Application of BCKDK inhibitors in the preparation of drugs for the prevention and treatment of osteoporosis

Abstract

This invention discloses the application of branched-chain ketoate dehydrogenase kinase (BCKDK) inhibitors in the preparation of drugs for the prevention and treatment of osteoporosis. This invention discovers that BCKDK can inhibit osteoblast differentiation, and reducing endogenous BCKDK expression in osteoblast precursor cells can promote osteoblast differentiation. The small molecule inhibitor BT2 (3,6-dichloro-2-benzothiocarboxylic acid), a BCKDK inhibitor, promotes osteoblast differentiation in ovariectomized mice, inhibits osteoclast differentiation, reduces ovariectomy-induced bone loss in mice, and increases bone mass. This invention is the first to propose that BCKDK inhibitors, by reducing BCKDK expression or inhibiting the biological function of the BCKDK protein, can promote osteoblast differentiation, inhibit osteoclast differentiation, and increase bone mass, thus having an effect on the prevention and treatment of osteoporosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of BCKDK inhibitors in the preparation of drugs for the prevention and treatment of osteoporosis. Background Technology

[0002] Osteoporosis is a systemic bone disease characterized by decreased bone mass and destruction of bone microstructure, leading to pain, spinal deformities, and fragility fractures. It is classified into primary and secondary osteoporosis. With the increasing aging of the world's population, the incidence of osteoporotic fractures is rising rapidly, becoming a significant public health issue.

[0003] In current clinical treatment of osteoporosis, commonly used drugs are mainly divided into two categories based on their core mechanisms of action: those that promote bone formation and those that inhibit bone resorption. Among these, the selection of drugs that effectively promote bone formation is very limited, with a narrow applicable population and strict restrictions on the duration of use, making it difficult to fully meet the personalized treatment needs of patients with different conditions and physical states. While drugs that inhibit bone resorption, which are more widely used clinically, can slow bone loss in the short term, long-term use can easily lead to various adverse reactions. Furthermore, long-term use of some drugs may increase the risk of mandibular osteonecrosis and atypical femoral fractures, and a few drugs have potential carcinogenic risks. These problems greatly limit their application value in long-term clinical treatment. Therefore, developing new osteoporosis treatment methods with higher safety, more precise mechanisms of action, and a balance between efficacy and long-term safety has become a key direction urgently needed in current clinical medicine and pharmaceutical research, and is of great significance for improving patient prognosis and enhancing the level of disease treatment.

[0004] Branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine, are essential amino acids and are necessary nutrients for mammalian life activities, playing a vital role in maintaining health. Branched-chain ketoacid dehydrogenase kinase (BCKDK) modifies the activity of the BCKDK complex through phosphorylation, preventing the catabolism of BCAs. The small molecule inhibitor BT2 (3,6-dichloro-2-benzothiophene carboxylic acid), upon binding to BCKDK, triggers the helical motion of the N-terminal domain of the BCKDK complex, leading to the dissociation of BCKDK from the complex. This activates the catabolism pathway of BCAs, reducing their levels in the body. Current reports indicate that BCKDK has multiple functions, including participation in metabolic compensation during embryonic development and regulating metabolites that influence tumor progression. Currently, there are no documented studies on the prevention and treatment of osteoporosis using BCKDK inhibitors. Summary of the Invention

[0005] This invention addresses the metabolic characteristics of osteoporosis by providing a pharmacological method to enhance the prevention and treatment of osteoporosis. The method is characterized by reducing BCKDK expression or inhibiting the biological function of BCKDK protein to promote BCAA catabolism, thereby preventing and treating osteoporosis, slowing its progression, and improving bone health.

[0006] The BCKDK encoding gene is located on human chromosome 16 and mouse chromosome 7. The mature polypeptide in both humans and mice contains 382 amino acids.

[0007] In response to the shortcomings of existing technologies and practical needs, this invention proposes the application of branched-chain ketoate dehydrogenase kinase (BCKDK) inhibitors in the preparation of drugs for the prevention and treatment of osteoporosis.

