A polypeptide for promoting osteogenic differentiation of mesenchymal stem cells and application thereof in preparation of a drug for treating osteoporosis

Abstract

This invention discloses a polypeptide that promotes osteogenic differentiation of mesenchymal stem cells and its application in the preparation of drugs for treating osteoporosis, belonging to the field of biomedical technology. The amino acid sequence of the polypeptide is shown in SEQ ID NO.1. This polypeptide was obtained by screening osteogenic-induced human bone marrow mesenchymal stem cells using ribosomal sequencing technology, and its coding ability was verified. Experiments show that this polypeptide can significantly upregulate the expression of osteogenic differentiation markers RUNX2 and COL1A1, and enhance alkaline phosphatase activity. In a hindlimb deloaded osteoporosis mouse model, overexpression of this polypeptide significantly increased bone mineral density, improved bone microstructure, promoted new bone formation, and improved bone biomechanical properties. This polypeptide is a human-derived natural polypeptide with low immunogenicity and good safety. This polypeptide can be used to prepare drugs that promote osteogenic differentiation and prevent / treat osteoporosis, showing broad clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drug technology, and specifically relates to a polypeptide that promotes osteogenic differentiation of mesenchymal stem cells and its application in the preparation of drugs for treating osteoporosis. Background Technology

[0002] Osteoporosis (OP) is a common systemic metabolic bone disease characterized by low bone mass, bone microarchitectural deterioration, increased bone fragility, and an increased risk of fractures. Based on its cause, it can be divided into primary osteoporosis and secondary osteoporosis. Primary osteoporosis is the most common, mainly including postmenopausal osteoporosis (type I), geriatric osteoporosis (type II), and idiopathic osteoporosis (juvenile type).

[0003] Currently, medications for treating osteoporosis can be categorized based on their mechanism of action into bone resorption inhibitors, bone formation promoters, dual-action drugs, and other mechanisms-of-action drugs. However, existing drugs all have limitations in clinical application. Regarding safety and adverse reactions, oral bisphosphonates can easily irritate the gastrointestinal mucosa, causing gastrointestinal reactions such as abdominal pain, nausea, and indigestion; denosumab can cause musculoskeletal pain and limb pain; romozolumab can cause joint pain, headache, muscle spasms, and peripheral edema. These adverse reactions directly affect patient adherence and even limit the clinical promotion of these drugs. In terms of cost-effectiveness, some newer drugs are expensive, and long-term use increases the financial burden on patients, while traditional drugs, although inexpensive, have relatively significant adverse reactions. In summary, considering that osteoporosis patients in my country are mostly middle-aged and elderly, have varying economic capabilities, and have high requirements for medication safety, there is an urgent need in clinical practice to find a drug with high safety, mild adverse reactions, strong cost-effectiveness, and the ability to effectively regulate bone metabolism and reduce fracture risk in the long term.

[0004] Due to their high activity, low toxicity, and ease of modification, peptide drugs have been widely used in antiviral, antitumor, cardiovascular disease, vaccine injection, and disease diagnosis, with good results. In recent years, they have also been explored for promoting bone formation. Deer antler peptides can upregulate osteogenic markers such as ALP and RUNX2 (Pilose antler aqueous extract promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells by stimulating the BMP-2 / Smad1,5 / Runx2 signaling pathway[J]. Chinese Journal of Natural Medicines, 2019, 17(10): 756-767); In addition, Chinese patent application CN116589562B extracted and synthesized an 11-peptide (sequence gdrgerggkge) from fish collagen, which can indirectly promote osteogenic differentiation of bone marrow mesenchymal stem cells by upregulating miR-21, and can be used to alleviate osteoporosis. However, the aforementioned peptides are all derived from exogenous biological materials (deer antler, fish collagen), and as xenogeneic active ingredients, they pose a potential immunogenicity risk when applied to humans. Furthermore, existing exogenous osteogenic peptides are mostly short-chain structures, their in vivo stability needs further verification, and their mechanisms of action are mostly indirect regulation; whether they can effectively regulate bone metabolism in the long term remains unclear. Therefore, developing a peptide drug with higher safety (especially lower immunogenicity), that can directly and effectively promote osteogenic differentiation, and that is suitable for long-term use remains a pressing technical problem to be solved in this field. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to propose a polypeptide that promotes osteogenic differentiation of mesenchymal stem cells and its application in the preparation of drugs for treating osteoporosis. This addresses the issues of existing osteoporosis treatments, such as significant adverse reactions and poor cost-effectiveness, while exogenous osteogenic polypeptides pose risks of immunogenicity, insufficient structural stability, and unclear side effects. This invention, for the first time, proposes a significant role for a polypeptide in promoting osteogenic differentiation of mesenchymal stem cells, increasing bone mineral density, and treating osteoporosis. Furthermore, as a naturally occurring polypeptide, it exhibits low immunogenicity. Therefore, this polypeptide X has broad application prospects as an active ingredient in the preparation of drugs or formulations for the prevention or treatment of osteoporosis.

