The Mechanism Of Bushen Huoxue Capsule On BMSCs Osteogenic And Adipogenic Differentiation In The Treatment Of Steroid-induced Osteonecrosis Of Femoral Head Via Hedgehog Signaling Pathway

Objective:In this study,the volume and distribution of necrotic tissue of femoral head in SONFH patients with ARCO?were observed by three-dimensional reconstruction of CT,and the possible targets and mechanisms of Bushen Huoxue capsule in preventing and treating ONFH were predicted based on network pharmacology,which can provide some reference for imaging evaluation of basic experiment and screening of key targets.To explore the mechanism of Bushen Huoxue capsule on osteogenic factors and adipogenic factors and Hedgehog signal pathway related factors in vitro and in vivo by observing the effect of Bushen Huoxue capsule on bone repair and fat formation in GC-related ONFH rats and by observing the effect of Bushen Huoxue capsule containing serum on osteogenesis and adipogenic differentiation of BMSCs treated with GC.Method:1.A three-dimensional reconstruction with CT datas of SONFH patients with ARCO?stage were performed by Mimics,and the femoral head was segmented into eight regions by 3-matic.The volume ratio of necrotic tissue in the femoral head were calculated and the distribution were analyzed.2.The action targets of Bushen Huoxue capsule in Traditional Chinese Medicine related databases and the potential targets of ONFH in disease related database were screened by network pharmacology.The possible targets and mechanism of Bushen Huoxue capsule in preventing and treating ONFH were analyzed by bioinformatics software.3.The SD rat model of GC related ONFH was established by MPS,and different doses of Bushen Huoxue capsule were given.After 6 weeks,Micro-CT and X-ray and histopathological staining and serum biochemical detection and molecular biological techniques were carried out to evaluate the effect of Bushen Huoxue capsule on bone repair and fat formation in GC-related ONFH rats.4.BMSCs were isolated from SD rats.Different doses of Bushen Huoxue capsule containing serum and Dex were used to observe the proliferation of cells.The osteogenesis and adipogenic differentiation of BMSCs in different groups were detected by alizarin red staining and oil red"O"staining.The changes of protein expression of osteogenic factors and adipogenic factors and Hedgehog signal pathway related factors of BMSCs in different groups were analyzed by Western blotting.Results:1.The average volume of femoral head was(48399.52±9408.90)mm~3,and the average volume of necrotic tissue was(20917.08±6566.94)mm~3,and the average volume ratio of necrotic tissue to femoral head was(44.75±15.72)%.The proportion of necrotic volume in different regions was different,and the necrotic tissues were mainly distributed in the anterior superior medial and anterior superior lateral and posterior superior medial regions.2.The key candidate targets for the treatment of ONFH with Bushen Huoxue capsule were identified by network pharmacology,and bioinformatics analysis showed that above targets were related to cell proliferation,chronic inflammation,bone formation,lipid metabolism and vascular regeneration.3.After 6 weeks,Micro-CT showed that the Bushen Huoxue capsule could improve bone loss caused induced by MPS in GC related ONFH rats.HE staining and immunohistochemical staining showed that compared with the model group,the pathological changes of bone trabeculae and fat accumulation were significantly decreased in the low dose group,middle dose group and high dose group of Bushen Huoxue capsule.Serum biochemical test showed that after MPS intervention,the total cholesterol content in serum increased significantly,while the content of ALP decreased significantly.Compared with the model group,Bushen Huoxue capsule could enhance the protein expression of bone morphogenetic factors(Runx2,Collagen I)and Hedgehog signal pathway related factors(Shh,Gli2)in femoral head of SD rats,and decrease the protein expression of fat formation related factors(PPAR?,C/EBP?).4.The Bushen Huoxue capsule containing serum not only could promote the cell proliferation of the normal BMSCs,but also could obviously increase the cell proliferation activity of the BMSCs treated by the dexamethasone.After 3 weeks of bone induction,the positive protein by alizarin red staining in the low dose group,middle dose group and high dose group of Bushen Huoxue capsule containing serum were significantly higher than that of the control group.After 2 weeks of lipid induction,the positive protein by oil red"O"staining in the low dose group,middle dose group and high dose group of Bushen Huoxue capsule containing serum were lower than that of the control group.Bushen Huoxue capsule containing serum and dexamethasone were used to interfere BMSCs for for 72h,and the results of Western blotting showed that Bushen Huoxue capsule could enhance the protein expression of osteogenic factors(Runx2,Collagen I)and Hedgehog signal pathway related factors(Shh,Gli2)in BMSCs,and weaken the protein expression of adipogenic factors(PPAR?,C/EBP?)in BMSCs.Conclusion:1.The volume and distribution of necrotic tissue in femoral head could be evaluated quickly and intuitively by three-dimensional reconstruction in Mimics software.2.The mechanism of Bushen Huoxue capsule in treating SONFH was clarified to a certain extent based on network pharmacology and bioinformatics,which was helpful to optimize the experimental design of subsequent molecular mechanism.3.Bushen Huoxue capsule could promote bone repair and inhibit fat formation in GC related ONFH rats based on Hedgehog signaling pathway.4.The Bushen Huoxue capsule containing serum could promote the osteogenic differentiation of BMSCs and inhibit the adipocyte differentiation of BMSCs through the Hedgehog signaling pathway.