Bone growth promoter and its application

Abstract

The present invention belongs to the field of medicine, and specifically relates to a bone growth promoter and its application in the medical field. The present invention, for the first time, discovered through high-throughput virtual screening the inhibitory effect of morugin on the osteoblastic and osteoporosis-fighting benchmark target, sclerostin protein, and verified this through experiments. At the same time, the osteoblastic differentiation-promoting effect of morugin was confirmed for the first time through in vitro and animal experiments. The present invention proposes morugin and its pharmaceutically acceptable salts as a bone growth promoter, which can be used alone or in combination to promote bone growth and fight osteoporosis. Its product form includes medicine.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a bone growth promoter, moringin, and its application in the field of medicine. Background Art

[0002] The high incidence of fractures and osteoporosis poses a significant threat to human health. Fractures lead to structural damage and dysfunction of the bone, affecting not only the bones themselves but also surrounding tissues, nerves, blood vessels, and organs throughout the body. Osteoporosis is characterized by decreased bone mass and increased bone fragility. The decreased motor function and high risk of fractures caused by osteoporosis present serious social problems and economic burdens. Therefore, the need for the prevention, diagnosis, and treatment of fractures and osteoporosis is becoming increasingly urgent.

[0003] There are many types of drugs that promote bone growth and fight osteoporosis, and their targets and mechanisms of action are also different (Chinese Pharmaceutical Association Hospital Pharmacy Committee, "Expert Consensus on the Rational Use of Osteoporosis Treatment Drugs (2023)" Editorial Group, Zhang Yu. Expert Consensus on the Rational Use of Osteoporosis Treatment Drugs (2023) [J]. Chinese Journal of Hospital Pharmacy, 2024, 44(9): 985-1006.), mainly in two aspects:

[0004] First, bone formation promoters. A representative example is sclerostin inhibitors (Marini F, Giusti F, Palmini G, et al. Role of Wnt signaling and sclerostinin bone and as therapeutic targets in skeletal disorders[J]. Osteoporosis International, 2022, 34(2):213-238. DOI:10.1007 / s00198-022-06523-7.). In 2019, the US FDA approved the new sclerostin inhibitor Evenity for marketing, making it the first new osteoporosis drug with the dual effects of increasing bone formation and reducing bone resorption.

[0005] Sclerostin is a glycoprotein encoded by the sost gene and secreted by osteogenic cells that inhibits bone formation. On the one hand, sclerostin competitively inhibits the Wnt signaling pathway by co-receptors LRP-5 / 6, thereby inhibiting this signaling pathway and, in turn, bone formation. On the other hand, after being embedded in matrix proteins, osteocytes begin to secrete sclerostin, which is then transported through the bone tissue vascular network to osteoblasts on the bone surface, thereby inhibiting osteoblast-mediated bone formation. Due to the inhibitory effect of sclerostin on bone formation, inhibiting its expression or activity can significantly increase bone mass and strength without significant adverse effects outside of bone, making it an effective therapeutic target for osteoporosis. Animal models and clinical studies have shown that sclerostin inhibitors can significantly increase bone density and strength, and compared with traditional drugs such as alendronate and teriparatide, sclerostin inhibitors are more effective in increasing bone density.

[0006] Second, bone resorption inhibitors. These include bisphosphonates and calcitonins. Bisphosphonates primarily act on osteoclasts, inhibiting their ability to dissolve bone, reducing bone loss and thereby increasing bone density. Drugs like alendronate and zoledronic acid are suitable for patients at risk of fracture or who have already experienced a fracture. Calcitonins target osteoclasts, rapidly inhibiting osteoclast activity and reducing bone dissolution. They also have analgesic properties and are suitable for relieving pain during acute attacks and preventing complications. Examples include eel calcitonin analogs and salmon calcitonin.

[0007] Currently, anti-osteoporosis sclerostin protein inhibitors have shortcomings such as high cost, poor injection compliance, and potential side effects. Therefore, there is an urgent need for anti-osteoporosis products with clear molecular mechanisms, easy use, good safety, and low price.