[0006] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention provides a polypeptide that promotes osteogenic differentiation of mesenchymal stem cells, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The amino acid sequence of the polypeptide X is Met–Gly–Gly-Pro-Leu-Leu-Glu-Gly-Arg-Glu-Ser-Ser-Phe-Trp-Gly-Lys-Leu-Arg-Gln-Trp-Gly-Arg-Gln-Ser-Arg-Met-Ser-Pro-His-Pro-Gly-Trp-Gly-Ser-Ser-Thr-Gly-Cys-Ile-Cys-Arg-Gly-Ser-Gln-Arg-Asn-Ala-Gly-Ala-Lys-Arg-Ser-Pro-Gly-Lys-Glu-Gly-Trp-Gly-Thr-Thr-Cys-Met.

[0008] (MGGPLLEGRESSFWGKLRQWGRQSRMSPHPGWGSSTGCICRGSQRNAGAKRSPGKEGWGTTCM, as shown in SEQ ID NO.1) The peptides are derived from mammals and are human-derived natural peptides. Compared to exogenous protein or peptide drugs, human-derived natural peptides have lower immunogenicity, significantly reducing the risk of the body developing drug-resistant antibodies and allergic reactions. They also have better biocompatibility and safety, which is beneficial for long-term administration.

[0009] The polypeptide includes functional variants that have more than 95% homology with its amino acid sequence and retain the same biological activity.

[0010] The derivatives are those obtained by hydroxylating, carboxylating, methylating, phosphorylating, acetylating, glycosylating, or esterifying the amino acid side chain groups, amino terminus, or carboxyl terminus of the polypeptide, while retaining its biological activity of promoting osteogenic differentiation of mesenchymal stem cells. Modification can improve the in vivo stability of the polypeptide (e.g., resistance to protease degradation), prolong its half-life, enhance tissue targeting, or improve bioavailability without affecting its osteogenic promoting activity, thus providing more technical options for the formulation development of polypeptide drugs.

[0011] The polypeptide X provided by this invention can be artificially synthesized through chemical synthesis or genetic engineering, or it can be obtained by separation and purification from cells.

[0012] Secondly, the present invention provides the use of the polypeptide that promotes osteogenic differentiation of mesenchymal stem cells, or the functional variant or derivative thereof, in the preparation of a drug or formulation that promotes osteogenic differentiation.

[0013] Thirdly, the present invention provides the use of the polypeptide that promotes osteogenic differentiation of mesenchymal stem cells, or the functional variant or derivative thereof, in the preparation of a medicament or formulation for the prevention or treatment of osteoporosis.

[0014] Fourthly, the present invention provides a pharmaceutical composition for the prevention or treatment of osteoporosis, comprising the said polypeptide, the said functional variant or the said derivative, and a pharmaceutically acceptable adjuvant or carrier.

[0015] Preferably, the carrier of the pharmaceutical composition includes inorganic carriers, bone-targeting nanocarriers, adhesives, wetting agents, excipients, fillers, absorption enhancers, surfactants, lubricants, etc., and microbial carriers, plasmids, cell vesicles, liposomes, lentiviral vectors, or adenovirus vectors.

[0016] The dosage form of the pharmaceutical composition is selected from at least one of the following: powder, inhaler, patch, tablet, injection, granule, pill, syrup, oral liquid, capsule, ointment, or suppository.

[0017] The pharmaceutical composition further comprises at least one adjunctive therapeutic agent selected from the group consisting of: bisphosphonates, selective estrogen receptor modulators, parathyroid hormone analogs, RANKL inhibitors, calcium preparations, or vitamin D preparations.

[0018] Fifthly, the present invention provides a method for screening polypeptides that promote osteogenic differentiation of mesenchymal stem cells. The method includes analyzing the translatome of human osteogenic-induced bone marrow mesenchymal stem cells using ribosomal sequencing technology to obtain candidate polypeptide sequences, and screening the polypeptides with the amino acid sequences shown in SEQ ID NO.1 by verifying osteogenic differentiation function.

[0019] Compared with the prior art, the present invention achieves the following technical effects: This invention provides a polypeptide with a specific amino acid sequence (SEQ ID NO.1) that promotes osteogenic differentiation of mesenchymal stem cells. This polypeptide can significantly upregulate the expression levels of osteogenic differentiation markers (RUNX2, COL1A1) and enhance alkaline phosphatase activity, thereby effectively promoting the differentiation of mesenchymal stem cells into osteoblasts, providing a novel active molecule for the treatment of osteoporosis and other bone metabolic diseases. Furthermore, polypeptide X is a naturally occurring polypeptide in vivo, and compared to large protein molecules, it has lower immunogenicity and fewer toxic side effects. Therefore, polypeptide X can be effectively used in the preparation of drugs or reagents that promote osteoblast differentiation; it can also be used as an active ingredient in the preparation of drugs for the prevention or treatment of osteoporosis.

