Use of cby1 inhibitors in the preparation of anti-osteoporosis drugs for promoting bone formation

Abstract

The application discloses application of a Chibby1 (CBY1) inhibitor in preparation of an anti-osteoporosis drug for promoting bone formation, and belongs to the technical field of biological medicines. It is found for the first time that CBY1 can inhibit differentiation of bone marrow stromal cells into osteoblasts, and that bone marrow stromal cells with CBY1 gene silencing have increased osteogenic differentiation. It is proposed for the first time that reducing CBY1 expression or inhibiting the function of CBY1 can promote differentiation of osteoblasts, and that a CBY1 inhibitor has the potential of increasing osteogenic activity and preventing and treating osteoporosis by inhibiting CBY1 expression or inhibiting the function of CBY1.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of CBY1 inhibitors in the preparation of anti-osteoporosis drugs that promote bone formation. Background Technology

[0002] Osteoporosis is a systemic bone disease characterized by low bone mass and destruction of bone tissue ultrastructure. As a result, bone fragility increases significantly, and even minor external forces can induce fractures, posing a serious threat to the health of patients.

[0003] With the accelerating aging of the world's population, the prevalence of osteoporosis and the incidence of osteoporotic fractures are rising rapidly. Related data shows that fractures of the hip, vertebrae, and other parts of the body caused by osteoporosis in middle-aged and elderly people not only lead to pain and limited mobility for patients, but also significantly increase the burden of family care and social medical costs, becoming a significant public health issue that urgently needs attention and response.

[0004] Currently, drugs used clinically to treat osteoporosis primarily fall into two categories based on their mechanisms of action: promoting bone formation and inhibiting bone resorption. Among these, the selection of drugs that effectively promote bone formation is relatively limited, making it difficult to meet the treatment needs of diverse patients. On the other hand, the more widely used drugs that inhibit bone resorption may cause various adverse reactions with long-term use, and some even pose a potential risk of increasing cancer risk, thus restricting their clinical application value. Against this backdrop, the limitations of existing treatments are becoming increasingly apparent, and developing new osteoporosis treatments with higher safety and more precise mechanisms has become a crucial direction urgently needing breakthroughs in both clinical and research fields.

[0005] Chibby 1 (CBY1) is an evolutionarily conserved small protein that directly interacts with the C-terminal region of β-catenin, preventing β-catenin from binding to Tcf / Lef transcription factors and thus inhibiting β-catenin-mediated transcriptional activation, thereby antagonizing β-catenin. Furthermore, CBY1 can interact with 14-3-3 scaffolding proteins to form a stable ternary complex, promoting β-catenin nuclear export and further regulating β-catenin's intracellular localization and function. Abnormal CBY1 expression is associated with various diseases. For example, in chronic myeloid leukemia, downregulation of CBY1 is associated with BCR-ABL1, and its reduced expression affects β-catenin activity, thus influencing disease development and progression. In addition, CBY1 gene deletion leads to ciliasis characterized by Joubert syndrome. However, research on CBY1's regulation of osteoblast differentiation and its impact on bone mass has not been reported in the literature. Currently, there are no research reports documenting CBY1's regulation of osteoblast differentiation in existing technologies. Summary of the Invention

[0006] In response to the shortcomings of existing technologies and practical needs, this invention proposes the application of CBY1 inhibitors in the preparation of anti-osteoporosis drugs that promote bone formation. These drugs can increase osteoblast differentiation and activity by targeting CBY1, thereby promoting bone formation and helping to prevent and treat osteoporosis.

[0007] This invention is the first to discover that CBY1 can inhibit osteoblast differentiation, and that osteoblast differentiation is increased in bone marrow stromal cells with CBY1 gene silencing. This invention is also the first to propose that reducing CBY1 expression or inhibiting its function can promote osteoblast differentiation. CBY1 inhibitors increase osteogenic activity by inhibiting CBY1 expression or function, and have the potential to prevent and treat osteoporosis.

[0008] The CBY1 encoding gene is located on human chromosome 22 and mouse chromosome 15. The mature polypeptide in humans and mice contains 126 (human) and 127 (mouse) amino acids, respectively, with amino acid sequences as shown in SEQ ID NO. 1 (human) or SEQ ID NO. 2 (mouse).