[0008] Drug screening and in vitro and animal studies are feasible approaches for discovering sclerostin inhibitors. Virtual screening, also known as in silico screening or virtual screening in computer-aided drug design, uses computer technology and specialized software to identify potentially effective drug candidates from a large pool of compounds. This approach can significantly reduce the time and cost of drug development and improve the efficiency of new drug discovery (Zeng J, Han J, Liu Z, et al. Pentagalloylglucose disrupts the PALB2-BRCA2 interaction and potentiatestumor sensitivity to PARP inhibitor and radiotherapy[J]. Cancer letters, 2022, 546:215851. DOI:10.1016 / j.canlet.2022.215851.). The results of virtual screening require in vitro cell and animal experiments to evaluate their efficacy. In in vitro cell research and evaluation, the MC3T3-E1 cell model is a commonly used cell model for studying bone diseases and mechanisms (Zheng W, Luo Q, Shi D, et al. FDPS-lnfluenced Transcriptome Alternations in MC3T3-E1 Cells are Associated with the Pathogenesis of Osteoporosis[J]. Science of advanced materials, 2023(8):15.), and has been used in studies such as osteogenic differentiation and osteoporosis (Liu Ning. Study on the role and mechanism of miR-148a-3p in osteogenic differentiation of MC3T3-E1 cells and osteoporotic bone remodeling[D]. China Medical University, 2023. DOI:10.27652 / d.cnki.gzyku.2023.001911.).

[0009] Thermal drift experiments have been an effective means of studying the binding of compounds to target proteins in recent years. They are used to detect the interaction between drugs and proteins in vitro and to screen specific protein candidate drugs in vitro. When cells are treated with drugs, the drug binds to the target protein, enhancing the thermal stability of the target protein and increasing the melting temperature (Wang Li, Liu Jingfang, Li Weilin, et al. Standard operating procedures for identifying drug targets in living cells based on cell-based thermal drift assay (CETSA) technology [J]. Acta Microbiologica Sinica, 2023, 63(6): 2488-2501.).

[0010] In animal studies and evaluations of sclerostin inhibitors, in situ osteogenesis models can be used to evaluate the effects of sclerostin inhibitors on bone tissue, providing a strong experimental basis. In situ osteogenesis models refer to inducing or promoting the formation and regeneration of bone tissue in a specific part of an animal through specific experimental methods. When establishing a model, bone cells can be induced to differentiate in vitro and injected or transplanted subcutaneously into mice or other animals. Bone tissue regeneration can be evaluated by imaging or histological examination (Ma D, Zhong C, Yao H, et.al. Engineering injectable bone using bone marrowstromal cell aggregates. Stem Cells Dev. 2011 Jun;20(6):989-99.). In addition to ALP, Runx2 indicators also play a vital role in the bone formation process. Their changes directly reflect the state of bone metabolism and the activity of bone formation.

[0011] Traditional Chinese Medicine (TCM) has a valuable foundation of thousands of years of evidence-based medicine. Exploring new anti-osteoporosis approaches from numerous empirical records and reports of effective applications in treating bone and joint diseases is a viable strategy. Mulberries, as a traditional Chinese medicinal material with a long history, have a wide range of applications. The Compendium of Materia Medica and Shennong's Herbal Classics record that mulberries can "benefit the five internal organs and joints." The Tang Dynasty's Newly Revised Materia Medica mentions that mulberries can treat "weakness of the waist and knees." The Suixiju Dietary Guide mentions that mulberries "nourish the liver and kidneys and strengthen walking." However, the specific active ingredients in mulberries that "benefit the five internal organs and joints" and "nourish the liver and kidneys and strengthen walking" remain unknown. Therefore, using modern biotechnology to study the active ingredients in mulberries that are closely related to bone growth is extremely valuable research for the prevention and treatment of bone diseases. Summary of the Invention

[0012] In response to the problems existing in the existing technology, the present invention provides a moringin that can inhibit sclerostin protein and has a bone growth promoter. It is used to promote bone growth, resist osteoporosis and other common bone problems, and clarifies its application value in the medical field.

[0013] The technical contents of the present invention are as follows:

[0014] The present invention provides a promoter with bone growth effect, the active ingredient of which is moringin or a pharmaceutically acceptable salt thereof.

