Application of orthosilicic acid in prevention and treatment of femoral head necrosis caused by glucocorticoid

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of orthosilicic acid to prevention and treatment of femoral head necrosis caused by glucocorticoid. The invention studies the influence of orthosilicic acid (OSA) on apoptosis and proliferation of human umbilical vein endothelial cells (HUVECs) induced by dexamethasone (Dex), and establishes an SONFH mouse model to evaluate the potential effect of OSA in vivo. The result shows that the OSA has no toxicity to the HUVECs when the concentration is less than 100 mu M, and can reverse Dex-induced HUVECs apoptosis. The OSA promotes the oxidative stress resistance of the HUVECs in vitro and promotes osteoblast differentiation. Dex can mediate HUVECs apoptosis by inducing endoplasmic reticulum stress, and OSA can antagonize Dex-related endoplasmic reticulum stress by promoting Akt phosphorylation. In vivo, the OSA can protect the HUVECs from being damaged by Dex, and the femoral head necrosis induced by Dex is reversed.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of orthosilicic acid in preventing and treating femoral head necrosis caused by glucocorticoid. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Femoral head necrosis, also known as avascular necrosis of the femoral head, is caused by a variety of reasons, resulting in poor local microcirculation due to ischemia of the femoral head, causing the femoral head to lose its normal shape, manifested as trabecular fracture and femoral head collapse. Glucocorticoid (GC)-induced femoral head necrosis (SONFH) is caused by long-term use of GC, leading to avascular necrosis of the femoral head. GC is the most widely used and effective anti-inflammatory and immunosuppressant in clinical practice, but GC can cause serious damage to bones (such as femoral head necrosis and osteoporosis). The mechanism by which GC induces femoral head necrosis is still unclear, but it is known to be related to impaired angiogenesis and weakened osteogenesis. Therefore, enhancing angiogenesis and osteogenesis may help prevent or treat SONFH.

[0004] Silicon (Si) is one of the essential trace elements for the human body. For humans, it mainly comes from food, among which orthosilicic acid (OSA, Si(OH)4) is the most valuable form. Studies have shown that Si has a positive effect on maintaining bone homeostasis and regeneration. It can enhance the osteogenic capacity of preosteoblasts for the treatment and prevention of osteoporosis and alleviate the damage of oxidative stress to cells. However, the pathophysiological role of Si in SONFH has rarely been studied. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the object of the present invention is to provide an application of orthosilicic acid in preventing and treating femoral head necrosis caused by glucocorticoids.

[0006] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0007] In a first aspect, the present invention provides the use of orthosilicic acid in the preparation of a drug for preventing and treating femoral head necrosis caused by glucocorticoids.

[0008] Preferably, the drug prevents and treats glucocorticoid-induced femoral head necrosis by promoting angiogenesis and osteogenic differentiation.

[0009] Preferably, the promotion of angiogenesis and osteogenic differentiation is achieved through the following pathways:

[0010] Antagonize the inhibitory effect of glucocorticoids on endothelial cell proliferation;

[0011] Reversing the inhibitory effect of glucocorticoids on endothelial cell migration;

[0012] Reversing the inhibitory effect of glucocorticoids on osteogenic differentiation;

[0013] Antagonizes glucocorticoid-induced endothelial cell apoptosis;

[0014] Promoting Akt phosphorylation alleviates glucocorticoid-associated endoplasmic reticulum stress-mediated cell apoptosis.

[0015] Preferably, the reversal of the inhibitory effect of glucocorticoid on endothelial cell migration includes one or more of increasing the migration area of ​​endothelial cells, increasing the number of endothelial cells that complete migration, and increasing the expression of VEGFA and CD31.

[0016] Preferably, the reversal of the inhibitory effect of glucocorticoid on osteogenic differentiation comprises increasing the expression of ALP and Runx 2.

[0017] Preferably, the antagonizing of glucocorticoid-induced endothelial cell apoptosis comprises inhibiting the expression of cleaved-caspase-3 and Bax.

[0018] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0019] Preferably, the dosage form of the drug is an oral preparation or an injection preparation.

