Application of salicin in preparation of medicine for preventing and / or treating osteoporosis

Abstract

The invention relates to application of salicin in preparation of a medicine for preventing and / or treating osteoporosis, and belongs to the technical field of medical treatment. The invention provides application of salicin in preparation of a medicine for preventing and / or treating osteoporosis. The salicin can promote in-vitro proliferation and migration of hBMSCs and hASCs, reduce the oxidative stress level of cells, promote osteogenic differentiation of the cells by influencing an AMPK signal channel, and inhibit apoptosis and bone loss by removing ROS in bone tissue and up-regulating expression of endogenous antioxidant enzyme, can be used for treating osteoporosis, and has good biological safety.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the use of salicin in the preparation of a drug for preventing and / or treating osteoporosis. Background Art

[0002] Osteoporosis is a systemic bone metabolic disease characterized by reduced bone mineral density, damaged bone microstructure, and increased fracture risk, which is highly prevalent in groups such as postmenopausal women and the elderly. Currently, clinical drugs for treating osteoporosis, such as bisphosphonates (BPs), receptor activator of nuclear factor-κB ligand (RANKL) inhibitors, parathyroid hormone (PTH) analogs, etc., mainly act by inhibiting osteoclast activity or promoting bone formation in the short term. However, long-term use can cause problems such as osteonecrosis of the jaw, increased risk of cardiovascular diseases, and attenuation of efficacy, and there is also a lack of a root control strategy for the imbalance between bone formation and resorption clinically.

[0003] Research has found that oxidative stress is one of the core mechanisms of osteoporosis pathogenesis. The excessive accumulation of reactive oxygen species (ROS) can activate the NF-κB signaling pathway. On the one hand, it can inhibit the expression of key transcription factors for osteoblast differentiation, such as Runx2 and Osterix, to hinder bone matrix mineralization; on the other hand, it can activate osteoclasts by upregulating the ratio of receptor activator of nuclear factor-κB ligand / osteoprotegerin (RANKL / OPG), thereby exacerbating bone resorption. Clinical results show that the levels of oxidative damage markers such as 8-hydroxy-2'-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) in the serum of osteoporosis patients are significantly increased, while the activities of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPX) are decreased, indicating a positive correlation between oxidative stress and bone metabolism disorder.

[0004] Existing technologies have tried to use antioxidants such as vitamin C (VC) and N-acetylcysteine (NAC) to intervene in osteoporosis models, but the antioxidants have poor targeting to bone tissue, short drug efficacy duration, and lack of a direct promoting effect on osteoblast differentiation, so it is difficult to achieve clinical transformation. In terms of regulating osteoblast differentiation, although drugs such as recombinant BMP-2 can directly activate the NF-κB signaling pathway, they have defects such as heterotopic ossification, high cost, and dependence on local administration. Summary of the Invention

[0005] The purpose of the present invention is to provide the use of salicin in the preparation of a drug for preventing and / or treating osteoporosis, so as to solve the problems of high treatment cost, poor efficacy, short drug efficacy duration, and large side effects in the prior art for the treatment of osteoporosis.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides the use of salicin in the preparation of a medicament for preventing and / or treating osteoporosis.

[0008] The present invention provides the use of salicin in the preparation of a medicament for inhibiting oxidative stress.

[0009] The present invention provides the use of salicin in the preparation of a medicament for promoting the proliferation and / or differentiation of stem cells, wherein the stem cells are human bone marrow mesenchymal stem cells and / or adipose mesenchymal stem cells.

[0010] Preferably, the concentration of salicin in the medicament is 5-20 mg / L.

[0011] The present invention provides a preparation, which comprises salicin and a pharmaceutically acceptable carrier.

[0012] Preferably, the concentration of salicin in the preparation is 5-20 mg / L.

[0013] The present invention provides the use of the above-mentioned preparation in the preparation of a product for preventing and / or treating osteoporosis.

[0014] The present invention provides the use of the above-mentioned preparation in the preparation of a product for inhibiting oxidative stress.

[0015] The present invention provides the use of the above-mentioned preparation in the preparation of a product for promoting the proliferation and / or differentiation of stem cells.

[0016] Preferably, the stem cells are human bone marrow mesenchymal stem cells and / or adipose mesenchymal stem cells.

[0017] The present invention has the following technical effects and advantages:

[0018] Salicin of the present invention can promote the in vitro proliferation and migration of human bone marrow mesenchymal stem cells (hBMSCs) and adipose mesenchymal stem cells (hASCs), reduce the level of oxidative stress of cells, promote osteogenic differentiation of cells by affecting the AMPK signaling pathway, inhibit cell apoptosis and bone loss by selectively scavenging ROS in bone tissue and up-regulating the expression of endogenous antioxidant enzymes, and can be used for the treatment of osteoporosis, with good biosafety. Description of the Drawings