[0015] The accelerator has the following functions:

[0016] (1) Inhibit the activity of sclerostin protein;

[0017] (2) Promote osteogenic differentiation of osteoblast precursor cells;

[0018] a. Enhances TCF / LEF1 transcriptional activity and activates the Wnt / β-catenin signaling pathway in MC3T3-E1 cells;

[0019] b. Enhanced alkaline phosphatase activity in MC3T3-E1 cells to induce osteoblast differentiation;

[0020] c. Promote the formation of calcified nodules in MC3T3-E1 cells;

[0021] (3) Promote bone formation in mice;

[0022] a. Promote the expression of ALP in mouse bone tissue;

[0023] b. Promote the expression of Runx2 in mouse bone tissue.

[0024] The present invention also provides the use of the promoter in the preparation of a drug for promoting bone growth. Specifically, the promoter can promote osteoblast differentiation and mineralization, thereby increasing bone density and bone toughness.

[0025] Furthermore, the promoter can be used to prepare drugs for promoting fracture healing and / or resisting osteoporosis.

[0026] The accelerators are used alone or in combination to prepare medicines.

[0027] The medicine comprises moringin or a pharmaceutically acceptable salt thereof.

[0028] The drug is composed of moringin and a pharmaceutically acceptable carrier; or, the drug further contains one or more of a disintegrant, a wetting agent, a binder, a filler, an absorption promoter, a solvent, a lubricant, a surfactant, a flavoring agent, a sweetener, an antioxidant, a preservative and a pigment, an ointment base, and a transdermal enhancer.

[0029] The dosage form of the drug includes one or more of tablets, capsules, injections, granules and suspensions.

[0030] Compared with the prior art, the present invention has the following beneficial values:

[0031] 1. The present invention discovered for the first time through virtual screening the inhibitory effect of moringin on the osteogenesis benchmark target sclerostin protein and confirmed it through in vitro experiments.

[0032] 2. The present invention, through MC3T3-E1 cell model experiments, confirmed for the first time the effect of moringin on promoting osteogenic differentiation of mouse osteoblast precursor cells MC3T3-E1. Specifically, moringin can enhance the TCF / LEF1 transcriptional activity in MC3T3-E1 cells, activate the Wnt / β-catenin signaling pathway, enhance the alkaline phosphatase activity that induces osteoblast differentiation in MC3T3-E1 cells, and promote the formation of calcified nodules in MC3T3-E1 cells.

[0033] 3. The present invention, by constructing an in situ osteogenesis model, confirmed for the first time the osteogenesis-promoting effect of moringin on mice. The changes in bone tissue size before and after the treatment of moringin and the differences in ALP and Runx2 expression in bone tissue confirmed that moringin can promote bone formation in mice.

[0034] 4. This invention proposes moringin as a bone growth promoter, which can be used alone or in combination to prepare drugs for preventing and treating fractures and osteoporosis. Moringin, a natural compound extracted from mulberries, does not rely on limited chemical raw materials and complex synthesis processes. It offers advantages such as safety, effectiveness, and a clear molecular mechanism. Compared to currently marketed drugs, it has potential advantages in long-term safety and compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 , molecular weight score distribution diagram of natural product compound library combined with sclerostin 2K8P structure screening results;

[0036] Figure 2 , luciferase assay of candidate compounds;

[0037] Figure 3 , the effective dose of moringin to enhance luciferase activity;

[0038] Figure 4 , Morin inhibits the activity of sclerostin protein

[0039] Figure 5 , the safety of moringin in MC3T3-E1 cells;

[0040] Figure 6 , ALP staining of MC3T3-E1 cells after 7 days of treatment with moringin;

[0041] Figure 7 , ALP activity of MC3T3-E1 cells after 7 days of treatment with moringin;

[0042] Figure 8 , fluorescence intensity of the moringin calcium ion probe;

[0043] Figure 9 , comparison of heterotopic transplant tissue size in mouse in situ osteogenesis experiment;

[0044] Figure 10 , immunofluorescence staining of ALP in mouse bone tissue sections;

[0045] Figure 11 , Immunofluorescence staining of Runx2 in mouse bone tissue sections. DETAILED DESCRIPTION

[0046] To make the technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be further described below in conjunction with specific embodiments. Unless otherwise specified, all raw materials used in the embodiments are commercially available.