[0020] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0021] The present invention established a SONFH mouse model to evaluate the potential effects of OSA in vivo. The results showed that OSA effectively improved the sparse and disordered trabeculae and damaged bone marrow microcirculation in the SONFH mouse model, and increased the positive counts of CD31, VEGFA, and Runx2.

[0022] The present invention uses human umbilical vein endothelial cells for cell experiments, and the results show that OSA is non-toxic to HUVECs at a concentration below 100 μM, and can reverse Dex-induced HUVECs apoptosis. OSA promotes the anti-oxidative stress ability of human umbilical vein endothelial cells in vitro and promotes osteoblast differentiation. Glucocorticoids can mediate HUVECs apoptosis by inducing endoplasmic reticulum stress, and OSA can antagonize Dex-related endoplasmic reticulum stress by promoting Akt phosphorylation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 : In the figure are high-resolution images of the femoral heads of the control group (Control), MPS group, and MPS+OSA group in Example 1, and comparison diagrams of the bone surface and bone volume (BS / BV), bone volume fraction (BV / TV), number (Tb.N), trabecular thickness (Tb.Th), and bone mineral density (BMD);

[0025] Figure 2 A is the H&E staining image of the femoral head of the control group (Control), MPS group, and MPS+OSA group in Example 1; B is the IHC staining image of the femoral head of the control group (Control), MPS group, and MPS+OSA group in Example 1;

[0026] Figure 3 A in the figure is the cell counting result in the culture medium containing different OSA concentrations in Example 2, and B is the cell counting result of the control group (Control), Dex group, Dex+OSA group, and OSA group in Example 2;

[0027] Figure 4 This is a graph showing the results of observing HUVECs migration in the scratch test in Example 2;

[0028] Figure 5 This is a diagram showing the results of observing HUVECs migration in the Transwell assay in Example 2;

[0029] Figure 6 A is a graph showing the results of the in vitro vascularization experiment in Example 2, and B is a graph showing the comparison of the expression levels of VEGFA and CD31 in Example 2;

[0030] Figure 7 A is an ALP staining image after osteogenic induction in Example 2, B is an Alizarin red staining image after osteogenic induction in Example 2, and C is a comparison of the expression levels of ALP and Runx 2 after osteogenic induction in Example 2;

[0031] Figure 8 A is a graph showing the results of mitochondrial membrane potential detection in Example 2, B is a graph showing the results of TUNEL test in Example 2, C is a graph showing the results of AnnexinV-FITC / PI double staining by flow cytometry in Example 2, and D is a graph showing the expression comparison of cleaved-caspase-3 and Bax in Example 2;

[0032] Fig. 9 A is a microscopic image of endothelial cells in Example 2, and B is a graph showing the determination of ROS content in endothelial cells in Example 2;

[0033] Fig.10A is a comparison chart of the expression levels of p-PERK, p-Eif-2α, and CHOP in Example 2, and B is a comparison chart of the phosphorylation level of Akt in Example 2;

[0034] Fig.11 A is the result of detecting the expression of p-Akt and VEGFA by cell immunofluorescence technology in Example 2, B is the result of Transwell test in Example 2 verifying the promoting effect of MK-206 in inhibiting OSA, and C is the result of angiogenesis test in Example 2 verifying the promoting effect of MK-206 in inhibiting OSA. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0036] Example 1

[0037] Thirty mice aged 8-10 weeks were purchased and raised under standard conditions. The Si concentration in the basal feed was about 2 μg Si / g, and the concentration soluble in drinking water was less than 0.01 mg / L. Methylprednisolone (MPS Ruihui USA) was used to establish the SONFH model. They were divided into a control group, an MPS group, and an MPS+OSA group. To establish the SONFH model, lipopolysaccharide (LPS) was first used for continuous intraperitoneal injection at 20 μg / kg for 3 days. Then 100 mg / kg of MPS was injected intraperitoneally for 4 consecutive weeks, and then allowed to progress naturally for 4 weeks. The MPS+OSA group was injected intragastrically with MPS+OSA (10 ml / kg / d monomethylsilyl triol (MMST) (LLR-G5 Ltd., Castlebar, Ireland) every day, in which the Si concentration was 0.117 mg / ml), and the MPS group was injected with an equal volume of PBS. After continuous injection of MPS+OSA for four weeks and then allowed to progress naturally for 4 weeks, the mice were euthanized. The proximal femur was carefully isolated and fixed for Micro-CT analysis, eosin (HE) staining, and immunohistochemistry (IHC) staining.