[0020] The application provided by this invention, based on sufficient in vitro functional validation data (including ALP staining, qPCR, and Western blotting results), clarifies the direct role of the peptide in promoting the differentiation of mesenchymal stem cells into osteoblasts, providing a new active ingredient for bone tissue engineering and bone regeneration medicine. In vivo efficacy validation in a hindlimb deloaded osteoporosis mouse model (including Micro-CT bone microstructure analysis, calcein double labeling experiment, and three-point bending biomechanical test) demonstrates that the peptide can significantly increase bone density, improve trabecular bone microstructure, promote new bone formation, and enhance bone biomechanical properties, thereby exhibiting a clear protective effect against osteoporosis. Attached Figure Description

[0021] Figure 1 This section verifies the coding ability and expression of peptide X of the present invention. A shows the Western blotting verification after transfecting HEK 293T cells with both full-length and mutant plasmids of peptide X. BHEK 293T cells were transfected with the full-length peptide X plasmid, and immunofluorescence was detected. Scale bar: 10 μm. Western blotting verification of ChBMSCs after transfecting with adenovirus of peptide X (n=3). D shows immunofluorescence detection of hBMSCs after transfecting with adenovirus of peptide X. Blue: DAPI-labeled cell nucleus; Green: Flag-labeled peptide X; Red: Phalloidin-labeled cytoskeleton; Merge: merged image. Scale bar: 20 μm.

[0022] Figure 2 This invention illustrates the expression changes of peptide X and osteogenic differentiation markers after osteogenic induction. In this study, A represents the protein expression levels of peptide X, RUNX2, and COL1A1 detected by Western blotting 3 days after osteogenic induction. CON: Uninduced control group; OSTEO: Osteogenic induction group. p <0.05 vs CON group. B represents the mRNA expression levels of peptide X, RUNX2, and COL1A1 detected by qRT-PCR 3 days after osteogenic induction. p <0.05 vs CON group.

[0023] Figure 3This invention validates the function of peptide X in promoting osteogenic differentiation of hBMSCs. A shows a volcano plot of hBMSC transcriptome sequencing after overexpression of peptide X, with red dots representing significantly upregulated genes (1819), blue dots representing significantly downregulated genes (349), and gray dots representing genes with no significant difference. B shows the KEGG pathway enrichment analysis of differentially expressed genes (first 30 pathways). C shows the protein expression levels of RUNX2, COL1A1, and peptide X after overexpression of peptide X, detected by Western blotting, with GAPDH as an internal control. D shows the mRNA expression levels of RUNX2 and COL1A1 after overexpression of peptide X, detected by qRT-PCR. p<0.05 vs NC group), E is the alkaline phosphatase activity of hBMSCs after ALP staining to detect the expression of peptide X (left: NC group, right: Ad-X group). Figure 4 This invention describes the protective effect of peptide X on osteoporotic mice. A represents Micro-CT three-dimensional reconstructed images showing the microstructure of the distal femur in each group of mice; B represents quantitative analysis of bone microstructure parameters, including BMD, BV / TV, BS / TV, and Tb.Sp. p <0.05 vs HLU+rAAV9-NC group), C is the calcein double-label staining image and quantitative analysis of the double-label spacing, D is the load-deflection curve of the three-point bending test, and E is the quantitative analysis of biomechanical parameters, including stiffness, moment of inertia of section and maximum load ( p <0.001 vs HLU+rAAV9-NC group). Detailed Implementation

[0024] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0025] The amino acid sequence listing of this invention is shown in SEQ ID NO.1. The amino acid sequence of polypeptide X is 63 amino acids in length, as detailed in the sequence listing file. The amino acid sequence of polypeptide X is: MGGPLLEGRESSFWGKLRQWGRQSRMSPHPGWGSSTGCICRGSQRNAGAKRSPGKEGWGTTCM (as shown in SEQ ID NO.1) For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0026] In the following examples, some reagent and instrument information is shown in Table 1.

[0027] Table 1: Experimental Instruments / Reagents

[0028] Example 1: Screening and validation of functional peptides in human osteogenic induction hBMSCs This embodiment is used to obtain the polypeptide of the present invention, the amino acid sequence of which is shown in SEQ ID NO.1 (hereinafter referred to as "polypeptide X"). (1) Screening of functional peptides in human osteogenic induction hBMSCs Human bone marrow mesenchymal stem cells (hBMSCs, purchased from Suzhou Cyagen Biotech Co., Ltd.) were seeded in a special medium (Oricell, HUXMA-90011) containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 60%-70%, the medium was replaced with osteogenic induction medium (containing 10% FBS, 10 mM β-glycerophosphate, 50 μM ascorbic acid, and 0.1 μM dexamethasone), and induced for 3 days, with 3 biological replicates per group.