[0047] Example 1 Virtual screening revealed the inhibitory effect of moringin on the osteoblastic target sclerostin protein

[0048] Virtual drug screening targeting sclerostin protein includes the following steps:

[0049] (1) Processing of sclerostin protein structure

[0050] The SOST_HΜMAN protein structure was processed as a target protein using the Protein Preparation Wizard module in the Schrödinger software, and the prepared structure was used as a receptor file for subsequent virtual screening.

[0051] (2) Preparation of small molecule compound library

[0052] The natural product compound library is processed to ensure the global conformation of small molecules during virtual screening. Conformations of small molecules are generated, with a maximum of 32 conformations generated for each small molecule. The prepared compound library is used as a ligand file for subsequent virtual screening.

[0053] (3) Molecular docking

[0054] The prepared receptor and ligand files were screened using the SP (Standard Precision) screening mode, with docking parameters set to default values ​​and a small molecule flexible docking method employed. Energy optimization was then performed after docking. Based on the docking results, the small molecules selected for the next step of screening, i.e., the candidate drugs, were then screened in vitro.

[0055] (4) Evaluation of docking results

[0056] Affinity scoring: Schrödinger software was used to perform molecular docking on each compound in the compound library and calculate the affinity score between the compound and the 2K8P structure of SOST_HΜMAN. The data generated by the docking process of each compound library is as follows Figure 1As shown, the compound library contains 279 compounds that bind to the 2K8P structure, and their affinities range from -11.8030 kcal / mol to -5.2366 kcal / mol (the lower the score, the better the evaluation result);

[0057] Structural diversity analysis: Structural diversity analysis was performed on 279 compounds in the compound library combined with the 2K8P structure. Structure-based clustering was performed using MOE software. The fingerprint clustering algorithm was the Jarvis-Patrick clustering method. The structural similarity was set to 70%. The 279 molecules in the compound library and the 2K8P structure were divided into 165 categories.

[0058] Visual binding mode analysis: The binding conformations of 279 molecules and proteins were visually inspected, and unreasonable structures were removed. A total of 74 candidate drugs, including morulaine, were retained for the next in vitro experimental screening.

[0059] Example 2 TCF / LEF1-Luc-Puro Reporter Lentivirus was used to construct the TCF / LEF1-Luc MC3T3-E1 cell line in vitro, and the activation effect of 10 μM moringin on the wnt signaling of MC3T3-E1 cells was verified.

[0060] The 74 drug candidates obtained from the virtual screening in Example 1 were screened in vitro. The compounds used for screening were derived from the natural product compound library of Taoshu Biotechnology Co., Ltd. and dissolved in DMSO to a 10 mM concentration. The specific procedures for the in vitro screening were as follows:

[0061] (1) Model construction

[0062] First, the TCF / LEF1-Luc-Puro Reporter plasmid was extracted, followed by TCF / LEF1 lentiviral packaging and titer determination. The purified, endotoxin-free lentiviral vector and its auxiliary packaging component plasmid were extracted and co-transfected into HEK-293T cells using HGTransgene™ Reagent. Enhancing buffer was added 10-12 hours after transfection, and fresh medium was replaced 8 hours later. Culture was continued for 48 hours, and the cell supernatant, rich in lentiviral particles, was collected and concentrated to obtain a high-titer lentiviral concentrate. Viral titer was then determined and calibrated in HEK-293T cells. MC3T3-E1 cells were then infected with the packaged TCF / LEF1-Luc lentivirus to generate a stable TCF / LEF1-Luc MC3T3-E1 strain. For cell samples of MC3T3-E1 cells infected with TCF / LEF1-Luc lentivirus, the overexpression effects of luciferase and puro were verified by Real Time PCR.