[0038] High-resolution images of the femoral head were obtained using a Quantum GX microCT scanner (PerkinElmer Inc. Waltham, MA). At the same time, bone surface area to bone volume (BS / BV), bone volume fraction (BV / TV), number (Tb.N), trabecular thickness (Tb.Th), and bone mineral density (BMD) values ​​were obtained using Analyze 12.0 (PerkinElmer Inc. Waltham, MA).

[0039] The harvested femurs were fixed with paraformaldehyde for 24 h at 4 °C and decalcified with 10% EDTA for 21 days at room temperature. The specimens were embedded in paraffin, cut into 5 μm sections, deparaffinized in xylene, rehydrated through a series of graded ethanol, and rinsed in distilled water. Masson and HE were performed and histological observations were performed. Analysis of osteocytes and lacunae was performed as described previously. Histological sections were observed under an Olympus VS120 (Olympus, Japan) using a 40× objective. Images were captured and analyzed using Image J software (NIH, Bethesda, MD, USA; https: / / imagej.nih.gov / ij / ), and the surface of the bone was manually outlined. The following primary antibodies were used for immunohistochemistry: anti-CD31 (1:400), anti-VEGFA (1:400), and anti-Runx2 (1:400).

[0040] like Figure 1 As shown in the figure, the MPS group developed typical subchondral cystic changes, and the bone mineral density (BMD), relative bone volume (BV / TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) indicators were severely reduced. Compared with the control group, the nucleus-trabecular separation (Tb.Sp) and bone surface / bone volume (BS / BV) in the MPS group increased. However, compared with the MPS group, the BMD, BV / TV, Tb.N and Tb.Th of the MPS+OSA group were higher, while Tb.Sp and BS / BV were lower. As shown in the figure, the MPS group had a lower bone density (BMD), relative bone volume (BV / TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th). Figure 2 Figure A shows that decalcified tibia sections were stained using H&E staining, and it was observed that the trabeculae in the MPS group were sparse and structurally disordered, and the microcirculation in the bone marrow cavity was impaired, while this condition was improved in the MPS+OSA group. Figure 2 As shown in Figure B, IHC staining also showed that the number of CD31, VEGFA, and Runx2 positive in the MPS group was significantly lower than that in the control group, while the number of positive in the MPS+OSA group was increased. These results indicate that OSA plays an important role in the treatment of MPS-induced femoral head necrosis.

[0041] Example 2

[0042] Human umbilical vein endothelial cells (HUVECs) were purchased from Zhongyang Yuanzhou Company, and mouse preosteoblastic cells (MC3T3-E1) were purchased from Zhongqiao Xinzhou Company. HUVECs were co-cultured with ECM medium, to which 5% fetal bovine serum, 1% endothelial cell growth supplement, and 1% penicillin / streptomycin solution were added. MC3T3-E1 was cultured in MEM medium, supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin solution. 10 μM Dex was added to the culture medium as the Dex group to detect the effects on cell proliferation, apoptosis, angiogenesis ability and osteogenesis ability. 10 μM Dex+20 μM OSA was added to the culture medium as the Dex+OSA group to detect the antagonistic effect of OSA on Dex damage. The culture medium without Dex or OSA was used as the control group, and 20 μM OSA was added to the culture medium as the OSA group.

[0043] 1. Orthosilicic acid is non-toxic to HUVECs and can antagonize the side effects of glucocorticoids on endothelial cells

[0044] Sodium silicate (Sigma-Aldrich, Germany) was diluted with ultra-high purity water to prepare orthosilicic acid (OSA) stock solution. MEM and ECM culture media containing different concentrations of orthosilicic acid (10μM, 20μM, 50μM, 80μm, 100μM) were prepared to observe the promoting effect of orthosilicic acid concentration on human umbilical vein endothelial cells. The pH value of the culture medium was adjusted to the normal range using hydrochloric acid.

