Study On The Mechanism Of Huo Xue Tong Luo Capsule Promoting The Repair Of Steroid-Induced Osteonecrosis Of Femoral Head Based On ERα-Wnt/β-Catenin Pathway

ObjectiveThe Huo Xue Tong Luo(HXTL)capsule,has been found to prevent the collapse and further deterioration of asymptomatic osteonecrosis of the femoral head(ONFH).At present,a large number of clinical studies have proved that HXTL has a good therapeutic effect,but the relevant basic research has not yet clarified the specific mechanism.This study aims to investigate whether HXTL can promote osteogenic differentiation of human bone marrow stromal cells(h BMSCs)through ERα-Wnt/β-catenin pathway,accelerate the repair of femoral head necrosis area,and prevent the occurrence and further deterioration of femoral head necrosis collapse.MethodsIn vitro: Flow cytometry(FCM)was used to detect the expression levels of CD73,CD44,CD29,CD11 b,CD45 and human leukocyte antigen-DR isotype(HLA-DR)in the experimental cells to determine whether the cells were h BMSCs;MTT assay was used to detect the proliferation and activity of h BMSCs in different concentrations of HXTL;Alizarin red staining and Alkaline phosphatase activity assay were used to detect the osteogenesis of h BMSCs under different concentrations of HXTL,so as to determine the optimal concentration of HXTL.In the presence or absence of DKK1,a specific inhibitor of Wnt signaling pathway,and ICI 182780,a high affinity inhibitor of ER: qPCR was used to detect the expression of osteogenic marker gene(Runx2、OPN、COL 1、DLX5、BGLAP and ERα),and the expression of Wnt/β-catenin pathway marker gene(β-catenin、TCF7、LEF1、C-MYC、CYCLIN D and C-JUN)at different stages of osteogenesis;Western-blot was used to detect the expression levels of p-β-catenin and β-catenin;Immunofluorescence staining was used to observe the nuclear translocation of β-catenin in h BMSCs.In vivo: Eighteen SD rats were randomly divided into three groups(n=6): the sham group,SIONFH group and experimental group(SIONFH rats treated with HXTL capsules by gavage).Rats of the SIONFH and experimental groups were modeled using methylprednisolone(MPS)(20 mg/kg/d),which was injected into both hips for 3 days per week for successive 3 weeks.The sham rats were injected with DMSO at the same concentration.Rats in the experimental group were given 0.63 g/kg/d HXTL capsules by gavage from day 3 to day 28,while rats in the sham group and SIONFH group were given the same dose of 0.9% normal saline(NS).All femurs were retrieved at day 28 for histological analysis HE staining and Micro-CT scanning.Rat blood was collected from the abdominal aorta for separating serum samples.ResultsIn vitro experiment: Initially,attached h BMSCs reached 90% confluence and exhibited spindle and flat shapes at 7 days.h BMSCs of passages 2 and 3 reached 90% confluence and were characterized by fish shape at day 3.The results of flow cytometry indicated that 99.9%of cells were positive for CD73,100.0% for CD44 and 99.0% for CD29,whereas 0.3% were negative for CD11 b,0.4% for CD45 and 0.4% for HLA-DR;hence,the majority of the isolated and purified cells were characterized as h BMSCs by marker analysis.To determine whether HXTL capsules have promotional effect on h BMSC proliferation and osteogenic differentiation,cells were cultured in basic medium with vary concentrations HXTL capsules(0,1,5 and 10 μg/ml)for 1,2,3,7 and 14 days.MTT assays indicated that HXTL(10 μg/ml)significantly promoted h BMSC proliferation at above time points(p<0.05).After 14 days culture,we assessed the osteogenic effect of HXTL capsules on h BMSCs.HXTL increased calcium deposition and nodules,especially at a concentration of 10 μg/ml.These results were consistent with those of the ALP activity assay.The results of q PCR showed that 10μg/ml HXTL promoted the m RNA expression of of osteogenic marker gene(Runx2、OPN、COL1、DLX5、BGLAP and ERα),as well as the expression of Wnt/β-catenin pathway marker gene(β-catenin、TCF7、LEF1、C-MYC、CYCLIN D and C-JUN).The expression level of p-β-catenin at three different time points was lower in the HXTL group than those in the control group.And their levels on days 3,7 and 14 could be upregulated by the pretreatment of DKK1 and ICI 182780.In summary,Western-blot results indicated that HXTL enhancedβ-catenin expression through the Wnt/β-catenin pathway.HXTL(10 μg/ml)facilitated the nuclear(blue)translocation of β-catenin(red)in h BMSCs,which was hampered when cells were treated with DKK1 and ICI 182780.In vivo experiment: Micro CT analysis revealed that HXTL significantly attenuated steroid-induced bone loss in the femoral head.In the quantitative analysis,BV/TV,Tb.N and Tb.Th were found to be evidently increased while Tb.Sp was reduced in the SIONFH +HXTL group compared with those in SIONFH group.Furthermore,H&E staining confirmed the suppressive effect of HXTL on osteocyte apoptosis in vivo.As shown by ELISA,HXTL promoted the serum expression of OPG,RANKL,β-catenin and platelet-derived growth factor-BB.In conclusion,HXTL promoted osteogenesis and inhibited osteocyte apoptosis in rats.ConclusionsHXTL capsules have promoting effect on h BMSCs proliferation and osteogenesis in vivo and in vitro,which may relate to the activation of ERα-Wnt signaling pathway along with the accumulation and nuclear translocation of β-catenin.Our work provided a foundation to inspire further clinical studies and facilitate the application of HXTL capsule in treating SIONFH.