[0008] Compared with the prior art, the present invention has the following beneficial effects:

[0009] This invention discloses for the first time a pharmacological method to enhance the prevention and treatment of osteoporosis. The drug prepared using this method can prevent and treat osteoporosis by targeting and inhibiting BCKDK expression or its protein biological function. This invention also reveals for the first time that BCKDK can inhibit osteoblast differentiation, and reducing endogenous BCKDK expression in osteoblast precursor cells can promote osteoblast differentiation. Furthermore, the small molecule inhibitor BT2 (3,6-dichloro-2-benzothiocarboxylic acid), which inhibits osteoclast differentiation in ovariectomized mice, reduces ovariectomy-induced bone loss in mice, and increases bone mass.

[0010] This invention is the first to propose that BCKDK inhibitors promote branched-chain amino acid catabolism by reducing BCKDK expression or inhibiting its protein biological function, thereby promoting osteoblast differentiation, inhibiting osteoclast differentiation, increasing bone mass, and thus having the effect of preventing and treating osteoporosis. Attached Figure Description

[0011] Figure 1 The results show the inhibition of osteogenic differentiation of mouse pre-osteoblast MC3T3-E1 cells by BCKDK. A shows the expression level of Bckdk mRNA in MC3T3-E1 cells after transfection with the Bckdk overexpression plasmid by qRT-PCR; B shows ALP staining 14 days after osteogenic induction; C shows the ALP activity of cells after osteogenic induction; D shows the expression of key osteogenic differentiation factors Runx2, Osterix, alkaline phosphatase (Alp), and Osteopontin (Opn) mRNA by qRT-PCR 3 days after osteogenic induction; E shows the expression of Runx2, Osterix, ALP, and OPN proteins by Western blotting 3 days after osteogenic induction.

[0012] Figure 2 This image shows the results of osteogenic differentiation of bone marrow stromal cells (BMSCs) after the BCKDK gene has been knocked out. Image A shows the knockout of Bckdk using Ad-Cre adenovirus. fx / fx The gene knockout efficiency of the BCKDK encoding gene in mouse BMSCs was detected by RT-qPCR. B shows ALP staining 14 days after osteogenic induction; C shows the ALP activity in cells after osteogenic induction; D shows the mRNA expression of key osteogenic differentiation factors Runx2, Osterix, Alp, and Opn by qRT-PCR 3 days after osteogenic induction; E shows the protein expression of Runx2, Osterix, ALP, and OPN by Western blotting 3 days after osteogenic induction.

[0013] Figure 3 The results of BT2-induced osteogenic differentiation of bone marrow stromal cells in sham-operated (Sham) and ovariectomized (OVX) mice are shown. A shows ALP staining 14 days after osteogenic induction; B shows the mRNA expression of key osteogenic differentiation factors Runx2, Osterix, alkaline phosphatase (Alp), and osteopontin (Opn) by qRT-PCR 3 days after osteogenic induction; C shows the protein expression of Runx2, Osterix, ALP, and OPN by Western blotting 3 days after osteogenic induction.

[0014] Figure 4 The results show the inhibition of osteoclast differentiation of bone marrow-derived macrophages (BMMs) in BT2-treated (Sham) and ovariectomized (OVX) mice. A shows TRAP staining 8 days after osteoclast induction; B shows the TRAP-positive osteoclast count; C shows the expression of osteoclast differentiation-related factors, activated T cell nuclear factor c1 (NFATc1), cathepsin K (CTSK), and matrix metalloproteinase 9 (MMP9), by Western blotting 3 days after osteoclast induction.

[0015] Figure 5 The results of μCT scans of the tibia in mice after continuous gavage administration of BT2 for 8 weeks are shown. A is a 3D longitudinal section (sagittal plane) of the tibial metaphysis; B is the trabecular bone volume fraction (Tb.BV / TV); C is the number of trabeculae (Tb.N); D is the trabecular bone mineral density (Tb.BMD); E is a 3D reconstruction of the cortical bone; and F is the cortical bone thickness (Ct.Th).