[0045] Cell counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was used to detect cell activity. 2000 HUVECs were seeded in each well of a 96-well plate, and the cells were cultured according to the pre-set groups (control group, Dex group, Dex+OSA group, and OSA group). At 0, 24, 48, and 72 hours, 10% CCK-8 reagent was added to the culture medium and incubated at 37°C for two hours. The optical density (OD) at 450 nm was measured using an ELISA reader (BioTek, USA), and the OD difference between the cell-containing culture medium and the blank culture medium reflected the cell viability.

[0046] The proliferation capacity of HUVECs was detected using Cell-LightTM EdU Apollo In Vitro Kit (Guangdong Ruibo). The cells were inoculated in ECM culture medium and given different interventions according to the previously set groups. After continuous culture for 48 hours, the cells were stained according to the instructions. Then they were counterstained with DAPI. The results were observed using a fluorescence microscope.

[0047] like Figure 3As shown in Figure A, it was observed that orthosilicic acid at a concentration below 100 μM had no toxicity to HUVECs, and OSA at a concentration of 30 μM had the strongest promoting effect on HUVECs at 48 hours. Figure 3 As shown in B, the control (NC) group, Dex group, Dex+OSA group, and OSA group were compared to test the effect of OSA on HUVECs after Dex induction. The results showed that Dex (10 μM) could significantly inhibit the proliferation of HUVECs, while orthosilicic acid (30 μM) could promote the proliferation of HUVECs and antagonize the inhibitory effect of Dex (10 μM) on the proliferation of HUVECs.

[0048] HUVECs were seeded in six-well plates and cultured to full confluence with different treatments. A 200 μL pipette tip was used to make scratches with a width of about 1 mm. The detached cells were rinsed with PBS, serum-free medium was added, and photos of the same position were taken under a microscope at 0, 6, and 12 hours.

[0049] After pretreatment, 2×10 4 Cells were seeded on the upper layer of the chamber, and complete culture medium was placed on the lower layer of the chamber as a chemical attractant. After incubation for 24 hours, the cells on the upper surface were fixed with 4% paraformaldehyde and wiped off. The cells on the lower surface were stained with crystal violet staining solution (Biyuntian China) for 10 minutes, and the excess dye was washed off. Five areas were randomly selected under a microscope for observation and photography.

[0050] Matrigel (Corning, China) was fixed in a 96-well plate according to the instructions. HUVECs were pretreated with different reagents and plated at 2 × 10 4 The cells were inoculated on the surface of matrix gel and the angiogenesis was observed under a microscope 3 hours later.

[0051] The main stage of vascularization is the migration of endothelial cells, so the effects of Dex and OSA on the migration of HUVECs were determined by scratch test and Transwell test. Figure 4 As shown in the figure, compared with the control group, Dex significantly inhibited the migration ability of HUVECs, resulting in a decrease in the migration area, and the migration area increased significantly after adding OSA. The results of the Transwell test were similar to those of the scratch test. Figure 5 As shown in Figure 2, compared with the control group, the number of cells that completed migration in the Dex group was significantly decreased, while OSA reversed this situation. Figure 6As shown in A, compared with the control group, the number of Dex-composed rings and nodes was significantly reduced, and the inhibition of angiogenesis by Dex was significantly rescued after co-treatment with OSA. CD31, as a specific marker of vascular endothelial cells, participates in maintaining the structural integrity of vascular endothelial cells. VEGFA is a specific mitogen for vascular endothelial cells, which can promote the growth of vascular endothelial cells in vitro and induce angiogenesis in vivo. The protein expression levels of VEGFA and CD31 in HUVECs were detected by Western blotting. Figure 6 As shown in B, the results showed that the expression of VEGFA and CD31 was significantly decreased after Dex induction, while the expression of VEGFA and CD31 was increased after OSA treatment.

[0052] 2. Orthosilicic acid can reverse the inhibitory effect of glucocorticoids on osteogenic differentiation

[0053] BMMSCs were seeded in 6-well plates and cultured in osteogenic differentiation medium containing different treatments. ALP activity was determined using an ALP staining kit (Beyotime, Shanghai, China) after 14 days of continuous culture. Alizarin Red S (Solabio, Beijing, China) was used for staining after 21 days of culture. Five areas were randomly selected under a microscope for photography.