[0009] SEQ ID NO. 1:

[0010] MPFFGNTFSPKKTPPRKSASLSNLHSLDRSTREVELGLEYGSPTMNLAGQSLKFENGQWIAETGVSGGVDRREVQRLRRRNQQLEEENNLLRLKVDILLDMLSESTAESHLMEKELDELRISRKRK

[0011] SEQ ID NO. 2:

[0012] MPLFGSIFSPKKTPPRKSASLSNLHSLDRSTRELELGLDYGTPTMNLAGQSLKFENGQWVADSVISGGVDRRETQRLRKRNQQLEEENNLLRLKVDILLDMLSETTAESHLKDKELDELKVTNRRRK

[0013] According to the present invention, CBY1 inhibitors can activate the Wnt / β-catenin signaling pathway and promote osteoblast differentiation.

[0014] This invention provides a potential drug for the prevention and treatment of osteoporosis. The drug targets the CBY1 encoding gene or its protein, regulates the expression of genes or proteins related to osteoporosis, and achieves the purpose of preventing and treating osteoporosis.

[0015] 1. Application of CBY1 inhibitors in the preparation of osteoporosis-preventing drugs that promote bone formation. The key feature is that it promotes osteogenic differentiation of mesenchymal stem / progenitor cells by targeting and inhibiting CBY1 expression or function.

[0016] 2. Furthermore, the drug is a CBY1 inhibitor, used to increase osteogenic differentiation capacity, thereby preventing and treating osteoporosis.

[0017] 3. Further, the drug includes at least one of an inhibitory drug targeting the CBY1 encoding gene or an inhibitory drug targeting the CBY1 encoded protein.

[0018] 4. Furthermore, the inhibitory drug for the CBY1 encoding gene includes siRNA, shRNA virus, or cells that downregulate CBY1 expression and target the CBY1 encoding gene.

[0019] 5. Furthermore, the inhibitory drug targeting CBY1 protein includes any one of small molecule compounds, nucleic acids, amino acids, and peptides.

[0020] 6. Furthermore, the dosage form of the drug includes oral liquid, injection, tablet, pill, dispersant, capsule, drops, granules, suspension or emulsion.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention is the first to discover that CBY1 can inhibit osteoblast differentiation, and that osteoblast differentiation is increased in bone marrow stromal cells with CBY1 gene silencing. This invention is also the first to propose that reducing CBY1 expression or inhibiting its function can promote osteoblast differentiation. CBY1 inhibitors increase osteogenic activity by inhibiting CBY1 expression or function, and have the potential to prevent and treat osteoporosis. Attached Figure Description

[0023] Figure 1 The results show that CBY1 overexpression inhibits osteogenic differentiation of the mouse stromal cell line ST2. A shows the CBY1 protein expression level after ST2 cells were transfected with the CBY1 overexpression plasmid by Western blotting; B shows alkaline phosphatase (ALP) staining 14 days after osteogenic induction; C shows the mRNA expression of key osteogenic differentiation factors Runx2, Osterix, Alp, and Opn by qRT-PCR 3 days after osteogenic induction; D shows the ALP and OPN protein expression by Western blotting 3 days after osteogenic induction.

[0024] Figure 2The results show that CBY1 gene silencing promotes osteogenic differentiation of the mouse stromal cell line ST2. A shows the knockdown of CBY1 gene expression in ST2 cells after transfection with CBY1 siRNA by Western blotting; B shows ALP staining 14 days after osteogenic induction; C shows the expression of key osteogenic differentiation factors Osterix, Alp, and OpnmRNA by qRT-PCR 3 days after osteogenic induction.

[0025] Figure 3 The results of overexpressing Rhotekin in ST2 cells to antagonize CBY1 function and promote osteoblast differentiation were investigated. A) IP was performed on lysates of untransfected BMSCs using a CBY1 antibody, and the levels of β-catenin and Rhotekin in the immunoprecipitates were detected by Western blotting. B) ST2 cells were transfected with different concentrations of Rhotekin-His, and the protein levels of CBY1, Rhotekin, and β-catenin in the cells were detected by Western blotting. C) Cell lysates from experiment B were IPed with a CBY1 antibody, and the protein levels of CBY1, Rhotekin, and β-catenin in the immunoprecipitates were detected by Western blotting. D) Changes in the expression levels of Wnt / β-catenin signaling pathway proteins were detected after ST2 cells were transfected with a Rhotekin overexpression plasmid. E) ST2 cells were transfected with a TCF / LEF reporter gene (14XTCF-rOC-Luc) or rOC-Luc and a Rhotekin overexpression plasmid, and luciferase activity was measured. F shows ALP staining in ST2 cells co-transfected with Rhotekin and CBY1 overexpression plasmids 14 days after osteogenic induction. G shows the expression of non-p-β-catenin, ALP, and OPN proteins detected by Western blotting 3 days after osteogenic induction.