[0029] Cells were collected separately, lysed, and non-ribosome-protected RNA fragments were digested with RNase I to extract ribosome-protected RNA fragments (approximately ~30 bp). Sequencing libraries were constructed, and single-end sequencing was performed using the Illumina NovaSeq 6000 platform. Raw data underwent quality control via FASTP to filter low-quality data, resulting in clean reads. The read alignment tool bowtie2 (v2.2.8) was used to remove ribosomal RNA, transfer RNA, snoRNA, snRNA, and miRNA. Read length distribution was statistically analyzed, retaining only reads with lengths between 20 bp and 40 bp. The filtered reads were then aligned to the reference gene sequence using Star. ORF scores and RRS were calculated based on the abundance and location distribution of each sORF. Fickett scores and Hexamer scores were calculated based on sORF sequence characteristics. These four values ​​were combined to screen for potentially translatable sORFs. Differential expression analysis was performed using DESeq2, with the selection criteria being FDR < 0.05 and |log2FC| > 1.

[0030] In the ORFs that showed significantly upregulated expression after osteogenic induction, candidate molecules located in non-coding RNA regions and encoding peptides between 20 and 100 amino acids in length were further screened. A peptide of 63 amino acids in length was finally obtained and named peptide X, whose amino acid sequence is shown in SEQ ID NO.1. Ribo-seq data showed that the translational abundance of this peptide was 1.7-fold higher than that of uninduced cells 3 days after osteogenic induction (p<0.01).

[0031] (2) Encoding ability and expression verification of peptide X To verify whether the ORF predicted by Ribo-seq ribosomal sequencing has real peptide coding ability, the following experiment was conducted.

[0032] ① Plasmid construction and HEK 293T cell transfection The complete ORF of peptide X (including the start codon ATG) was cloned into the pcDNA3.1(+) vector, and a Flag tag was fused to the C-terminus to construct an overexpression plasmid. The start codon was deleted by site-directed mutagenesis (ATG→Δ) to construct a mutant plasmid as a negative control.

[0033] HEK 293T cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. When the cell confluence reached 90%-100%, the old medium was discarded, and 3 mL of 0.25% trypsin was added. The cells were then incubated at 37°C for 2 minutes for digestion. After observing the cells under a microscope and confirming they had become rounded, 5 mL of complete medium was added to stop the digestion. The cell suspension was then aspirated into a 15 mL centrifuge tube and centrifuged (1000 rpm for 5 min). The supernatant was discarded, and the cells were resuspended in complete medium. The cell suspension was then added to six-well plates for further culture. The 293T cells in the six-well plates were observed. When cell confluence reached 60%-70%, the overexpression plasmid and mutant plasmid were transfected separately using Lipofectamine 2000 (5 ng plasmid per well). 5 μL of Lipofectamine-2000 was added to 150 μL of Opti-mem medium and mixed thoroughly (Solution A). Separately, 5 ng of the overexpression plasmid (carrying the Flag tag) and 5 ng of the mutant plasmid Mut (carrying the Flag tag) were added to 150 μL of Opti-mem medium and mixed thoroughly (Solution B). Solution B was then mixed with Solution A and allowed to stand for 20 min. 1.7 mL of serum- and antibiotic-free DMEM high-glucose medium was added to each well of a six-well plate, and the mixture was added to each well. Six hours after transfection, the medium was replaced with medium containing 10% fetal bovine serum but without antibiotics. Once cell confluence reached 100%, total protein was extracted and analyzed by Western blotting (using anti-Flag antibody, with GAPDH as an internal control).

[0034] like Figure 1 As shown in Figure A, a specific Flag band was detected at approximately 15 kDa in the overexpression plasmid transfection group, while no such band was observed in the mutant plasmid transfection group (start codon deleted). This indicates that the ORF of peptide X must depend on the start codon ATG to encode the peptide, confirming that the ORF has true coding ability. The expression of the GAPDH internal control was consistent in all groups. Figure 1 (B) ②Adenovirus transfection of hBMSCs A recombinant adenovirus (AdX) expressing the peptide X-Flag fusion protein was constructed. hBMSCs were cultured in a dedicated medium. When cell confluence reached 60%-70%, 2 ng of peptide X-overexpressing adenovirus (carrying the Flag tag) was added to 300 μL of Opti-mem medium and mixed thoroughly (Solution C). 1.7 mL of hBMSC-specific medium containing serum and without antibiotics was added to each well of a six-well plate, followed by Solution C. The plate was gently shaken to ensure thorough mixing. When cell confluence reached 100%, RNA and protein were extracted, and either an empty virus control (NC) or peptide X-overexpressing adenovirus (AdX, MOI=50) was added, respectively. Forty-eight hours after transfection, cell lysates were collected for Western blotting and immunofluorescence staining.