[0063] Wnt proteins are secreted, lipid-modified glycoproteins that activate diverse intracellular signaling pathways. They regulate cell growth, function, differentiation, and cell death and are essential for normal embryonic development. Wnt proteins activate Frz receptors, leading to the translocation of β-catenin into the nucleus. Binding to TCF / LEF transcription factor binding sites activates downstream pathways, leading to the expression of luciferase. Luciferase readouts represent signaling pathway activation and can be used to evaluate the in vitro efficacy of Wnt pathway-related drugs. Using a Wnt-β-specific TCF / LEF-β-driven reporter system optimized for Wnt 3a responses, a natural product library was screened in vitro. Cell-based screening identified several sclerostin inhibitors with the ability to enhance Wnt / β-catenin signaling.

[0064] (2) Compound treatment

[0065] Remove the cells from the incubator, digest and centrifuge to collect the cell pellet, resuspend in complete medium and count. Then adjust the cell concentration to 1.5×10 5 cells / mL. Use a pipette to add 100 μL of cells / well to the center well. Add 100 μL of PBS to the surrounding wells. Cover the plate and incubate in a 37°C cell culture incubator. After 24 hours, discard the complete medium, add 0.02 μg / mL of Wnt3a protein and 10 μM of drug to each well, and incubate in a 37°C cell culture incubator for 24 hours.

[0066] (3) Fluorescence detection

[0067] Equilibrate the 96-well plate at room temperature for 5 minutes. Then, add 100 μL of luciferase assay reagent to each well to lyse the cells. Pipet up and down the wells five times with a pipette to ensure complete cell lysis. Wait 5 minutes to allow for complete cell lysis. Pipette 100 μL of the mixture (assay reagent + cell culture medium) into each well of the white assay plate. Fluorescence is measured at 570 nm using a Molecular Devices Spectra Max L microplate reader.

[0068] The results are as follows Figure 2 Among the candidate compounds, the luciferase value of moringin was significantly higher than that of the blank group, indicating that moringin can bind to the TCF / LEF transcription factor binding site and activate the classic Wnt signaling pathway. The Wnt signaling pathway can promote osteoblast differentiation and help delay osteoporosis. Figure 3 We studied the effective dose of moringaine and the results showed that 5, 10, and 20 μM moringaine could enhance luciferase activity in a dose-dependent manner.

[0069] Example 3 In vitro experiments demonstrate the inhibitory effect of moringin on sclerostin protein

[0070] To verify that morulaine activates Wnt signaling by binding to sclerostin and inhibiting its activity, a thermal shift experiment was performed, including the following steps:

[0071] (1) Cell stimulation and treatment

[0072] MC3T3-E1 cells were stimulated with 10 μM moringin for 24 h. After washing with PBS buffer, the cells were trypsinized and processed into a suspension using PBS solution containing protease inhibitors.

[0073] (2) Thermal drift experiment

[0074] The cell suspension was heated at 37, 42, 47, 52, and 57°C using a PCR instrument. The binding ability of moringin to sclerostin protein was detected by Western blotting.

[0075] (3) Analysis of binding ability and inhibition

[0076] The grayscale value of the protein band was quantified by ImageJ software, and the binding of moringin to sclerostin protein (inhibiting the activity of sclerostin protein) was quantified and analyzed by Graphpad prism9.0.

[0077] The results are as follows Figure 4 Thermal shift experiments confirmed that 10 μM moringin could bind to sclerostin and reduce its activity. Moringin has the function of a sclerostin inhibitor.

[0078] Example 4 Cytotoxicity experiments demonstrate that moringin has no cytotoxicity

[0079] To study the toxicity of moringin in MC3T3-E1 cells, a CCK8 assay was performed. The specific procedures are as follows:

[0080] Cell viability was detected using the Cell Counting Kit-8 (Meilun Biotech) according to the manufacturer's instructions. 100 μL of MC3T3-E1 cell suspension was seeded into a 96-well plate at a density of 5 × 10 cells per well. 3 Cells were cultured at 37°C for 24 hours. Then, moringin was added sequentially to the culture medium. At the designated time points (48 hours), 100 μL of 10% CCK-8 solution was added to each well. After incubation at 37°C for 1 hour, absorbance was measured at 450 nm on a microplate reader, and cell viability (%) was calculated using untreated control cells. Each experiment was performed in triplicate.