[0016] Figure 6The results of staining on tibial tissue sections from mice that underwent continuous gavage with BT2 for 8 weeks are shown. A shows HE staining of the tibial metaphysis; B shows osteoblast count; C shows tartrate-resistant acid phosphatase (TRAP) staining; and D shows TRAP-positive osteoclast count. Detailed Implementation

[0017] 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.

[0018] 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.

[0019] The materials and methods involved in the embodiments of the present invention are described below.

[0020] (1) Culture and induction of differentiation of MC3T3-E1 cells

[0021] Mouse pre-osteoblasts MC3T3-E1 were cultured in α-MEM medium containing 10% fetal bovine serum (FBS) and transfected when the cell density reached 60%. After transfection, when the cell confluence reached approximately 80%, the medium was replaced with complete α-MEM medium containing osteogenic inducers (10% FBS, 50 µg / ml ascorbic acid, 5 mmol / L sodium β-glycerophosphate), and 50 μM BT2 or vehicle solvent was added. The medium was replaced every 3 days. After 72 h of osteogenic induction, mRNA and protein were collected, and alkaline phosphatase (ALP) staining was performed after 14 days of induction.

[0022] (2) Cell transfection

[0023] PCR primers were designed based on the coding sequence (CDS) of mouse Bckdk: Forward Primer: 5'-TTGGTACCGAGCTCGGATCCGCCACCATGATACTGACTTCAGTGCTG-3' (SEQ ID NO. 1); Reverse Primer: 5'-GCTGGATATCTGCAGAATTCTTAGATTCTGAAGCTTTCCTCCCGAC-3' (SEQ ID NO. 2). PCR amplification was performed using mouse cDNA as a template. The amplified CDS fragment was ligated from the BamHI / EcoRI site into the pcDNA3.1(+) vector using the pEASY®-Basic Seamless Cloning Kit (TransGen Biotech, Beijing, China) to construct the Bckdk overexpression plasmid (Bckdk Construct).

[0024] Plasmid transfection: When the cell density reaches 60%, 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.

[0025] (3) Establishment of mouse ovariectomy (OVX) model

[0026] Twelve-week-old wild-type female C57BL / 6J mice were weighed. After anesthesia, the mice were placed abdomen-down on a surgical board. Hair was removed from their backs using a shaver. An incision of approximately 0.5-1 cm was made along the midline of the back using ophthalmic scissors. The perirenal white fat pad was located. The muscle layer was bluntly dissected to reveal a cauliflower-shaped ovary encased in white fat. The fat was gently pulled out of the incision. The ovary and surrounding white fat were grasped with forceps, and the ovary was removed using a high-frequency electrocautery device. The ovary on the other side was removed using the same method. The skin was sutured shut, and antibiotics and iodine were applied to the suture site to prevent infection. The sham surgery group underwent the same procedure, but the ovary was not removed.

[0027] (4) BT2 gavage treatment of mice

[0028] C57BL / 6 mice were purchased from Spiefol Biotechnology Co., Ltd. (Beijing, China) and housed in an SPF-grade barrier environment with free access to food and water. BT2 solution was prepared using a vehicle medium of 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% physiological saline. Mice were randomly divided into four groups: Sham / Vehicle, Sham / BT2, OVX / Vehicle, and OVX / BT2. At 12 weeks of age, mice underwent either OVX surgery or sham surgery. Three days post-surgery, BT2 was administered via gavage at a dose of 40 mg / kg. The control group received the vehicle medium (5% DMSO) for 8 weeks. Animal experiments were approved by the Animal Ethics Committee of Zhu Xianyi Memorial Hospital, Tianjin Medical University.

[0029] (5) μCT analysis

[0030] After euthanasia, tibial specimens were isolated and scanned using a Scanco viva CT80 (Swiss Scanco Medical AG) with scanning parameters of 55 kVp and 145 μA. Three-dimensional reconstruction was performed using Scanco software. The region of interest in cancellous bone was selected as a 1 mm region starting 0.1 mm below the growth plate, and the region of interest in cortical bone was selected as a 0.5 mm region starting 2 mm below the growth plate.