[0035] Western blotting results: as follows Figure 1 As shown in Figure C, a specific flag band was detected at approximately 15 kDa in the AdX transfection group, while this band was not observed in the NC group. The expression of the GAPDH internal control was consistent across all groups. Figure 1 The presence of C indicates that peptide X can be effectively expressed in hBMSCs.

[0036] ③ Immunofluorescence staining 48 h after transfection, the culture medium was discarded, and the cells were washed twice with PBS. Paraformaldehyde was added and the cells were incubated at room temperature for 40 min for fixation. After discarding the paraformaldehyde, the cells were washed twice with PBS. 100 μl of Triton × 100 was added to each well and the cells were incubated at room temperature for 10 min. After discarding the solution, the cells were washed twice with PBS. 200 μl of goat serum was added to each confocal dish for blocking for 2 h. After discarding the goat serum, diluted primary antibody was added directly, and the cells were incubated overnight at 4°C.

[0037] After recovering the antibody, wash twice with PBS, add diluted fluorescent secondary antibody, and incubate at 37°C in the dark for 30 min.

[0038] After discarding the solution, wash twice with PBS. Add 100 μl of mounting medium (containing DAPI) to each confocal dish and store at 4°C protected from light or take photos.

[0039] Immunofluorescence staining results: such as Figure 1 As shown in Figure D, the AdX transfection group exhibited a clear Flag green fluorescent signal, co-localizing with the phalloidin-labeled cytoskeleton. DAPI staining of the nuclei showed normal nuclear morphology in all groups. These results further confirm that peptide X can be successfully expressed in hBMSCs.

[0040] The above results indicate that peptide X has real coding ability and can be effectively expressed in HEK 293T cells and hBMSCs.

[0041] Example 2: Detection of peptide X and osteogenic differentiation markers in human bone marrow mesenchymal stem cell lines after induction. This embodiment is used to detect the expression changes of peptide X during the osteogenic induction differentiation of hBMSCs and its correlation with osteogenic differentiation markers.

[0042] (1) Culture and osteogenic induction of human mesenchymal stem cells Human bone marrow mesenchymal stem cells (hBMSCs, purchased from OriCell) were resuscitated and seeded into 10cm cell culture dishes using OriCell's mesenchymal stem cell-specific medium, and cultured in a 37°C cell culture incubator with 5% CO2. When the cell confluence reached 90%-100%, the old medium was discarded, and 3mL of 0.25% trypsin was added. After digestion in a 37°C cell culture incubator for 2 min, the cells were observed to be rounded under a microscope. After stopping digestion, 5mL of complete medium was added, and the cell suspension was aspirated into a 15mL centrifuge tube and centrifuged (1000 rpm, 5 min). The supernatant was discarded, and the cells were resuspended in complete medium. The cell suspension was then added to six-well plates for culture. A special induction medium containing 100nM dexamethasone, 10mM β-glycerophosphate sodium, and 50μg / mL ascorbic acid was prepared. After cell attachment, the experimental group was cultured in the special induction medium. Total RNA and protein were extracted from the cells after three days of induction.

[0043] (2) qRT-PCR detection of osteogenic-related gene mRNA expression Total RNA was reverse transcribed into cDNA using the PrimeScript™ RT Master Mix kit. The composition of the reaction system is shown in Table 2.

[0044] Table 2: Reverse Transcription Reaction System

[0045] Reverse transcription program: 37℃ for 15 min, 85℃ for 5 s, store at 4℃.

[0046] Use SYBR® Premix Ex Taq TMII. qPCR amplification was performed using the kit. The PCR reaction system, reaction conditions, and primers are shown in Tables 3-5. Table 3: Reverse Transcription Reaction System

[0047] The GAPDH gene was used as an internal reference gene, and the data were obtained using 2... -△△Ct Gene expression levels were analyzed.

[0048] Table 4: PCR reaction conditions

[0049] Table 5: Primer Sequences

[0050] (3) Western blotting to detect protein expression Cells were collected after 3 days of induction, and total protein was extracted. Protein concentration was determined using the BCA method. Equal amounts of protein were subjected to SDS-PAGE electrophoresis, transferred to a membrane, blocked, and incubated overnight at 4°C with anti-RUNX2 antibody, anti-COL1A1 antibody, anti-peptide X antibody (or anti-Flag antibody), and anti-GAPDH antibody (internal control). The next day, the corresponding secondary antibodies were added and incubated at room temperature for 2 hours. ECL chemiluminescence imaging was performed, and the band gray values ​​were analyzed using ImageJ software. Changes in the expression levels of peptide X and osteogenic differentiation markers RUNX2 and COL1A1 were detected after 3 days of osteogenic induction.