[0026] Figure 4This study presents the results of silencing Rhotekin expression in bone marrow stromal cells (BMSCs) to activate CBY1 function, thereby reducing osteoblast differentiation and cancellous bone mass in mice. A shows the protein expression levels of Rhotekin and the Wnt / β-catenin signaling pathway detected by Western blotting in BMSCs transfected with Rhotekin siRNA or control siRNA in the mouse tibial medullary cavity. B and C show ALP staining results 14 days after osteogenic induction and qRT-PCR detection of key osteogenic differentiation factors Osterix, Alp, and Opn mRNA 3 days after osteogenic induction in BMSCs transfected with Rhotekin siRNA or control siRNA. D and E show images of ALP immunohistochemical staining on bone sections and quantitative results of osteoblasts on bone trabeculae. FI shows μCT analysis and statistical results of histological parameters of bone mass in the proximal metaphysis of the mouse tibia after transfection with Rhotekin siRNA or control siRNA in the mouse tibial medullary cavity. Detailed Implementation

[0027] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides further illustrative information. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit its scope.

[0028] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0029] The materials and methods involved in the embodiments of the present invention are described below.

[0030] (1) Cell culture and induced differentiation

[0031] The mouse stromal cell line ST2 was cultured in α-MEM medium containing 10% fetal bovine serum (FBS).

[0032] Osteogenic induction of ST2 cells: When the cell confluence reached approximately 80%, the medium was replaced with α-MEM complete medium containing osteogenic inducers (10% FBS, 50 µg / ml ascorbic acid, 5 mmol / L sodium β-glycerophosphate). The medium was replaced every 3 days. mRNA and protein were collected 72 h after osteogenic induction, and ALP staining was performed 14 days after induction.

[0033] (2) Cell transfection

[0034] PCR primers were designed targeting the coding region (CDS) of CBY1, namely: Forward Primer: 5'-TTGGTACCGAGCTCGGATCCGCCACCATGCCTCTCTTTGGCAGCA-3' (SEQ ID NO. 3); Reverse Primer: 5'-GCTGGATATCTGCAGAATTCTCAGAGCAGAAACTCATCTCTGAAGAGGATCTGCTTCCTCCTGCGGTTGGTAA-3' (SEQ ID NO. 4).

[0035] PCR primers were designed targeting the coding region (CDS) sequence of Rhotekin, namely Forward Primer: 5'- TTGGTACCGAGCTCGGATCCGCCACCATGTTCTCTCGAAACCATC-3' (SEQ ID NO. 5); Reverse Primer: 5'- GCTGGATATCTGCAGAATTCTCAATGGTGATGGTGATGATGCACTGGGGACTGGAGCCATG-3' (SEQ ID NO. 6).

[0036] Using mouse cDNA as a template, PCR amplification was performed. The obtained CDS amplification fragment was ligated from the BamHI / EcoRI site into the pcDNA3.1(+) vector using the pEASY®-Basic Seamless Cloning Kit (TransGenBiotech, Beijing, China) to construct Cby1 or Rhotekin overexpression plasmids.

[0037] Plasmid transfection: When the cell density reaches 70%, transfection is performed using PEI transfection reagent (MCE, Shanghai, China). Taking a 24-well plate as an example, 0.5 μg of plasmid DNA is added to a 1.5 mL EP tube, 0.625 μL of PEI Transfection Reagent is added, mixed, and allowed to stand for 3 minutes. Then, 100 μL of serum-free medium is added, gently mixed, and allowed to stand for 30 minutes. Finally, 150 μL of serum-free medium is added to form the transfection reagent-nucleic acid complex. After completely discarding the medium in the well plate, 250 μL of the mixed reagent is added to each well of the cells, gently mixed, and placed in a CO2 cell culture incubator. The medium is changed 6 hours after transfection.

[0038] (3) Quantitative real-time polymerase chain reaction (qRT-PCR)

[0039] Total RNA was extracted from cells using a total RNA extraction kit (Omega, America). Reverse transcription was performed using a first-strand cDNA synthesis kit (PerfectStart Uni RT&qPCR Kit, TransGen, Beijing, China). Real-time quantitative PCR was performed using the SYBR Green fluorescent PCR method, with β-actin as an internal control. －∆ ∆ Ct The relative expression levels of each target gene were calculated as follows: ∆Ct = Ct value of target gene - Ct value of internal reference gene; ∆∆Ct = ∆Ct of experimental group - ∆Ct of control group. All experiments were repeated 3 times.