[0019] Figure 1Among them, a is the growth trend of hASCs measured by the CCK-8 kit, b is the growth trend of hBMSCs measured by the CCK-8 kit, c is the quantitative analysis of the migration number of hASCs under different concentrations of salicin determined by the Transwell experiment, d is the quantitative analysis of the migration number of hBMSCs under different concentrations of salicin determined by the Transwell experiment, e is the microscopic image of the migration of hASCs under different concentrations of salicin in the Transwell experiment, f is the microscopic image of the migration of hBMSCs under different concentrations of salicin in the Transwell experiment, g is the quantitative analysis of the migration area of hASCs in the scratch experiment, h is the quantitative analysis of the migration area of hBMSCs in the scratch experiment, i is the morphological details of hASCs in the scratch experiment, j is the morphological details of hBMSCs in the scratch experiment; In the figure, the scale bar of the Transwell experiment image is 100μm, and the scale bar of the scratch experiment image is 100μm;

[0020] Figure 2 Among them, a is the microscopic image of the ROS level of hASCs, b is the microscopic image of the ROS level of hBMSCs, c is the quantitative result of the ROS level of hASCs, d is the quantitative result of the ROS level of hBMSCs, e is the RT-PCR detection result of the relative mRNA expression level of the CAT gene in hASCs, f is the RT-PCR detection result of the relative mRNA expression level of the ROS gene in hASCs, g is the RT-PCR detection result of the relative mRNA expression level of the SOD1 gene in hASCs, h is the RT-PCR detection result of the relative mRNA expression level of the SOD2 gene in hASCs, i is the RT-PCR detection result of the relative mRNA expression level of the CAT gene in hBMSCs, j is the RT-PCR detection result of the relative mRNA expression level of the ROS gene in hBMSCs, k is the RT-PCR detection result of the relative mRNA expression level of the SOD1 gene in hBMSCs, l is the RT-PCR detection result of the relative mRNA expression level of the SOD2 gene in hBMSCs, m is the activity of superoxide dismutase in hASCs, n is the activity of superoxide dismutase in hBMSCs, o is the content of superoxide dismutase in hASCs, p is the content of superoxide dismutase in hBMSCs, q is the content of catalase in hASCs, r is the content of catalase in hBMSCs, s is the immunofluorescence detection result of the relative expression of ROS protein in hASCs, t is the immunofluorescence detection result of the relative expression of ROS protein in hBMSCs; In the figure, merge is the result after the overlap of the two stains;

[0021] Figure 3In it, a is the ALP staining result of hBMSCs, b is the ARS staining result of hBMSCs, c is the ARS quantification result of hBMSCs, d is the ALP quantification result of hBMSCs, e is the qRT-PCR detection result of the relative mRNA expression levels of RUNX2 and ALP genes in hBMSCs at 7 days of culture, f is the immunofluorescence detection result of the relative expression of ALP protein in hBMSCs at 7 days of culture, g is the qRT-PCR detection result of the relative mRNA expression levels of RUNX2 and BGLAP genes in hBMSCs at 14 days of culture, h is the immunofluorescence detection result of the relative expression of RUNX2 protein in hBMSCs at 7 days of culture; in the figure, the scale bar of the immunofluorescence detection image is 100 μm, and merge is the result after the overlap of the two stains;

[0022] Figure 4 In it, a is the ALP staining result of hASCs, b is the ARS staining result of hASCs, c is the ALP quantification result of hASCs, d is the ARS quantification result of hASCs, e is the qRT-PCR detection result of the relative mRNA expression levels of RUNX2 and ALP genes in hASCs at 7 days of culture, f is the qRT-PCR detection result of the relative mRNA expression levels of RUNX2 and BGLAP genes in hASCs at 14 days of culture, g is the immunofluorescence detection result of the relative expression of ALP protein in hASCs at 7 days of culture, h is the immunofluorescence detection result of the relative expression of RUNX2 protein in hASCs at 7 days of culture; in the figure, the scale bar of the immunofluorescence detection image is 100 μm, and merge is the result after the overlap of the two stains;

[0023] Figure 5 In it, a is the HE staining result of hBMSCs subcutaneously implanted in the back of nude mice in each group, b is the Masson staining result of hBMSCs subcutaneously implanted in the back of nude mice in each group, c is the immunohistochemical staining result of hBMSCs subcutaneously implanted in the back of nude mice in each group; in the figure, the scale bar of the 40× field of view is 100 μm, and the scale bar of the 100× field of view is 50 μm;

[0024] Figure 6Experimental results of mice with bilateral ovariectomy model. Among them, a is the micro-CT scan images of the femoral horizontal plane and coronal plane of mice in each group, b is the HE staining results of the femoral coronal plane of mice in each group, c is the Masson staining results of the femoral coronal plane of mice in each group, d is the immunohistochemical staining results of the femurs of mice in each group, e is the bone mineral density level of the femurs of mice in each group, f is the bone volume fraction level of the femurs of mice in each group, g is the trabecular bone thickness level of the femurs of mice in each group, h is the trabecular bone number level of the femurs of mice in each group, i is the bone surface area / bone volume level of the femurs of mice in each group, j is the trabecular bone spacing level of the femurs of mice in each group, k is the trabecular bone pattern factor level of the femurs of mice in each group, l is the expression level of bone alkaline phosphatase in the sera of mice in each group, m is the expression level of type 1 procollagen N-terminal propeptide in the sera of mice in each group, n is the relative concentration of catalase in the sera of mice in each group, o is the relative concentration of superoxide dismutase in the sera of mice in each group; in the figure, the scale bar of HE staining, Masson staining and immunohistochemical staining images is 500 μm;