[0081] The results are as follows Figure 5 Compared with the control group, after MC3T3-E1 cells were treated with 5, 10, and 20 μM moringin for 48 hours, the cell viability of the moringin groups was greater than 90%, indicating that moringin had no cytotoxicity at this dose and could be used to evaluate osteogenic differentiation experiments.

[0082] Example 5 ALP staining was used to verify the effect of moringin on alkaline phosphatase activity in MC3T3-E1 cells, thereby proving that moringin promotes osteogenic differentiation of MC3T3-E1 cells.

[0083] ALP staining and enzyme activity determination, the specific procedures are as follows:

[0084] (1) MC3T3-E1 cell suspension was prepared at a concentration of 1×10 4 Cells were seeded at a density of 100 cells / well in a 12-well plate and incubated at 37°C. After 24 hours, the complete culture medium was replaced with osteogenic differentiation medium, and then orange peel was added in sequence for 7 days. ALP staining and ALP activity were detected using an ALP kit (Beyotime) and an ALP activity kit (Beyotime). The cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 30 minutes, and then BCIP / NBT working solution was added. Incubate at room temperature in the dark for 5-30 minutes, wash 3 times with PBST, 10 minutes each time, and observe the pictures under a light microscope. The results are as follows Figure 6 .

[0085] (2) For ALP activity determination, cells were collected using cell lysis buffer. The lysed cells were mixed with pNPP (5 mM pNPP, 1 mM MgCl2, 1 mM ZnCl2) substrate solution (0.1 M Mglycine buffer, pH = 10.4) at room temperature for 30 min, and the enzymatic reaction was stopped by adding 4 M NaOH solution. The absorbance value was measured at 405 nm, and the ALP activity was calculated based on the p-nitrophenol (pNP) standard curve and calibrated with the total protein concentration determined by the BCA method. The results are shown in Figure 2. Figure 7 .

[0086] Osteoblasts (OBs) are the primary functional cells responsible for bone formation, responsible for new bone formation. The fundamental biological characteristics of osteoblast differentiation are bone matrix synthesis, secretion, mineralization, and maturation. Osteoblasts first synthesize extracellular matrix (ECM) components such as type I collagen (COL-I), osteocalcin (OC), and osteopontin (OPN). They then release calcium ions and enzymes such as alkaline phosphatase (ALP) through matrix vesicles. Calcium ions, activated by ALP, are deposited on collagen fibrils, completing the matrix mineralization process and ultimately forming bone tissue. Type I collagen expression begins during the proliferation phase and reaches its peak during matrix synthesis. Alkaline phosphatase, an early marker of osteoblast differentiation, appears during the matrix synthesis phase and reaches its peak during the mineralization phase.

[0087] We treated MC3T3-E1 cells with 5, 10, and 20 μM of moringin for 7 days and then performed ALP staining. Figure 6 and Figure 7 Compared with the control group, blue precipitates appeared in the cytoplasm of the three doses of moringin groups, indicating that osteoblast differentiation was promoted.

[0088] Example 6 Alizarin red staining was used to demonstrate that moringin promoted osteoblast matrix deposition and mineralization.

[0089] After 21 days of culture, the cell samples were discarded from the culture medium, washed 1-2 times with PBS, fixed with 4% paraformaldehyde for 10 minutes, washed again 1-2 times with PBS, and stained with 0.1% Alizarin Red-S (Beyotime) solution for 10 minutes. Finally, they were rinsed with PBS and observed under an inverted light microscope. OD values ​​were measured at 562 nm using 10% hexadecylpyridine (Aladdin).

[0090] Osteocalcin is a marker of osteoblast differentiation and maturation. Its expression generally begins early during mineralization and reaches its peak after the mineralized nodules mature. Mineralized nodules are a hallmark of osteoblast differentiation and maturation, and are also the primary morphological characteristic of osteoblasts performing their osteogenic function. Observing osteoblast mineralized nodules is a commonly used technique for studying osteoblast differentiation. Furthermore, current research on the pathophysiology of osteoporosis relies heavily on 2D in vitro cell culture. However, gene expression in human osteoblasts cultured in this 2D format is limited and does not reflect the complexity of in vivo biophysical cues. Differentiated human osteoblasts cultured in 3D (using biphasic calcium phosphate particles) exhibit osteocyte-selective gene expression (E11 / GP38) comparable to that of mature human cortical osteoblasts. Therefore, we further evaluated the ability of moruginin to promote osteoblast mineralization in a 3D organoid model using a calcium ion probe.