[0040] (4) Western blotting

[0041] Total protein was extracted from cells using RIPA lysis buffer and separated by SDS-PAGE gel electrophoresis. The protein was then transferred to a nitrocellulose membrane, blocked with 5% skim milk powder at room temperature for 1 h, and incubated overnight at 4°C with primary antibody. After incubation with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature, the expression of the target protein was detected using an ECL chemiluminescence kit (ABclonal, Wuhan, China).

[0042] (5) Synthesis and transfection of siRNA

[0043] Cby1 siRNA was synthesized by Anhui General Biotechnology [sense strand: 5'-AAUAUGCUGCCAAAGAGAGGC-3' (SEQ ID NO. 7), antisense strand: 5'-CUCUCUUUGGCAGCAUAUUCA-3' (SEQ ID NO. 8)]. Rhotekin siRNA-1 [(sense strand: 5'-UGUGGAAGGAUACAGAGUAUUUCAA-3' (SEQ ID NO. 9), antisense strand: 5'-UUGAAAUACUCUGUAUCCUUCCACAUG-3' (SEQ ID NO. 10)] was dissolved in sterile DEPC water to a final concentration of 20 nM before use. After aliquoting, it was stored at -80°C for later use. 24 hours before transfection, cells were digested with 0.25% trypsin and seeded into 24-well plates. When the cell confluence reached 30-60%, siRNA transfection was prepared. Cby1 siRNA was transfected into ST2 cells and Rhotekin siRNA was transfected into BMSCs using an RNA transfection kit (Getico Biotech, Shanghai, China). The culture medium was replaced with complete medium 24 hours after transfection. The expression efficiency of the corresponding genes could be analyzed approximately 48 hours after transfection, or further treatment could be performed.

[0044] (6) Co-immunoprecipitation (Co-IP)

[0045] Cells were lysed using RIPA buffer containing protease inhibitors. After lysis, 500 μg of each protein sample was incubated with 4 μg of anti-CBY1 antibody (FineTest, Wuhan, China) or IgG at 4 °C. Protein A / G magnetic beads were added to capture the immune complexes, and the mixture was incubated at 4 °C for 2 h. The beads were washed with PBS supplemented with 0.5% Tween-20 to remove any non-specifically bound proteins. The immunoprecipitated complexes were then eluted, and the presence of CBY1, Rhotekin, and β-catenin was detected by Western blot analysis.

[0046] (7) Alkaline phosphatase staining

[0047] After osteogenic induction and differentiation for 14 days, alkaline phosphatase (ALP) staining was performed. Cells were washed with 1×PBS buffer, fixed with 4% paraformaldehyde for 15 min, and incubated with BCIP / NBT staining working solution (Beyotime, Shanghai, China) at room temperature in the dark for 15 min.

[0048] (8) In vivo transfection of siRNA

[0049] 2'-ome modified Rtkn siRNA-1, Rtkn siRNA-2, and control siRNA were synthesized by Genepharma (Shanghai, China). Rhotekin siRNA-1 [(sense strand: 5'-UGUGGAAGGAUACAGAGUAUUUCAA-3' (SEQ ID NO. 9), antisense strand: 5'-UGAAAUACUCUGUAUCCUUCCACAUG-3' (SEQ ID NO. 10)]; Rhotekin siRNA-2 [(sense strand: 5'-UGCAAGAUGGAUUCCGUATT-3' (SEQ ID NO. 11), antisense strand: 5'-UACGGAAUCCAUCUUGCACTT-3' (SEQ ID NO. 12)]. Ten-week-old female wild-type C57BL / Six-year-old mice were housed in a specific pathogen-free (SPF) facility within the same room, with a 12-hour light:12-hour dark cycle and controlled room temperature (22±2°C) and humidity (55±5%). Mice were divided into groups of five and housed in standard individually ventilated cage systems (Tecniplast, Italy). After one week of acclimatization, mice were randomly assigned to two groups: a control group and an Rtkn siRNA group. 11 μg of control siRNA or Rtkn siRNA (a combination of Rtkn siRNA-1 and Rtkn siRNA-2, 5.5 μg each) and 1.5 μL of in vivo JetPEI (Polyplus, Illkirch, France), diluted with 5% glucose, were combined at an N / P ratio of 6. After anesthetizing the mice, the prepared mixture was injected into the tibial medullary cavity. Three days after intratibial transfection, BMSCs were extracted from a portion of the mouse tibia for Rtkn detection. The knockdown efficiency of siRNA in vivo was assessed. The remaining mice underwent three additional transfections at weeks 2, 6, and 10 following the initial transfection. Mice were sacrificed 12 weeks after the initial transfection, and tibial samples were collected for histological and immunohistochemical analysis.