[0051] Experimental results are as follows Figure 2 As shown, 3 days after osteogenic induction, the mRNA expression levels of RUNX2 and COL1A1 in hBMSCs were significantly higher than those in the uninduced control group (CON). The protein expression levels of RUNX2 (57 kDa) and COL1A1 (220 kDa) were significantly upregulated compared to the uninduced control group; simultaneously, the protein expression level of peptide X (15 kDa) was also significantly increased, consistent with the expression trends of osteogenic differentiation markers. The expression of the internal control GAPDH (37 kDa) was consistent across all groups.

[0052] The above results indicate that the expression level of peptide X was significantly upregulated during the osteogenic induction and differentiation of hBMSCs, consistent with the expression trends of osteogenic differentiation markers RUNX2 and COL1A1, suggesting that peptide X may be involved in regulating the osteogenic differentiation process of mesenchymal stem cells.

[0053] Example 3: Functional validation of peptide X in mesenchymal stem cells This embodiment uses overexpression of peptide X, combined with transcriptome sequencing, Western blotting, qRT-PCR, and ALP staining experiments, to clarify the promoting effect of peptide X on osteogenic differentiation of hBMSCs.

[0054] (1) Cell culture and adenovirus transfection Human bone marrow mesenchymal stem cells (hBMSCs, purchased from OriCell) were resuscitated and seeded in 10 cm culture dishes using OriCell mesenchymal stem cell-specific medium, and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 60%-70%, either an empty virus control (NC) or a peptide-X overexpressing adenovirus (Ad-X, MOI=50) was added. Forty-eight hours after transfection, cells from each group were collected for the following assays.

[0055] (2) Transcriptome sequencing and KEGG pathway analysis After transfecting hBMSCs with peptide X adenovirus for 48 h, hBMSCs from the NC and Ad-X groups were collected, and total RNA was extracted for mRNA sequencing. Differentially expressed genes were screened using |log2FC| ≥ 1 and adjusted p-value < 0.05 as the screening criteria. KEGG pathway enrichment analysis was performed on the differentially expressed genes to explore the biological processes and signaling pathways that peptide X may be involved in.

[0056] like Figure 3 As shown, after overexpression of polypeptide X, the expression of 1819 genes was significantly upregulated, and the expression of 349 genes was significantly downregulated. Figure 3 (A). KEGG pathway enrichment analysis ( Figure 3 The top 30 differentially expressed gene enrichments in the study (B) included the PI3K-Akt signaling pathway, MAPK signaling pathway, JAK-STAT signaling pathway, and TNF signaling pathway, which are closely related to osteogenic differentiation regulation. This suggests that peptide X may promote osteogenic differentiation by regulating the above signaling pathways.

[0057] (3) qRT-PCR detection of osteogenic-related gene mRNA expression Forty-eight hours after transfection, hBMSCs from the NC and Ad-X groups were collected, total RNA was extracted, and qRT-PCR was performed according to the method described in Example 3. GAPDH was used as an internal control to detect the mRNA expression levels of RUNX2 and COL1A1.

[0058] Figure 3 As shown in Figure D, compared with the NC group, the mRNA expression levels of RUNX2 and COL1A1 in the Ad-X group (overexpressing peptide X) were significantly increased (p<0.05), indicating that peptide X can upregulate the transcriptional level of osteogenic differentiation marker genes.

[0059] (4) Western blotting detection of osteogenic-related protein expression Forty-eight hours after transfection, hBMSCs from the NC and Ad-X groups were collected, total protein was extracted, and Western blotting was performed according to the method described in Example 3, using anti-peptide X antibody (approximately 15 kDa), anti-RUNX2 antibody (approximately 57 kDa), anti-COL1A1 antibody (approximately 139 kDa), and anti-GAPDH antibody (approximately 37 kDa, internal control).

[0060] like Figure 3 As shown in Figure C, the protein expression level of peptide X (15 kDa) in the Ad-X group was significantly higher than that in the NC group, confirming the effectiveness of the adenovirus overexpression system. Simultaneously, the protein expression levels of osteogenic differentiation markers RUNX2 (57 kDa) and COL1A1 (139 kDa) in the Ad-X group were significantly upregulated compared to the NC group. The expression of the GAPDH internal control was consistent across all groups.

