012)76.

[0602]

[74] D.O’Neil,H.Glowatz,M.Schlumpberger,Ribosomal RNA depletion forefficient use of RNA-seq capacity,Curr Protoc Mol Biol Chapter 4(2013)Unit 419.

[0603]

[75] S.L ê,J.Josse,F.Husson,FactoMineR:An R Package for MultivariateAnalysis,Journal of Statistical Software 25(1)(2008)25(1),1–18.

[0604]

[76] F.J.Rios,R.M.Touyz,A.C.Montezano,Isolation and Differentiation ofMurine Macrophages,Methods Mol Biol 1527(2017)297-309.

[0605]

[78] Kubi,J.A.,A.S.Brah,K.M.C.Cheung,Y.L.Lee,K.F.Lee,S.C.W.Sze,W.Qiao,and K.W.Yeung.2023.‘A new osteogenic protein isolated from Dioscorea oppositaThunb accelerates bone defect healing through the mTOR signaling axis’,BioactMater,27:429-46.

Claims

1. A protein comprising an amino acid sequence according to SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, or any combination thereof.

2. A composition comprising the protein according to claim 1.

3. The composition of claim 2, further comprising at least one carrier or excipient.

4. The composition of claim 2, wherein the protein is HKUOT-S2.

5. The composition of claim 4, wherein HKUOT-S2 is 32 kDA.

6. A method of bone formation, comprising: An effective amount of a composition comprising a protein comprising an amino acid sequence according to any one of SEQ ID NOs: 81-97, or a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 81-97, or a peptide according to any one of SEQ ID NOs: 81-97, or a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 81-97, is administered to a subject.

7. The method of claim 6, further comprising increasing the expression of Ep4, Ptges, Cox2, Eno2, Calca, or any combination thereof in the subject.

8. The method of claim 6, further comprising activating mTOR / 4E-BP1, AMPK, and BMP signaling pathways in the subject.

9. The method of claim 8, further comprising increasing the expression of Prkaa1, Prkaa2, Prkab1, Prkab2, Prkag1, Prkag2, Prkag3 and Mtor or any combination thereof in the subject.

10. The method of claim 6, further comprising inhibiting ovariectomy (OVX)-induced and glucocorticoid-induced osteoporosis.

11. The method of claim 6, wherein the protein or the peptide is present in the composition at about 0.1 mg kg -1 About 10 mg kg -1 A dose of is administered to the subject.

12. The method of claim 11, wherein the dose is about 2.18 mg kg -1 .

13. The method of claim 6, wherein the composition further comprises at least one carrier or excipient.

14. A method for treating inflammatory diseases, spinal cord injuries, liver or kidney diseases, cardiovascular diseases, diabetes, postmenopausal syndrome, infertility or hematopoietic diseases, or enhancing wound healing, the method comprising: An effective amount of a composition comprising a protein comprising an amino acid sequence according to any one of SEQ ID NOs: 81-97, or a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 81-97, or a peptide according to any one of SEQ ID NOs: 81-97, or a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 81-97, is administered to a subject.

15. The method of claim 14, further comprising increasing the expression of Ep4, Ptges, Cox2, Eno2, Calca, or any combination thereof in the subject.

16. The method of claim 14, further comprising activating the mTOR / 4E-BP1, AMPK and / or BMP signaling pathway in the subject.

17. The method of claim 16, further comprising increasing the expression of Prkaa1, Prkaa2, Prkab1, Prkab2, Prkag1, Prkag2, Prkag3, and Mtor, or any combination thereof, in the subject.

18. The method of claim 14, further comprising inhibiting ovariectomy (OVX)-induced and glucocorticoid-induced osteoporosis.

19. The method of claim 14, wherein the protein or the peptide is present in the composition at about 0.1 mg kg -1 About 10 mg kg -1 A dose of is administered to the subject.

20. The method of claim 11, wherein the dose is about 2.18 mg kg -1 .

21. The method of claim 14, wherein the liver or kidney disease is hepatitis, cirrhosis, or nephropathy; the cardiovascular disease is hypertension or cardiomyopathy; the inflammatory disease is osteoarthritis; the hematopoietic disease is hemolytic anemia; and the postmenopausal syndrome is osteoporosis.

Images