[0050] (9) μCT analysis

[0051] Mouse tibial specimens were scanned using a Scanco viva CT80 (Scanco Medical AG, Switzerland) with scanning parameters of 55 kVp and 145 μA. Three-dimensional reconstruction was performed using Scanco software. The region of interest was selected as a 1 mm region starting 0.1 mm below the growth plate.

[0052] (10) Immunohistochemistry

[0053] Immunohistochemical (IHC) staining was performed by dewaxing sections in xylene, rehydrating them in gradually decreasing concentrations of ethanol, and then digesting them with 0.05% trypsin at 37°C for 20 minutes for antigen retrieval. Endogenous peroxidase was quenched with 3% hydrogen peroxide for 15 minutes at room temperature. After blocking nonspecific binding with 1% BSA, sections were incubated with anti-ALP (Abcam, Cambridge, MA, USA) antibodies at 37°C for 2 hours. After washing, sections were incubated with HRP-conjugated secondary antibody at 37°C for 1 hour. Immunoreactivity was observed using the chromogenic substrate DAB for 30 minutes, and cell nuclei were counterstained with hematoxylin for 1–3 seconds. The number of ALP-positive cells on the trabeculae was quantified. Analysis was performed on a region extending 1 mm from 0.1 mm below the growth plate.

[0054] (11) Statistical analysis

[0055] Statistical analysis was performed using GraphPad Prism 8.0 software. All data are expressed as mean ± standard deviation. Two-tailed unpaired independent Student's t-tests were used to compare differences between two groups, while one-way ANOVA or two-way ANOVA was used to compare multiple groups. Post-hoc comparisons were performed using the Dunnett test or the Least Significant Difference test, respectively. A p-level significance level of <0.05 was considered statistically significant.

[0056] Example 1

[0057] This study analyzed the effect of CBY1 on osteogenic differentiation of the stromal cell line ST2. ST2 cells were transfected with a CBY1 overexpression plasmid (CBY1 Construct), and changes in osteogenic differentiation after CBY1 overexpression were analyzed. Western blotting showed that the CBY1 Construct group significantly increased CBY1 protein (…). Figure 1 A). After osteogenic induction, ALP staining was found to be lighter in the CBY1Construct group ( Figure 1 B), mRNAs of key osteogenic differentiation factors Runx2, Osterix, ALP, and OPN ( Figure 1 C) and ALP and OPN proteins ( Figure 1 D) The expression levels were all reduced, indicating that overexpression of CBY1 inhibited osteogenic differentiation of ST2 cells.

[0058] Example 2

[0059] This study analyzed the effect of CBY1 gene silencing on osteogenic differentiation of the stromal cell line ST2. CBY1 siRNA (CBY1 siRNA) and its control siRNA (Ctrl siRNA) were transfected into ST2 cells. Western blotting revealed a significant decrease in CBY1 mRNA levels in ST2 cells transfected with CBY1 siRNA. Figure 2 A). After osteogenic induction, compared with the control, ST2 cells transfected with CBY1 siRNA showed darker ALP staining ( Figure 2 B), mRNAs of key osteogenic differentiation factors Osterix, Alp, and Opn ( Figure 2 C) Significantly increased expression indicates that knockdown of CBY1 promotes osteogenic differentiation of ST2 cells.