[0031] (6) HE staining of bone tissue

[0032] Immediately after euthanizing mice, tibial tissue was fixed in 10% neutral formalin (NBF) at 4°C for 72 hours. It was then washed with phosphate-buffered saline (PBS) and transferred to 14% EDTA (pH 7.2) for decalcification at 4°C for 3 weeks. After decalcification, the tissue was washed with PBS, then subjected to graded ethanol dehydration, xylene clearing, and finally embedded in paraffin. The paraffin blocks were then cut into 5 μm thick sagittal sections, and hematoxylin-eosin (H&E) staining was performed on the sections. The dewaxed and hydrated tibial sections underwent the following steps: hematoxylin staining for 3 seconds, washing with water until the waste liquid was colorless, separation with 1% hydrochloric acid ethanol for 3 seconds, eosin staining for 3 minutes, washing with water until the waste liquid was colorless, dehydration, mounting, microscopic observation, photographing, and counting of osteoblasts on the trabecular bone surface.

[0033] (7) TRAP staining of bone tissue

[0034] Osteoclast staining was performed on sections using tartrate-resistant acid phosphatase (TRAP) staining. The staining solution consisted of 0.1 mol / L acetate buffer (pH 5.0), 0.1 mg / mL naphthol AS-MX phosphate, 0.6 mg / mL solid red violet LB salt, and 0.05 mol / L sodium tartrate. Sections were incubated at 37°C for 30 min, rinsed with double-distilled water, and stained with hematoxylin for 1 min. After rinsing with double-distilled water, sections were separated by 1% hydrochloric acid ethanol for 3 s, rinsed again with double-distilled water, and then mounted with glycerol gelatin. The target observation area was defined as a region extending 1.0 mm distally from 0.1 mm below the growth plate.

[0035] (8) Isolation, culture and osteogenic differentiation of bone marrow stromal cells (BMSCs)

[0036] Primary bone marrow mesenchymal stem cells (BMSCs) were isolated using the whole bone marrow adherent culture method. Mice treated by gavage for 8 weeks were sacrificed, and the femurs and tibias of both hind limbs were aseptically removed. The epiphyses were removed to expose the medullary cavity. The medullary cavity was flushed multiple times with 5 mL of DMEM culture medium using a syringe, and cells were collected and then seeded into α-MEM containing 10% fetal bovine serum. The medium was changed for the first time after 3 days of culture to remove non-adherent cells. After washing with PBS, the medium was replaced with fresh medium. The medium was changed every 3 days. Cells were passaged when they reached 80% confluence. Third-generation cells were used for subsequent experiments.

[0037] Osteogenic induction of BMSCs: When 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 alkaline phosphatase (ALP) staining was performed 14 days after induction.

[0038] (9) Alkaline phosphatase staining

[0039] After osteogenic induction and differentiation for 14 days, alkaline phosphatase 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.

[0040] Isolation and culture of osteogenic precursor cells from skull bone

[0041] Skull bones from newborn mice within 3 days of birth were collected. Connective tissue was aseptically dissected and minced. After rinsing with PBS, 1 ml of 0.25% trypsin + 1 ml of 0.1% type I collagenase digestion solution was added. The cells were initially digested at 37°C with shaking for 10 min, and the supernatant was discarded to remove contaminating cells. The digestion was repeated twice with an equal volume of digestion solution, 20 min each time. The supernatant from each digestion was collected, and α-MEM containing 10% fetal bovine serum was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 min, resuspended in complete culture medium, and incubated at 37°C with 5% CO2. The medium was changed every 3 days, and the cells were passaged when 80% confluence was reached. Cells from passages 3-6 were used for experiments.

[0042] (10) In vitro knockout of the Bckdk gene in osteogenic precursor cells of the calcaneus

[0043] Bckdk fx / fx Mice were donated by Professor Sun Haipeng of Zhu Xianyi Memorial Hospital, Tianjin Medical University. Ad-Cre adenovirus or Ad-GFP adenovirus was purchased from Hanheng Biotechnology Co., Ltd. (Shanghai, China). Newly hatched Bckdk mice within 3 days of isolation were used. fx / fx Mouse cranial osteogenic precursors were infected with Ad-Cre or Ad-GFP adenovirus at an MOI of 20. 72 hours after infection, cells were collected for RNA extraction, and the mRNA expression level of Bckdk was detected by qRT-PCR. When cell confluence reached approximately 80%, osteogenic induction was performed. ALP staining and ALP activity assays were conducted 14 days after induction. On day 3 of osteogenic induction, cells were collected for total RNA and protein extraction, and the expression levels of osteogenic differentiation-related factors were detected by qRT-PCR and Western blotting.