[0061] (5) ALP staining Forty-eight hours after transfection, ALP staining was performed to detect early osteogenic differentiation activity of the cells. The specific steps were as follows: remove the cell culture medium, wash twice with PBS, fix with 4% paraformaldehyde at room temperature for 3 hours, and wash twice more with PBS. Prepare the staining working solution (3 mL of alkaline phosphatase chromogenic buffer with 10 μL of BCIP solution and 20 μL of NBT solution), add 1 mL of the staining working solution to each well, incubate at room temperature in the dark for about 30 minutes, remove the staining working solution, wash 1-2 times with PBS, and then take pictures.

[0062] The results are as follows Figure 3 As shown in Figure E, compared with the NC group, the Ad-X group (overexpressing peptide X) showed significantly stronger ALP staining, indicating that peptide X can enhance the alkaline phosphatase activity of hBMSCs and promote early osteogenic differentiation.

[0063] In summary, overexpression of peptide X significantly upregulated the mRNA and protein expression levels of osteogenic differentiation markers RUNX2 and COL1A1, enhanced ALP staining intensity, and transcriptome sequencing revealed that peptide X could affect osteogenic differentiation-related signaling pathways such as PI3K-Akt, MAPK, and JAK-STAT. These results fully demonstrate that peptide X can promote osteogenic differentiation of hBMSCs.

[0064] Example 4: Protective effect of polypeptide X on osteoporotic mice In this embodiment, a mouse osteoporosis model was constructed by hindlimb unloding (HLU). Adeno-associated virus was used to overexpress peptide X in the deloaded hindlimb bone of mice. The protective effect of peptide X on the osteoporosis model was evaluated by combining methods such as micro-CT, calcein double labeling experiment and biomechanical detection.

[0065] (1) Animal grouping and model construction Thirty-two male 6-month-old C57BL / 6J mice were randomly divided into four groups: CON group: wild type, normally raised, without any treatment; HLU group: Hind limb deload model, without virus injection; HLU+rAAV9-NC group: hindlimb deloading model + knee joint injection of rAAV9-NC control; HLU+ rAAV9-X group: hindlimb deload model + knee joint injection of peptide X to overexpress AAV.

[0066] (2) Intra-articular injection of mouse knee joint After anesthetizing the mice using a gas anesthesia machine, hold them in a supine position. Bend the knee of one hind leg with your left hand to 90°, fully exposing the patellar plane. Tighten the syringe needle and draw up 10 μL of adeno-associated virus (AAV) solution, expelling all air from the syringe tip. Insert the needle at a 90° angle directly above the patellar ligament. After feeling a slight loss of air, advance the needle about 1-2 mm, then tilt the needle to 120° and inject the drug upwards and backwards. During injection, another experimenter pushes the needle slowly and steadily, using their index and middle fingers to stabilize the empty syringe. After injection, wait 1-2 seconds, then slowly withdraw the syringe needle. Gently move the mouse's hind limbs several times before returning it to its cage.

[0067] (3) Mouse hindlimb unloading osteoporosis model A hind limb unloading model was established using the tail suspension method. A supersaturated benzoin tincture solution prepared with anhydrous ethanol was applied to the mouse tail, followed by rosin resin. Medical tape was used to attach the tail to both sides, exposing the base of the tail for blood circulation observation. A paperclip was threaded through the tape at the base of the tail and then suspended from the crossbar of the cage, adjusting the height so that the mouse's body formed a 30° angle with the ground, with the hind limbs off the ground. Food and water were changed regularly, and benzoin tincture was applied to the mouse's perineum daily. After 21 days of suspension, the femurs of the hind limbs were collected for MicroCT scanning analysis.

[0068] (4) Micro CT scan analysis After collection, mouse hind limb femurs were fixed in 4% paraformaldehyde for 48 hours after muscle and connective tissue removal. They were then scanned using a three-dimensional dynamic X-ray microscopy platform. The samples were rotated 360° in 0.5° increments. The scanning area was 2.5 × 2.5 × 3 mm, approximately 1.5 mm from the proximal epiphyseal plate. 3 Cube. The bone microstructure parameters selected for analysis after scanning were: bone mineral density (BMD), bone volume fraction (BV / TV), bone surface area to bone volume ratio (BS / TV), and trabecular separation (Tb.sp).

[0069] like Figure 4 As shown, compared with the CON group, the HLU group showed significant bone loss, manifested as bone microstructure destruction, sparse trabeculae, and poor connectivity; while after peptide X overexpression treatment, compared with the HLU+rAAV9-NC group, the femoral bone mass of mice in the HLU+rAAV9-X group (peptide X overexpression) was significantly increased. Figure 4 (A). Quantitative analysis ( Figure 4 The results showed that BMD, BV / TV, and BS / TV were significantly increased and Tb.Sp was significantly decreased in the peptide X overexpression group (p<0.05), indicating that peptide X can effectively improve bone microstructure in osteoporosis model mice.