[0060] Example 3

[0061] This embodiment analyzed the binding of Rhotekin to CBY1 and its antagonism of CBY1 function, thereby antagonizing the inhibitory effect of CBY1 on osteoblast differentiation. Lysates of untransfected BMSCs were subjected to immunoprecipitation (IP) using a CBY1 antibody. Western blotting was used to detect β-catenin and Rhotekin in the immunoprecipitates. The results showed that CBY1 can bind to both β-catenin and Rhotekin in BMSCs. Figure 3 A). ST2 cells were transfected with different concentrations of Rhotekin-His, and the protein levels of CBY1, Rhotekin, and β-catenin in the cells were then detected by Western blotting. The results showed that the protein level of Rhotekin increased with the increase of the concentration of Rhotekin-His recombinant plasmid, but the increase of Rhotekin protein did not significantly affect the protein levels of CBY1 and β-catenin. Figure 3 B). Using CBY1 antibody against Figure 3 In experiment B, cell lysates were subjected to immunoprecipitation (IP), and the protein levels of CBY1, Rhotekin, and β-catenin in the immunoprecipitates were detected by Western blotting. The results showed that as the amount of Rhotekin protein bound to CBY1 gradually increased, the level of β-catenin protein bound to CBY1 gradually decreased. This indicates that Rhotekin competes with β-catenin for binding to CBY1. When Rhotekin protein increases and its binding to CBY1 increases, the amount of β-catenin protein bound to CBY1 decreases, thereby reducing the inhibition of β-catenin by CBY1 and activating the Wnt / β-catenin signaling pathway. Figure 3C). Changes in the expression levels of Wnt / β-catenin signaling pathway proteins were detected after ST2 cells were transfected with a Rhotekin overexpression plasmid. The results showed that Rhotekin overexpression activated the Wnt / β-catenin signaling pathway. Figure 3 D). ST2 cells were co-transfected with the TCF / LEF reporter gene (14XTCF-rOC-Luc) or rOC-Luc and the Rhotekin overexpression plasmid, and luciferase activity was measured. The results showed that Rhotekin overexpression increased luciferase activity in the TCF / LEF reporter gene vector, and the Wnt / β-catenin signaling pathway was activated. Figure 3 E). ST2 cells co-transfected with Rhotekin and CBY1 overexpression plasmids were stained with ALP 14 days after osteogenic induction. Figure 3 F), after 3 days of osteogenic induction, the expression of non-p-β-catenin, ALP, and OPN proteins was detected by Western blotting. Figure 3 The results of G, F, and G indicate that Rhotekin overexpression can antagonize the inhibitory effect of CBY1 on osteoblast differentiation.

[0062] Example 4

[0063] This study analyzed the changes in bone mass in mice after knocking down Rhotekin in BMSCs. The constructed Rhotekin siRNA and control siRNA were transfected into the tibial bone marrow cavity of mice. Primary BMSCs were harvested, and Western blotting was used to detect changes in the expression of Rhotekin and Wnt / β-catenin signaling pathways. The results showed that compared with the control siRNA group, the Rhotekin siRNA group had reduced Rhotekin protein, and reduced activity of non-p-β-catenin and TCF7L2 protein. Figure 4 A). After osteogenic induction, ALP staining in BMSCs cells from the Rhotekin siRNA group became lighter ( Figure 4 B), the mRNA expression of key osteogenic differentiation factors Osterix, Alp, and Opn was significantly reduced, indicating that knockdown of Rhotekin inhibits osteogenic differentiation of BMSCs (BMSCs). Figure 4 C).

[0064] Three months after transfection, mouse tibias were analyzed. Immunohistochemistry (IHC) revealed a significant decrease in the number of ALP-positive osteoblasts in the Rhotekin siRNA group compared to the control group. Figure 4D, E). μCT analysis showed that the tibial trabecular bone volume fraction (Tb. BV / TV), trabecular bone thickness (Tb. Th), and trabecular bone mineral density (Tb. BMD) were all significantly decreased in the RhotekinsiRNA group mice. Figure 4 FI). The above results indicate that knocking down Rhotekin in BMSCs in mice inhibits osteoblast differentiation and reduces trabecular bone mass.

[0065] In summary, drugs prepared by targeting the CBY1 gene or its encoded protein and inhibiting its expression or function have the potential to promote bone formation and prevent osteoporosis.

[0066] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. The application of a CBY1 inhibitor in the preparation of an anti-osteoporosis drug that promotes bone formation, characterized in that, The CBY1 inhibitor is an siRNA that targets the CBY1 encoding gene, and its sequences are SEQ ID NO. 7 and SEQ ID NO.

8.

2. The application according to claim 1, characterized in that, The inhibitors promote osteogenic differentiation of mesenchymal stem / progenitor cells and enhance osteogenic differentiation capacity, thereby preventing and treating osteoporosis.

3. The application according to claims 1-2, characterized in that, The dosage form of the drug is oral liquid, injection, tablet, pill, dispersant, capsule, drops, granules, suspension or emulsion.

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