[0054] Impaired osteoblast differentiation is an important part of SONFH. In order to evaluate the potential effect of OSA on osteogenic differentiation after Dex induction, osteogenic induction solution was used for induction according to the previous grouping. Figure 7 As shown in A and B in Figure 1, ALP staining and Alizarin red staining results showed that osteogenic differentiation ability was impaired after Dex induction, while OSA treatment reversed this effect. Figure 7 As shown in (C), similar results were obtained by western blotting, where Dex reduced the expression of ALP and Runx 2, while OSA reversed the effect.

[0055] 3. Orthosilicic acid can antagonize glucocorticoid-induced endothelial cell apoptosis

[0056] HUVECs were seeded in 24-well plates and, after pretreatment, the integrity of the mitochondrial membrane was detected using a mitochondrial membrane potential detection kit (JC-10) (Solaibao, China) according to the instruction manual. The results were observed using a fluorescence microscope.

[0057] Cells were seeded in 24-well plates and cultured with different interventions. TUNEL Bright Red Apoptosis Detection Kit (Vazyme, China) was used to detect cell apoptosis levels. Five areas were randomly selected under an inverted fluorescence microscope and photographed, and the images were analyzed using image J software.

[0058] Cells were cultured in 6-well plates with different interventions, and the cells were collected and washed with phosphate-buffered saline (PBS). The apoptosis of HUVECs was determined using the Annexin V-FITC apoptosis detection kit (Vazyme, China) according to the instruction manual. Flow cytometry was used for analysis.

[0059] The primary antibodies used in western blotting experiments were anti-Runx2 (1:1000), anti-ALP (1:1000), anti-caspase-3 (1:1000), anti-Bad (1:1000), anti-p-Bad (1:1000), anti-Bax (1:1000), anti-Bcl-2 (1:1000), anti-Cleaved-caspase-3 (1:1000), anti-PERK (1:1000), anti-p-PERK (1:1000), anti-p-eIF2α (1:1000), anti-CHOP (1:1000), anti-Akt (1:1000), anti-p-Akt (1:1000), anti-CD31 (1:1000), and anti-β-actin (1:10000) (Cell Signaling Technology, USA), anti-VEGFA (Abcam, Cambridge, UK).

[0060] Dex-induced apoptosis of HUVECs and the antagonistic effect of OSA were evaluated by detecting mitochondrial membrane potential, TUNEL assay, annexin VFITC / PI double staining, flow cytometry, and protein immunoblotting. The decrease in mitochondrial membrane potential is a landmark event in the early stage of apoptosis, and the transition from red fluorescence to green fluorescence is used as a detection indicator of early apoptosis. Figure 8 As shown in Figure A, compared with the control group, the green fluorescence ratio in the Dex group was significantly higher and the red fluorescence ratio was lower, while OSA significantly improved the decline in mitochondrial membrane potential. Figure 8 As shown in Figure B, the TUNEL assay showed that the number of apoptotic cells in the Dex group was significantly higher than that in the control group. On the contrary, the treatment of OSA antagonized the apoptotic effect. Figure 8 As shown in Figure C, similar results were obtained by flow cytometry using AnnexinV-FITC / PI double staining. The apoptosis in the Dex group was more significant than that in the control group, while the number of apoptotic cells was significantly reduced after the addition of OSA. Figure 8 As shown in D, western blotting showed that Dex activated the expression of cleaved-caspase-3 and Bax, and the addition of OSA inhibited the expression of cleaved-caspase-3 and Bax.

[0061] 5. OSA promotes Akt phosphorylation to alleviate glucocorticoid-related endoplasmic reticulum stress-mediated apoptosis

[0062] Endothelial cells were collected after being given different treatments according to the previous groups. According to previous studies, the samples were fixed, dehydrated, embedded, polymerized, sliced, and stained in sequence, and then observed under an electron microscope to capture the subcellular structure of the cells.

[0063] After pretreatment, the endothelial cells were tested for intracellular reactive oxygen species using a reactive oxygen species detection kit (Biyuntian China) and observed under a fluorescence microscope. Five areas were randomly selected for photography.