[0025] Figure 7 Experimental results of mice with suspension model. Among them, a is the micro-CT scan images of the femoral horizontal plane and coronal plane of mice in each group, b is the HE staining results of the femoral coronal plane of mice in each group, c is the Masson staining results of the femoral coronal plane of mice in each group, d is the immunohistochemical staining results of the femurs of mice in each group, e is the bone mineral density level of the femurs of mice in each group, f is the bone volume fraction level of the femurs of mice in each group, g is the trabecular bone thickness level of the femurs of mice in each group, h is the trabecular bone number level of the femurs of mice in each group, i is the bone surface area / bone volume level of the femurs of mice in each group, j is the trabecular bone spacing level of the femurs of mice in each group, k is the trabecular bone pattern factor level of the femurs of mice in each group, l is the expression level of bone alkaline phosphatase in the sera of mice in each group, m is the expression level of type I procollagen N-terminal propeptide in the sera of mice in each group, n is the relative concentration of catalase in the sera of mice in each group, o is the relative concentration of superoxide dismutase in the sera of mice in each group; in the figure, the scale bar of HE staining, Masson staining and immunohistochemical staining images is 500 μm;

[0026] Figure 8 Among them, a is the HE staining results of the heart, liver, spleen, lung and kidney of mice in each group in the in vivo experiment of mice with bilateral ovariectomy model, and b is the HE staining results of the heart, liver, spleen, lung and kidney of mice in each group in the in vivo experiment of mice with suspension model; in the figure, the scale bar of HE staining images is 500 μm;

[0027] Figure 9Among them, a is the Venn diagram of each group of hBMSCs, where the intersection area represents the common genes and the non-intersection area represents the differentially expressed genes; b is the up-regulated and down-regulated genes of each group of hBMSCs; c is the scatter plot of the GO gene enrichment degree; d is the scatter plot of the KEGG gene enrichment degree; e is the qRT-PCR detection result of the relative mRNA expression level of the PPKAA1 gene in each group of hBMSCs; f is the qRT-PCR detection result of the relative mRNA expression level of the PPKAA2 gene in each group of hBMSCs; g is the qRT-PCR detection result of the relative mRNA expression level of the PPKAB1 gene in each group of hBMSCs; h is the qRT-PCR detection result of the relative mRNA expression level of the ULK1 gene in each group of hBMSCs; i is the Western blot detection result of alkaline phosphatase, UNC-51-like kinase 1, phosphorylated adenosine monophosphate-activated protein kinase, adenosine monophosphate-activated protein kinase, and glyceraldehyde-3-phosphate dehydrogenase; j is the expression level of ALP protein in each group of hBMSCs; k is the expression level of ULK1 protein in each group of hBMSCs; l is the expression level of p-AMPK / AMPK protein in each group of hBMSCs. Detailed implementation mode

[0028] The present invention provides the application of salicin in the preparation of a drug for preventing and / or treating osteoporosis.

[0029] The present invention provides the application of salicin in the preparation of a drug for inhibiting oxidative stress.

[0030] The present invention provides the application of salicin in the preparation of a drug for promoting the proliferation and / or differentiation of stem cells, and the stem cells are human bone marrow mesenchymal stem cells and / or adipose mesenchymal stem cells.

[0031] In the present invention, the concentration of salicin in the drug is 5-20 mg / L, preferably 10 mg / L.

[0032] The present invention provides a preparation, and the preparation includes salicin and a pharmaceutically acceptable carrier.

[0033] In the present invention, the concentration of salicin in the preparation is 5-20 mg / L, preferably 10 mg / L.

[0034] The present invention provides the application of the described preparation in the preparation of a product for preventing and / or treating osteoporosis.

[0035] The present invention provides the application of the described preparation in the preparation of a product for inhibiting oxidative stress.

[0036] The present invention provides the application of the described preparation in the preparation of a product for promoting the proliferation and / or differentiation of stem cells.

[0037] In the present invention, the stem cells are human bone marrow mesenchymal stem cells and / or adipose mesenchymal stem cells.

[0038] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] In the cells of the present invention, human bone marrow mesenchymal stem cells (hBMSCs) and adipose mesenchymal stem cells (hASCs) are purchased from ScienCell Research Laboratories, USA;

[0040] In the test animals of the present invention, 6-week-old female BALB / C nude mice and 8-week-old SPF-grade female C57BL / 6N mice (body weight 25 ± 2.5 g) are purchased from the Animal Center of Peking University Health Science Center;