[0070] (5) Double labeling experiment of calcium chlorophyll After preparing a fresh calcein solution under light-protected conditions, the dosage was calculated at 8 μl / g, and intraperitoneal injections were administered 3 days and 10 days before the end of the HLU experiment. After sampling, the samples were fixed, embedded, sectioned, and photographed using a confocal microscope. Measurements and calculations were performed using ImageJ software.

[0071] Compared with the CON group, the double label spacing in the HLU group was significantly reduced, confirming that HLU reduces the rate of new bone formation in mice; compared with the HLU+rAAV9-NC group, the double label spacing in the HLU+rAAV9-X group was significantly widened, indicating that overexpression of peptide X can effectively restore the rate of new bone formation. Figure 4 (C)

[0072] (6) Three-point bending test The mouse femur was placed on the lower support, the span was adjusted to 10 mm, and the indentation speed was set to 0.01 mm / s. The machine was started, and the indentation head was pressed vertically downwards at the set speed until the femur fractured. By measuring the thickness of the mouse femoral cortex and the outer diameter of the long and short axes at the fracture site, and combining the results acquired by the instrument, various biomechanical parameters were analyzed.

[0073] By plotting the load-deflection curve ( Figure 4In the study (D), it was found that the biomechanical properties of the HLU group were significantly lower than those of the CON group. Compared with the HLU+NC group, the biomechanical properties were significantly improved after overexpression of peptide X. Further analysis of the experimental parameters revealed that, compared with the CON group, the HLU group had significantly lower stiffness, moment of inertia, and maximum load. Figure 4 The levels of E and P were significantly reduced (P<0.001); after overexpression of peptide X, compared with the HLU+NC group, the above three biomechanical performance indicators were significantly restored to different degrees (P<0.001).

[0074] In summary, the experimental results, using Micro-CT, calcein double labeling, and biomechanical experiments, demonstrate that in vivo overexpression of peptide X can significantly improve bone mass and bone microstructure in hindlimb deloaded osteoporosis model mice, promote new bone formation, and improve bone biomechanical properties, thus confirming that peptide X has a clear protective effect against osteoporosis.

[0075] Statistical analysis was performed using SPSS Statistical Analysis 22.0 and GraphPad Prism 10.0. Experimental data are presented as mean ± standard deviation. Student's t-test was used for comparisons between two samples; one-way ANOVA was used for statistical analysis of three or more groups. A p-value < 0.05 was considered statistically significant after three replicates.

[0076] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A polypeptide that promotes osteogenic differentiation of mesenchymal stem cells, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. The polypeptide for facilitating osteogenic differentiation of mesenchymal stem cells according to claim 1, characterized in that, The polypeptide is derived from mammals and is a human-derived natural polypeptide.

3. The polypeptide for promoting osteogenic differentiation of mesenchymal stem cells according to claim 1 or 2, characterized in that, The polypeptide includes functional variants that have more than 95% homology with its amino acid sequence and retain the same biological activity.

4. The derivative of the polypeptide promoting osteogenic differentiation of mesenchymal stem cells according to claim 1, characterized in that, The derivative is a derivative of the polypeptide that retains its biological activity of promoting osteogenic differentiation of mesenchymal stem cells after being modified by hydroxylation, carboxylation, methylation, phosphorylation, acetylation, glycosylation or esterification of the amino acid side chain groups, amino terminus or carboxyl terminus.

5. The polypeptide for promoting osteogenic differentiation of mesenchymal stem cells according to claim 1 or 2, characterized in that, The polypeptide is prepared by solid-phase polypeptide synthesis or recombinant expression.

6. The use of the polypeptide for promoting osteogenic differentiation of mesenchymal stem cells as described in claim 1 or 2, or the functional variant of claim 3 or the derivative of claim 4, in the preparation of a drug or formulation for promoting osteogenic differentiation.

7. The use of the polypeptide of claim 1 or 2 that promotes osteogenic differentiation of mesenchymal stem cells, or the functional variant of claim 3, or the derivative of claim 4, in the preparation of a medicament or formulation for the prevention or treatment of osteoporosis.

8. A pharmaceutical composition for the prevention or treatment of osteoporosis, characterized in that, It comprises the polypeptide of claim 1, the functional variant of claim 3, or the derivative of claim 4, and a pharmaceutically acceptable adjuvant or carrier.

9. The pharmaceutical composition according to claim 7, characterized in that, The dosage form of the pharmaceutical composition is selected from at least one of the following: powder, inhaler, patch, tablet, injection, granule, pill, syrup, oral liquid, capsule, ointment, or suppository.

10. The pharmaceutical composition according to claim 7 or 8, characterized in that, The pharmaceutical composition further comprises at least one adjunctive therapeutic agent selected from the group consisting of: bisphosphonates, selective estrogen receptor modulators, parathyroid hormone analogs, RANKL inhibitors, calcium preparations, or vitamin D preparations.

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