[0064] The primary antibodies used in western blotting experiments were anti-Runx2 (1:1000), anti-ALP (1:1000), anti-caspase-3 (1:1000), anti-Bad (1:1000), anti-p-Bad (1:1000), anti-Bax (1:1000), anti-Bcl-2 (1:1000), anti-Cleaved-caspase-3 (1:1000), anti-PERK (1:1000), anti-p-PERK (1:1000), anti-p-eIF2α (1:1000), anti-CHOP (1:1000), anti-Akt (1:1000), anti-p-Akt (1:1000), anti-CD31 (1:1000), and anti-β-actin (1:10000) (Cell Signaling Technology, USA), anti-VEGFA (Abcam, Cambridge, UK).

[0065] In order to study the mechanism of Dex-induced endothelial cell damage and OSA-induced endothelial cell rescue, the microstructure of endothelial cells was observed under transmission electron microscopy. Fig. 9 As shown in A, compared with the control (NC) group, the ER in the Dex group was significantly swollen, while the ER morphology in the Dex+OSA group was improved. Changes in ER morphology are often accompanied by increased intracellular reactive oxygen species (ROS) content. Fig. 9 This was demonstrated in Figure B. To determine whether Dex causes ER stress, protein immunoblotting was used to detect ER stress marker proteins p-PERK, p-Eif-2α, and CHOP. Fig.10 As shown in A, the above marker proteins were significantly increased in the Dex group, while OSA reversed this effect. As an effective inhibitory signal of cell apoptosis, Akt can antagonize apoptosis in many ways after being phosphorylated. Therefore, the changes of p-Akt when HUVECs underwent ERS were detected. Fig.10As shown in B, after Dex induction, there was no significant change in the phosphorylation level of Akt, but the ERS marker protein was upregulated. This effect could be reversed by GSK2656157 (PERK inhibitor). When OSA was added, the phosphorylation level of Akt increased, the expression of p-PERK and CHOP decreased, the expression of its downstream Bcl-2 increased, and cell apoptosis was inhibited. After the addition of MK-206 (Akt inhibitor), the therapeutic effect of OSA was reversed. This shows that OSA antagonizes endoplasmic reticulum stress-mediated apoptosis by promoting Akt phosphorylation. In addition, in order to verify the role of Akt in angiogenesis, the expression of p-Akt and VEGFA was detected by cell immunofluorescence technology. As shown Fig.11 As shown in A, after using OSA, the expression of p-Akt increased, and the expression of VEGFA also increased. Transwell test ( Fig.11 B) and angiogenesis assay ( Fig.11 Similar results were obtained in C). The promoting effect of OSA could be inhibited by MK-206. In summary, Dex can mediate HUVECs dysfunction by inducing ER stress in HUVECs, while OSA can reverse apoptosis and enhance HUVECs function by activating Akt.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of orthosilicic acid in the preparation of drugs for preventing and treating femoral head necrosis caused by glucocorticoids.

2. The use according to claim 1, characterized in that The drug prevents and treats femoral head necrosis caused by glucocorticoid by promoting angiogenesis and osteogenic differentiation.

3. The use according to claim 2, characterized in that The promotion of angiogenesis and osteogenic differentiation is achieved through the following pathways: Antagonize the inhibitory effect of glucocorticoids on endothelial cell proliferation; Reversing the inhibitory effect of glucocorticoids on endothelial cell migration; Reversing the inhibitory effect of glucocorticoids on osteogenic differentiation; Antagonizes glucocorticoid-induced endothelial cell apoptosis; Promoting Akt phosphorylation alleviates glucocorticoid-associated endoplasmic reticulum stress-mediated cell apoptosis.

4. The use according to claim 3, characterized in that The reversal of the inhibitory effect of glucocorticoid on endothelial cell migration includes one or more of increasing the migration area of ​​endothelial cells, increasing the number of endothelial cells that complete migration, and increasing the expression of VEGFA and CD31.

5. The use according to claim 3, characterized in that The reversal of the inhibitory effect of glucocorticoid on osteogenic differentiation includes increasing the expression of ALP and Runx 2.

6. The use according to claim 3, characterized in that The antagonism of glucocorticoid-induced endothelial cell apoptosis includes inhibiting the expression of cleaved-caspase-3 and Bax.

7. The use according to claim 1, characterized in that The drug further comprises pharmaceutically acceptable excipients.

8. The use according to claim 1, characterized in that The dosage form of the drug is an oral preparation or an injection preparation.

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