[0041] In the reagents of the present invention, PM cell culture medium, serum-free PM cell culture medium, OM cell culture medium, BCA protein assay kit, P1NP ELISA detection kit, BALP ELISA detection kit, CAT ELISA detection kit, SOD ELISA detection kit were purchased from Thermo Fisher Scientific, USA; CCK-8 kit was purchased from Dojindo Laboratories, Japan; alkaline phosphatase (ALP) staining kit was purchased from Kangwei Century Biotechnology Co., Ltd., Jiangsu; alkaline phosphatase (ALP) detection kit was purchased from Nanjing Jiancheng Bioengineering Institute; alizarin red S staining solution, β-actin (product number: A1978, 1:5000), HRP-labeled goat anti-rabbit IgG (1:5000), HRP-labeled goat anti-mouse IgG (1:5000) were purchased from Sigma-Aldrich, USA; TRIzol reagent was purchased from Invitrogen, USA; Prime SCRIPT RT kit was purchased from Takara, Japan; protease inhibitor (product number: P002) was purchased from Suzhou Xinsaimai Biotechnology Co., Ltd.; reactive oxygen species detection kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; GAPDH rabbit polyclonal antibody (product number: HX1832) was purchased from Beijing Huaxing Biotechnology Co., Ltd.; anti-p-AMPK Anti-AMPKα1(pThr183)+AMPKα2(pThr172) antibody [EPR5683] (product number: ab133448), anti-ALP antibody (product number: ab224335), anti-AMPK antibody (product number: ab32047), rabbit anti-human antibody (product number: ab16731, 1:1000) were purchased from abcam, UK; ULK1 antibody (product number: ab133747) was purchased from abmart, China; mouse anti-human antibody (product number: sc-17767, 1:800) was purchased from Santa Cruz, USA; NOX4 antibody (product number: 14347, 1:1000) was purchased from Cell Signaling, USA;

[0042] In the equipment of the present invention, CT analysis equipment GANTRY-STD CT and Inveon Research Workplace were purchased from Siemens, Germany.

[0043] Experimental Example 1: Stem cell proliferation experiment

[0044] Salicin was dissolved in 1‰ dimethyl sulfoxide (DMSO) to obtain a salicin concentrate with a concentration of 100 mg / L. The salicin concentrate was diluted with PM cell culture medium to obtain PM cell culture medium with salicin concentrations of 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L, respectively. Human bone marrow mesenchymal stem cells (hBMSCs) and adipose-derived mesenchymal stem cells (hASCs) were seeded into 96-well plates at a seeding size of 2000 cells / well. 100 μL of PM cell culture medium containing salicin at concentrations of 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L was added to each well, respectively. PM cell culture medium without salicin (0 mg / L) was used as a control. Each 96-well plate was placed at 37°C and 5% CO2 for cell culture. On day 0, 1, 3, 5, 7, and 14 of cell culture, the hBMSCs and hASCs in each well were counted using a CCK-8 kit. The results are shown in FIG. Figure 1 shown.

[0045] The results showed that low concentrations of salicin could promote the proliferation of hBMSCs and hASCs in vitro and inhibit cell apoptosis, among which 10 mg / L salicin had the best promoting effect.

[0046] Experimental Example 2: Transwell experiment

[0047] Salicin was dissolved in 1‰ DMSO to obtain a salicin concentrate with a concentration of 100 mg / L. The salicin concentrate was diluted with serum-free PM cell culture medium to obtain serum-free PM cell culture medium with salicin concentrations of 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L, respectively. hBMSCs and hASCs were added to the upper compartment of a Transwell chamber with an 8 μm pore filter at a volume of 1×10 5 Cells / chamber were then added with 600 μL of serum-free PM cell culture medium containing salicin at concentrations of 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L, respectively. Serum-free PM cell culture medium without salicin was used as a control (0 mg / L). 600 μL of PM cell culture medium was added to the lower compartment. Each Transwell chamber was incubated at 37°C and 5% CO2 for 24 hours, then rinsed with PBS buffer. The upper compartment was removed and fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 10 minutes. The number of migrating hBMSCs and hASCs was then counted. The results are shown in Figure 2. Figure 1 shown.

[0048] The results showed that low-concentration salicin could promote the in vitro migration of hBMSCs and hASCs, and the promoting effect of 10 mg / L salicin was the best.

[0049] Experimental Example 3: Scratch test

[0050] hBMSCs and hASCs were respectively inoculated into 6-well plates at an inoculation amount of 3×10 5 cells / well. When the confluence rate reached 90%, a cell scratch was made using the tip of a pipette tip. After washing with PBS buffer, 2.5 mL of serum-free PM cell medium with salicin concentrations of 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L prepared in Experimental Example 2 was added to each inoculation well, and serum-free PM cell medium without salicin was used as a control (0 mg / L). Each 96-well plate was placed in an incubator at 37 °C and 5% CO2 for cell culture; the migration images of hBMSCs and hASCs were observed at 0 h, 12 h, and 24 h of cell culture, and the cell migration rate was calculated. The results are as Figure 1 shown;

[0051] The calculation formula for the cell migration rate is:

[0052] In the formula: A0 is the scratch area before adding each serum-free PM cell medium, and A t is the scratch area after corresponding cell culture time.

[0053] The results showed that low-concentration salicin could promote the in vitro migration of hBMSCs and hASCs and inhibit cell apoptosis, and the promoting effect of 10 mg / L salicin was better.