[0044] (11) Isolation, culture and osteoclast differentiation of bone marrow-derived macrophages (BMMs)

[0045] Mouse whole bone marrow cells were seeded into 10 cm cell culture dishes and cultured in α-MEM containing 10% fetal bovine serum (FBS) and 5 ng / mL macrophage colony-stimulating factor (M-CSF). After 24 hours of culture, non-adherent cells were collected and cultured at 2.5 × 10⁻⁶ cells / mL. 5Cells were seeded at a density of 10 cells / well into 48-well plates. Bone marrow-derived macrophages (BMMs) were first cultured in medium containing 25 ng / mL M-CSF for 3 days, followed by the addition of 25 ng / mL M-CSF and 50 ng / mL soluble nuclear factor κB receptor activator ligand (sRANKL, purchased from ABclonal, Wuhan, China) to induce cell differentiation for 8 days. The differentiated osteoclasts were then stained with TRAP according to the kit instructions (Sigma-Aldrich, USA). Osteoclasts were observed and counted under a microscope; cells with TRAP-positive staining and ≥3 nuclei were considered osteoclasts.

[0046] (12) Reverse transcription-quantitative PCR (qRT-PCR)

[0047] Total RNA was extracted from cells using a total RNA extraction kit (Omega, USA). 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 SYBR Green fluorescence PCR, with β-actin as an internal control. -ΔΔCt The relative expression levels of each target gene were calculated using the following method: ΔCt = Ct value of target gene - Ct value of internal reference gene; ΔΔCt = ΔCt of experimental group - ΔCt of control group. All experiments were repeated at least 3 times.

[0048] (13) Western blotting

[0049] 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 assay kit (ABclonal, Wuhan, China).

[0050] (14) Statistical analysis

[0051] 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.

[0052] Example 1

[0053] This study analyzed the effect of BCKDK on osteogenic differentiation of MC3T3-E1 cells. MC3T3-E1 cells were transfected with the BCKDK overexpression plasmid (BckdkConstruct), and changes in osteogenic differentiation after BCKDK overexpression were analyzed. qRT-PCR detected a significant increase in Bckdk mRNA in the Bckdk Construct group (…). Figure 1 A). After osteogenic induction, ALP staining was found to be lighter in the Bckdk Construct group ( Figure 1 B), ALP activity decreased ( Figure 1 C), mRNAs of key osteogenic differentiation factors Runx2, Osterix, ALP, and OPN ( Figure 1 D) and protein ( Figure 1 The expression levels of E were reduced, indicating that overexpression of BCKDK inhibited osteogenic differentiation of MC3T3-E1 cells.

[0054] Example 2

[0055] This embodiment analyzes the effect of BCKDK gene knockout on osteogenic differentiation of cranial calcaneal osteogenic progenitor cells. BCKDK cells were infected with Ad-Cre and Ad-GFP adenoviruses. fx / fx Mouse cranial osteogenic progenitor cells were analyzed to examine changes in osteogenic differentiation following knockout of the BCKDK gene. qRT-PCR detected a significant reduction in Bckdk mRNA in the Ad-Cre group (…). Figure 2 A). After osteogenic induction, ALP staining was found to be more pronounced in the Ad-Cre group ( Figure 2 B), ALP activity increased ( Figure 2 C), mRNAs of key osteogenic differentiation factors Runx2, Osterix, and ALP ( Figure 2 D) and protein ( Figure 2 E) The expression levels were elevated, indicating that knocking out the BCKDK-encoding gene promoted osteogenic differentiation of osteogenic precursor cells in the calcaneus.