[0091] The results are as follows Figure 8 Compared with the blank group, the fluorescence intensity of the calcium ion probe of 10μM moringin was significantly enhanced. And on the 2D model, the mineralized nodules of moringin were significantly stronger than those of the blank group, indicating that moringin promoted the mineralization of osteoblast matrix, thereby promoting bone formation.

[0092] Example 7 Construction of an in situ osteogenesis model in mice to confirm that moringin promotes bone formation in mice

[0093] Three-week-old NOD / SCID mice were used for in situ osteogenesis experiments. MC3T3 cells were pretreated one week before ectopic osteoblast transplantation: all groups were treated with osteogenic differentiation induction culture medium. The moringin group was treated with osteogenic differentiation induction medium combined with moringin 10 μM, and the medium was changed every 3 days. DMSO (1 / 1000) was added as a negative control group. The dorsal skin of the mice was selected as the injection site, and 2×10 6 Cells / 200 μL were added. Photos were taken at one and two weeks to record the longest diameter (L), the longest transverse diameter perpendicular to the longest diameter (W), and the tumor height (H) of the transplanted tissue. Tissue volume was calculated using the formula V = π / 6 × L × W × H. After tissue collection, paraffin sections were prepared after fixation, dehydration, and embedding. Immunofluorescence was used to detect changes in ALP and Runx2 proteins.

[0094] The results are as follows Figure 9 After measuring the volume of tissue 14 days after transplantation, it was found that the in situ tissue of mice in the moringa-treated group was significantly larger than that in the blank group, confirming that moringa-treated group can not only promote the proliferation of osteoblasts, but also protect the activity of osteoblasts.

[0095] ALP is produced by osteoblasts and plays an important role in the growth and development of bones. Its main physiological function is to hydrolyze phosphates during osteogenesis, providing the necessary phosphate for the deposition of hydroxyapatite. At the same time, it hydrolyzes pyrophosphates, relieving their inhibitory effect on bone salt formation, which is beneficial to osteogenesis. The enhanced expression activity of ALP is a significant feature of osteoblast differentiation. Figure 10 The results of paraffin section staining showed that the expression of ALP was significantly enhanced in the in situ bone tissue of mice in the moringin group, confirming that moringin has the ability to promote the mineralization of mouse bone tissue.

[0096] Runx2 is a specific transcriptional regulator required for osteoblast differentiation. Gene mutation and deletion can lead to bone development disorders and affect intramembranous ossification and endochondral ossification. Runx2 can upregulate the synthesis of extracellular matrix of osteoblasts, accelerate cell proliferation and differentiation, and promote anabolism and osteogenic response. During osteoblast differentiation, Runx2 is upregulated in preosteoblasts, reaches the highest level in immature osteoblasts, and is downregulated in mature osteoblasts. Figure 11 The results of paraffin section staining showed that the expression of Runx2 was significantly enhanced in the in situ bone tissue of mice in the moringin group, indicating that the level of osteogenic differentiation in the tissue was enhanced under the induction of moringin.

Claims

1. Use of moringin in the preparation of a drug for inhibiting the activity of sclerostin protein to promote bone growth, wherein the bone growth drug is a drug for fracture healing and / or anti-osteoporosis.

2. The use according to claim 1, characterized in that Morin is used alone or in combination to prepare medicine.

3. The use according to claim 2, characterized in that The medicine consists of moringin and a pharmaceutically acceptable carrier.

4. The use according to claim 2, characterized in that The medicine contains one or more of a disintegrant, a wetting agent, a binder, a filler, an absorption accelerator, a solvent, a lubricant, a surfactant, a flavoring agent, a sweetener, an antioxidant, a preservative, a pigment, and a transdermal accelerator.

5. The use according to claim 2, characterized in that The dosage form of the medicine is one or more of tablets, capsules, injections, and granules.

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