[0054] Experimental Example 4: Oxidative stress determination experiment

[0055] (1) hBMSCs and hASCs were inoculated into 6-well plates at an inoculation amount of 3×10 5Cells were cultured for 24 hours until adherent. Serum-free PM cell culture medium containing 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, or 40 mg / L salicin was then added to each well. Serum-free PM cell culture medium containing H2O2 was used as a control. The cells were incubated at 37°C for 24 hours. After washing the cells twice with PBS, 200 μL of RIPA lysis buffer containing 1% protease inhibitors was added. The cells were lysed on ice for 30 minutes and centrifuged at 4°C at 12,000 rpm for 15 minutes. The supernatant was used as the total protein sample. The protein concentration of each total protein sample was determined using a BCA protein assay kit and adjusted to 2 μg / μL. ROS levels were then measured using the DCFH-DA fluorescent probe assay. The specific steps were: 10 μM DCFH-DA was added to each total protein sample, incubated at 37°C in the dark for 30 minutes, washed three times with PBS, and the fluorescence intensity of each total protein sample was measured using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0056] (2) 30 μg of each protein sample was separated by 12% SDS-PAGE and transferred to a 0.22 μm PVDF membrane at 300 mA for 90 min. The membrane was then blocked with 5% skim milk at room temperature for 1 h, incubated overnight with mouse anti-human antibody at 4°C, and then incubated with HRP-labeled goat anti-mouse IgG at room temperature for 1 h. After ECL development, the grayscale value of the bands was quantified using ImageJ software to obtain the SOD level of each total protein sample.

[0057] 30 μg of each protein sample was separated by 12% SDS-PAGE and transferred to a 0.22 μm PVDF membrane at 300 mA for 90 min. The membrane was then blocked with 5% skim milk at room temperature for 1 h, incubated overnight with rabbit anti-human antibody at 4°C, and then incubated with HRP-labeled goat anti-rabbit IgG at room temperature for 1 h. After ECL development, the grayscale value of the bands was quantified using ImageJ software to obtain the CAT level of each total protein sample.

[0058] 30 μg of each protein sample was separated by 12% SDS-PAGE and transferred to a 0.22 μm PVDF membrane at 300 mA for 90 min. The membrane was then blocked with 5% skim milk at room temperature for 1 h, incubated with NOX4 antibody overnight at 4°C, and then incubated with HRP-labeled goat anti-mouse IgG at room temperature for 1 h. After ECL development, the grayscale value of the bands was quantified using ImageJ software to obtain the ROX level of each total protein sample.

[0059] (3) Using β-actin as the internal reference gene, RT-PCR was used to detect the relative mRNA expression levels of CAT, ROS, SOD1, and SOD2 genes in hBMSCs and hASCs. The results are shown in Figure 3. Figure 2 shown.

[0060] The results showed that low-concentration salicin could reduce the oxidative stress levels of hBMSCs and hASCs, and among them, 10 mg / L salicin had a better effect on reducing the protein expression levels of CAT, SOD, and ROS in hBMSCs and hASCs.

[0061] Experimental Example 5: Alkaline Phosphatase Staining

[0062] (1) Dissolve salicin in 1‰ DMSO to obtain a salicin concentrate with a concentration of 100 mg / L. Dilute the salicin concentrate with OM cell culture medium to obtain OM cell culture media with salicin concentrations of 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L respectively. Inoculate hBMSCs and hASCs into PM cell culture medium, OM cell culture medium, and OM cell culture media with salicin concentrations of 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L respectively, and culture them at 37 °C and 5% CO2 for 7 days. After washing with PBS buffer, fixing with 95% cold ethanol, and washing with PBS buffer, stain hBMSCs and hASCs with an alkaline phosphatase (ALP) staining kit, and measure the ALP activity of hBMSCs and hASCs with an alkaline phosphatase (ALP) detection kit. The results are as Figures 3 - 4 shown;

[0063] (2) After washing the hBMSCs and hASCs cultured in step (1) for 7 days with PBS buffer and lysing them on ice with 1% Triton X-100, centrifuge them at 4 °C and 13362×g for 30 min, and measure the total protein concentration of hBMSCs and hASCs with a BCA protein analysis kit. The results are as Figures 3 - 4 shown.

[0064] The results showed that salicin at concentrations of 5 mg / L, 10 mg / L, and 20 mg / L could all promote the in vitro osteogenic differentiation of hBMSCs and hASCs cultured for 7 days, and among them, 10 mg / L salicin had the most obvious promoting effect. It shows that low-concentration salicin can promote the in vitro osteogenic differentiation of hBMSCs and hASCs.

[0065] Experimental Example 6: Alizarin Red Staining

[0066] hBMSCs and hASCs were respectively inoculated into PM cell culture medium, OM cell culture medium, and the OM cell culture medium containing salicin at concentrations of 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L prepared in Experimental Example 5. After culturing for 14 d at 37 °C and 5% CO2, 2% alizarin red S staining solution (ARS) was used for staining at room temperature for 20 min. After adding 100 mmol / L cetylpyridinium chloride to dissolve hBMSCs and hASCs, the OD of hBMSCs and hASCs was measured by spectrophotometry. 490nm value, and the results are as Figures 3 - 4 shown.