[0056] Example 3

[0057] This study analyzed the effect of BT2 treatment on osteogenic differentiation of mouse bone marrow mesenchymal stem cells (BMSCs). Mouse BMSCs were isolated and cultured after 8 weeks of gavage treatment for osteogenic induction. After osteogenic induction, compared with the Sham / Vehicle group, ALP staining was deeper in the Sham / BT2 group and lighter in the OVX / Vehicle group; compared with the OVX / Vehicle group, ALP staining was deeper in the OVX / BT2 group. Figure 3 A), mRNAs of key osteogenic differentiation factors Runx2, Osterix, ALP, and OPN ( Figure 3 B) and protein ( Figure 3 C) The expression changes were consistent with the staining results, indicating that BT2 can promote osteogenic differentiation of BMSCs in Sham group and OVX mice.

[0058] Example 4

[0059] This study analyzed the effect of BT2 treatment on osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs). Mice BMMs treated by gavage for 8 weeks were induced to undergo osteoclastogenesis. TRAP staining revealed that compared to the Sham / Vehicle group, osteoclast differentiation was reduced in the Sham / BT2 group and increased in the OVX / Vehicle group; compared to the OVX / Vehicle group, osteoclast differentiation was reduced in the OVX / BT2 group (…). Figure 4 The protein changes of osteoclast-related factors NFATc1, CTSK, and MMP9 in A and B were consistent with the staining results, indicating that BT2 can inhibit osteoclast differentiation of BMMs in Sham and OVX mice. Figure 4 C).

[0060] Example 5

[0061] This study analyzed the effect of BT2 on tibial bone mass in OVX mice. After 8 weeks of gavage administration of BT2 or Vehicle, tissue samples were collected for μCT analysis and histological staining. Compared with the Sham / Vehicle group, the OVX / Vehicle group showed significantly reduced cancellous and cortical bone mass, as evidenced by decreased static bone biometric parameters Tb.BV / TV, Tb.N, Tb.BMD, and Ct.Th, indicating successful OVX mouse model establishment. Figure 5 AF). Compared with the Sham / Vehicle group, the Sham / BT2 group mice showed significantly increased cancellous and cortical bone mass; compared with the OVX / Vehicle group, the OVX / BT2 group mice showed significantly increased cancellous and cortical bone mass, as evidenced by elevated static bone biometric parameters Tb.BV / TV, Tb.N, Tb.BMD, and Ct.Th. Figure 5 AF).

[0062] Example 6

[0063] We identified and counted osteoblasts using HE staining. Compared with the Sham / Vehicle group, the OVX / Vehicle group had fewer osteoblasts, while the Sham / BT2 group had more; compared with the OVX / Vehicle group, the OVX / BT2 group had more osteoblasts. Figure 6 AB). Osteoclasts were identified and counted by TRAP staining. Compared with the Sham / Vehicle group, the OVX / Vehicle group showed an increase in osteoclasts, while the Sham / BT2 group showed no significant change. Compared with the OVX / Vehicle group, the OVX / BT2 group showed a significant decrease in osteoclasts. These results indicate that BT2 can promote osteoblast differentiation and inhibit osteoclast differentiation in OVX mice, alleviate OVX-induced bone loss in mice, and increase bone mass (AB). Figure 6 CD).

[0064] In summary, BCKDK can inhibit osteoblast differentiation, thereby reducing endogenous BCKDK expression in osteoblast precursor cells and promoting osteogenic differentiation. The small molecule inhibitor BT2 of BCKDK can promote osteoblast differentiation in ovariectomized mice, inhibit osteoclast differentiation, alleviate bone loss in ovariectomized mice, and increase bone mass. BCKDK inhibitors have the potential to prevent and treat osteoporosis.

[0065] 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 branched-chain ketoate dehydrogenase kinase kinase (BCKDK) inhibitors in the preparation of drugs for the prevention and treatment of osteoporosis, characterized in that: The BCKDK inhibitor is BT2, namely 3,6-dichloro-2-benzothiophene carboxylic acid. It increases bone mass by targeting and inhibiting the biological function of BCKDK protein, thereby promoting osteoblast differentiation and inhibiting osteoclast differentiation, and thus preventing and treating osteoporosis.

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

3. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients.

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