[0067] The results showed that salicin at concentrations of 5 mg / L, 10 mg / L, and 20 mg / L could all promote the osteogenic differentiation of hBMSCs and hASCs cultured for 14 d in vitro, and the promoting effect of 10 mg / L salicin was the most obvious. It indicated that low-concentration salicin could promote the osteogenic differentiation of hBMSCs and hASCs in vitro.

[0068] Experimental Example 7: qRT-PCR and immunofluorescence detection

[0069] (1) Template acquisition: TRIzol reagent was used to extract the total RNA of hBMSCs and hASCs cultured for 7 d and 14 d according to the method described in Experimental Example 5. Then, Prime SCRIPT RT kit was used for reverse transcription to obtain the cDNA of hBMSCs and hASCs, which was used as the template for qRT-PCR detection;

[0070] (2) Primer design: Based on glyceraldehyde-3-phosphate gene GAPDH (NCBI ID: 2597), alkaline phosphatase gene ALP (NCBI ID: 249), runt-related transcription factor 2 gene RUNX2 (NCBI ID: 860), and bone γ-carboxyglutamic acid protein gene BGLAP (NCBI ID: 632) respectively, qRT-PCR primers were designed to detect the relative mRNA expression levels of ALP, RUNX2, and BGLAP genes in hBMSCs and hASCs. The GAPDH gene was used as an internal reference gene, and it was commissioned to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. The nucleotide sequences of each primer are shown in Table 1;

[0071] Table 1 Nucleotide sequences of qRT-PCR primers

[0072]

[0073] (3) qRT-PCR assay: The qRT-PCR assay system is 10 μL in volume, including 5 μL of SYBR Green, 1 μL of cDNA, 0.5 μL each of F / R primers, and the balance of ddH2O; the qRT-PCR assay conditions are: pre-denaturation at 95°C for 10 min; then denaturation at 95°C for 30 s → annealing at 60°C for 60 s, for a total of 40 cycles; then use ΔΔ the Ct method to calculate the relative mRNA expression levels of ALP, RUNX2, and BGLAP genes in hBMSCs and hASCs, and use 2-ΔΔ the Ct method to calculate the relative mRNA expression levels of ALP, RUNX2, and BGLAP genes in hBMSCs and hASCs relative to the GAPDH gene;

[0074] (4) Immunofluorescence assay: Immunofluorescence assay was performed on the relative expression of ALP protein and RUNX2 protein in hBMSCs and hASCs cultured for 7 d, and the results are as Figures 3 - 4 shown.

[0075] The qRT-PCR assay results showed that 10 mg / L salicin could better promote the expression of osteogenesis-related genes such as ALP and RUNX2 in hBMSCs and hASCs after 7 d of in vitro osteogenic induction, and the expression of osteogenesis-related genes such as BGLAP and RUNX2 in hBMSCs and hASCs after 14 d of in vitro osteogenic induction;

[0076] The immunofluorescence assay results showed that after 7 d of in vitro osteogenic induction, salicin at concentrations of 5 mg / L, 10 mg / L, and 20 mg / L could all increase the relative expression levels of osteogenesis-related proteins such as ALP protein and RUNX2 protein in hBMSCs and hASCs, and the improvement effect of 10 mg / L salicin was the most obvious;

[0077] It shows that low-concentration salicin can effectively promote the osteogenic differentiation of hBMSCs and hASCs in vitro.

[0078] Experimental Example 8: In Vivo Subcutaneous Transplantation Experiment

[0079] Twelve female BALB / C nude mice were divided into PM group and PM+Salicin group, with 6 nude mice in each group. hBMSCs were inoculated into PM cell culture medium without salicin and PM cell culture medium containing 10 mg / L salicin, respectively, and cultured at 37°C and 5% CO2 for 10 days. After harvesting, hBMSCs were mixed with tricalcium phosphate carriers and then implanted subcutaneously on the back of BALB / C nude mice. The nude mice in PM group were implanted with hBMSCs cultured in PM cell culture medium without salicin, and the nude mice in PM+Salicin group were implanted with hBMSCs cultured in PM cell culture medium containing 10 mg / L salicin. After the nude mice in each group were routinely cultured for 8 weeks, the hBMSCs implanted subcutaneously on the back of the nude mice in each group were collected for HE staining, Masson staining, and immunohistochemical staining with osteocalcin (OCN) as a marker to examine the osteogenesis. The results are shown as follows: Figure 5 shown.

[0080] HE staining results showed that compared with the PM group, nude mice in the PM+Salicin group had more new bone formation in vivo. Massom staining results showed that nude mice in the PM+Salicin group had more new bone formation in vivo, while nude mice in the PM group had more collagen. Immunohistochemical staining results showed that compared with the PM group, nude mice in the PM+Salicin group had more new bone formation in vivo, and the OCN content was significantly higher than that in the PM group. This indicates that 10 mg / L salicin can promote the osteogenic differentiation of hBMSCs in vivo.

[0081] Experimental Example 9: Construction of bilateral ovariectomized mouse model and in vivo experiments

[0082] (1) Model construction: SPF female C57BL / 6N mice were anesthetized and intraperitoneally injected with sodium pentobarbital at a dose of 50 mg / kg of mouse body weight. Bilateral ovariectomy (OVX) surgery was then performed. Specifically, the mice were fixed in a prone position, their backs were shaved and disinfected with iodine and alcohol alternately. Then, bilateral skin incisions of 1 cm were made at the level of the lumbar vertebrae L1 to L3. The muscle layer was separated from the incisions to open the abdominal cavity. The connection between the ovary and the fallopian tube was ligated, and the ovaries were removed, hemostasis was stopped, and sutures were performed to obtain bilateral ovariectomy (OVX) model mice.

[0083] (2) Reagent preparation: Dissolve salicin in PBS buffer to obtain a salicin solution with a concentration of 10 mg / L;

[0084] (3) In vivo experiment: Sixteen SPF-grade female C57BL / 6N mice were divided into the Sham+PBS group and the Sham+Salicin group, and another sixteen OVX model mice were divided into the OVX+PBS group and the OVX+Salicin group, with 8 mice in each group. Mice in the Sham+Salicin group and the OVX+Salicin group were intraperitoneally injected with a 10 mg / L salicin solution, and mice in the Sham+PBS group and the OVX+PBS group were intraperitoneally injected with PBS buffer. The dosage was 100 mL / kg of mouse body weight. At this time, the single-dose concentration of salicin in the blood of mice in the Sham+Salicin group and the OVX+Salicin group was 10 mg / L. They were intraperitoneally administered once a day for 30 consecutive days. Then, the femurs of mice in each group were taken for HE staining, Masson staining, and immunohistochemical staining with OCN as a marker to examine osteogenesis. The hearts, livers, spleens, lungs, and kidneys of mice in each group were removed for HE staining. The results are as Figure 6 and Figure 8 shown.

[0085] The results of mouse femur staining showed that after administration of 10 mg / L salicin, the bone loss in the femurs of OVX model mice was significantly improved. The results of mouse organ staining showed that no significant inflammatory reaction was observed in the organs of each group of mice, indicating that 10 mg / L salicin can improve the osteoporosis condition of OVX model mice and inhibit bone loss, and has good biosafety.

[0086] Experimental Example 10: Construction of a suspension model mouse and in vivo experiment

[0087] (1) Model construction: The tails of SPF-grade female C57BL / 6N mice were suspended, keeping the head tilted downward at 30°, the hind limbs elevated, and allowing the mice to move freely 360° in the cage. After continuous treatment for 14 days, suspension model mice were obtained;

[0088] (2) In vivo experiment: Sixteen SPF-grade female C57BL / 6N mice were divided into the Sham+PBS group and the Sham+Salicin group, and another sixteen suspended model mice were divided into the Suspended+PBS group and the Suspended+Salicin group, with 8 mice in each group. Mice in the Sham+Salicin group and the Suspended+Salicin group were intraperitoneally injected with the salicin solution prepared in Experimental Example 9 at a concentration of 10 mg / L, and mice in the Sham+PBS group and the Suspended+PBS group were intraperitoneally injected with PBS buffer. The dosage was 100 mL / kg of mouse body weight. At this time, the single-dose concentration of salicin in the blood of mice in the Sham+Salicin group and the Suspended+Salicin group was 10 mg / L. They were intraperitoneally administered once a day for 14 consecutive days. Then, the femurs of mice in each group were taken for HE staining, Masson staining, and immunohistochemical staining with OCN as the marker to examine osteogenesis. The hearts, livers, spleens, lungs, and kidneys of mice in each group were removed for HE staining. The results are as Figures 7 - 8 shown.

[0089] The staining results of mouse femurs showed that after administration of 10 mg / L salicin, the bone loss in the femurs of suspended model mice was significantly improved. The staining results of mouse organs showed that no significant inflammatory reaction or toxic reaction was observed in the organs of mice in each group, indicating that 10 mg / L salicin can improve the osteoporosis condition of suspended model mice and inhibit bone loss under weightlessness, and has good biosafety.

[0090] Experimental Example 11: micro-CT analysis [[ID=1)]

[0091] The femurs of mice in each group in Experimental Example 9 and Experimental Example 10 were taken, fixed with 10% formalin for 24 h, washed with PBS buffer, and then scanned at the proximal end of the femurs of mice in each group using a CT analysis device GANTRY-STD CT under the conditions of a scanning resolution of 8.82 μm, a tube voltage of 60 kV, a tube current of 500 μA, and a scanning time of 1500 ms. The bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone thickness (Tb.th), trabecular bone number (Tb.N), bone surface area / bone volume (BS / BV), trabecular bone spacing (Tb.Sp), and trabecular bone pattern factor (Tb.Pf) of the femurs of mice in each group were calculated using Inveon Research Workplace. The results are as Figures 6 - 7 shown.

[0092] Micro-CT analysis results showed that after administration of 10 mg / L salicin, the bone loss in the femurs of OVX model mice and suspension model mice was significantly improved, with a denser bone structure and more trabecular bone. The BMD, BV / TV, Tb.th, and Tb.N levels in the femurs of OVX model mice and suspension model mice all increased significantly, while the Tb.Sp, Tb.Pf, and BS / BV levels decreased significantly. After administration of 10 mg / L salicin to healthy mice, there were no significant differences in various parameters. This indicates that 10 mg / L salicin can improve the osteoporosis status of OVX model mice and suspension model mice and inhibit bone loss.

[0093] Experimental Example 12: Serum Biomarker Analysis

[0094] Blood samples of each group of mice in Experimental Example 9 and Experimental Example 10 were collected. After centrifugation at 3000 rpm for 15 min, the serum was separated. The levels of serum biomarkers such as bone alkaline phosphatase (BALP), procollagen type 1 N-terminal propeptide (P1NP), catalase (CAT), and superoxide dismutase (SOD) in the serum of each group of mice were detected using P1NP ELISA detection kit, BALP ELISA detection kit, CAT ELISA detection kit, and SOD ELISA detection kit respectively. The results are as Figures 6 - 7 shown.

[0095] The results showed that administration of 10 mg / L salicin could increase the expression levels of BALP and PINP in the serum of OVX model mice and suspension model mice, thereby inhibiting bone loss. At the same time, it could increase the relative concentrations of SOD and CAT in the serum of OVX model mice, thereby reducing the body's oxidative stress response.

[0096] Experimental Example 13: RNA-seq Analysis

[0097] hBMSCs were divided into a PM group, an OM group, and an OM+Sacilin group, and were respectively inoculated into PM cell culture medium, OM cell culture medium, and the OM cell culture medium with a salicin concentration of 10 mg / L prepared in Experimental Example 5. After culturing for 7 d at 37°C and 5% CO2, the total RNA of each group of hBMSCs was extracted as a sample according to the method described in Experimental Example 7, and entrusted to Novogene Co., Ltd. in Beijing for sample quality control, library construction, Illumina sequencing, and RNA-seq analysis. The results are as Figure 9 shown.

[0098] The results showed that there were differential genes in hBMSCs of the OM + Sacilin group compared with those of other groups. Among them, the AMPK signaling pathway had a significant difference and was closely related to the osteogenic function. Compared with hBMSCs of the OM group, the genes of PPKAA1, PPKAA2, PPKAB1, and ULK1 in hBMSCs of the OM + Sacilin group showed significantly up-regulated expression. It was indicated that salicin could promote the osteogenic differentiation of hBMSCs by affecting the AMPK signaling pathway.

[0099] Experimental Example 14: Western blot detection

[0100] Take the hBMSCs of each group cultured for 7 days in Experimental Example 13, add them to a radioimmunoprecipitation assay (RIPA) buffer containing 1% phosphatase and protease inhibitors, and lyse for 30 min. Then centrifuge at 4°C and 14,000 rpm for 20 min to obtain the supernatant. Use a BCA protein assay kit to measure the total protein concentration of hBMSCs in each supernatant, and perform SDS-PAGE electrophoresis and Western blot detection on the protein expression of alkaline phosphatase (ALP), UNC-51-like kinase 1 (ULK1), phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), adenosine monophosphate-activated protein kinase (AMPK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results are as Figure 9 shown.

[0101] The results showed that compared with hBMSCs of the PM group and the OM group, the protein expression levels of ALP, ULK1, and p-AMPK / AMPK in hBMSCs of the OM + Sacilin group increased. It was indicated that salicin could promote the osteogenic differentiation of hBMSCs by affecting the AMPK signaling pathway.

[0102] From the above examples, it can be seen that the present invention provides the application of salicin in the preparation of drugs for preventing and / or treating osteoporosis. The salicin of the present invention can promote the proliferation and migration of hBMSCs and hASCs in vitro, reduce the oxidative stress level of cells, promote the osteogenic differentiation of cells and improve the osteoporosis condition by affecting the AMPK signaling pathway, and inhibit apoptosis and bone loss by selectively scavenging ROS in bone tissue and up-regulating the expression of endogenous antioxidant enzymes, and has good biosafety.

[0103] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. The use of salicin in the preparation of a medicament for preventing and / or treating osteoporosis.

2. The use of salicin in the preparation of a medicament for inhibiting oxidative stress.

3. Use of salicin in the preparation of a drug for promoting the proliferation and / or differentiation of stem cells, characterized in that, The stem cells are human bone marrow mesenchymal stem cells and / or adipose mesenchymal stem cells.

4. The application according to any one of claims 1 to 3, characterized in that, The concentration of salicin in the medicament is 5 - 20 mg / L.

5. A preparation, characterized in that, The preparation comprises salicin and a pharmaceutically acceptable carrier.

6. The preparation according to claim 5, characterized in that, The concentration of salicin in the preparation is 5 - 20 mg / L.

7. The use of the preparation according to claim 5 or 6 in the preparation of a product for preventing and / or treating osteoporosis.

8. The use of the preparation according to claim 5 or 6 in the preparation of a product for inhibiting oxidative stress.

9. The use of the preparation according to claim 5 or 6 in the preparation of a product for promoting stem cell proliferation and / or differentiation.

10. The application according to claim 9, characterized in that, The stem cells are human bone marrow mesenchymal stem cells and / or adipose mesenchymal stem cells.

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