| Objective:1.To observe the clinical efficacy of Bushenhuoxue capsule in the treatment of SONFH by clinical efficacy evaluation methods and imaging evaluation methods.2.To explore the mechanism of Bushenhuoxue capsule in promoting the repair of SONFH by the differences in gene and protein expression of the master related factors and the downstream specific transcription factors of Wnt/?-Catenin signaling pathway.These transcription factors can promote proliferation(Cyclin D1),osteogenic-adipogenic differentiation(Runx2?Osterix?PPAR?),angiogenesis(VEGF)and so on.These transcription factors and proteins are derived from the Sprague-Dawley rats' femoral heads of intervention by dexamethasone and Bushenhuoxue capsule,and from the bone marrow mesenchymal stem cells in vitro and vivo of these SD rats.Methods:1.Clinical research:80 patients who were from orthopedic clinic of Shandong University of Traditional Chinese Medicine Hospital and in ARCO stage I or II of SONFH were randomly divided into control group(CG)and treatment group group(TG).40 cases were in each group.The CG was treated with Xianlinggubao capsule,and the TG with Bushenhuoxue capsule.To evaluate the clinical efficacy and drug safety by the clinical evaluation methods(Harris scale? WOMAC scale)? imaging evaluation methods(femoral head necrosis area evaluation ? femoral head collapse evaluation method)and liver-kidney function tests.These evaluation occurred before treatment and 3 months? 6 months? 9months and 12 months after treatment.2.Animal experimental research:1)In-vivo experiment:120 SD rats were randomly divided into control group? model group and treatment group and 40 in each group.The animal model of SONFH was established by injecting dexamethasone sodium phosphate into gluteal muscles.The treatment group was intervened with Bushenhuoxue decoction gavage,the other two groups were intervened with normal saline.After 6 weeks,the femoral head of 5 rats were randomly selected to observe the changes in each group by HE staining.After 12 weeks,all rats were sacrificed to obtain specimens(blood,femoral head tissue,bone marrow mesenchymal stem cells)for detection.The efficacy of Bushenhuoxue capsule could be further observed by the differences of triglyceride?cholesterol?serum calcium?alkaline phosphatase and bone trabecula?empty bone lacuna rate?fat vacuole contents.To observe the gene and protein expression differences of the Wnt/?-Catenin signaling pathway related downstream factors(Wnt10b??-Catenin?Runx2?VEGF?Osterix?PPAR?)by the RT-PCR and Western Blot.The proliferation of P3 r BMSCs of three groups rats was detected by CCK-8.The differences of osteogenic-adipogenic differentiation were observed by alizarin red staining ?oil red O staining and ALP quantitative detection after inducted.And the differences of gene and protein expression were detected by the RT-PCR and Western Blot.The mechanism of Bushenhuoxue recipe promoting bone repair of SONFH,which was based on Wnt/ ?-Catenin signaling pathway,was revealed and explored from tissue?cell?protein and nucleic acid levels.2)In-vitro experiment:The r BMSCs were cultured?purified and identified in vitro.The P3 generation r BMSCs were used in the experiment.And they were intervened with10-6mol/L dexamethasone sodium phosphate and different concentrations of Bushenhuoxue decoction serum.They were divided into eight groups(CG?LG?MG?HG?DG?LDG?MDG?HDG).The proliferation of P3 r BMSCs of 8 groups was detected by CCK-8,and the differences of osteogenic differentiation were observed by alizarin red staining and alkaline phosphatase quantitative detection.And the differences of specific gene and protein expression were detected by the RT-PCR and Western Blot.The mechanism could be further revealed and explored.Results:1.The results of clinical experiments:Before treatment,The results of two groups were no significant difference(P>0.05)by Harris and WOMAC scales.After 3months' treatment,they were no significant difference(P>0.05).And they were significant difference(P<0.05)after 6?9?12 months' treatment.There were statistical significance before and after treatment in each group after 3? 6?9?12 months' treatment.There was no significant difference in the calculated area of necrosis between the two groups before treatment(P> 0.05).There was no significant difference between the two groups of MRI calculation of the necrosis area after 3 months?6 months?9 months and 12 months of treatment(P> 0.05).There was no significant difference in the both group after 3 ? 6?9and 12 months compared with before treatment(P> 0.05).At the end of the experiment,the result of the evaluation of femoral head collapse was significant difference(P<0.05)by Chi-square and Log-rank tests.The drugs were safty.2.The results of animal experiments:1)In-vivo results:(1)The results of blood biochemical test:Compared with CG,the results of TC?TG?Ca and ALP in MG were obviously difference(P<0.05).Compared with MG,the results of TC?TG?Ca and ALP in TG were obviously difference(P<0.05).(2)The results of femoral head tissue test:HE staining:Compared with CG,the results of trabecular bone area?fat vacuole area and the ratio of empty lacuna in MG were obviously difference(P<0.05).Compared with MG,the results of trabecular bone area ?fat vacuole area and the ratio of empty lacuna in TG were obviously difference(P<0.05).RT-PCR and Western Blot:Comparing with CG,the m RNA and protain of specific transcription factors(Wnt10b??-Catenin ?Runx2?Osterix?VEGF)were obviously difference(P<0.05)in MG.Comparing with MG,the m RNA and protain of specific transcription factors(Wnt10b??-Catenin ?Runx2?Osterix?VEGF)were obviously difference(P<0.05)in TG.(3)The results of r BMSCs function test:Identification results of surface antigen:Compared with CG,the results of surface antigens(CD45 ? CD73 ? CD90 ? CD105)in MG were obviously difference(P<0.05).Compared with MG,the results of surface antigens in TG were obviously difference(P<0.05).The test method was FITC staining. The results of r BMSCs Proliferation: Comparing MG with CG and TG with MG,they were obviously difference(P<0.05)by CCK-8.And the expression of gene and protain of the specific factors(Wnt3a??-Catenin ?Cyclin D1)were obviously difference (P<0.05)by RT-PCR and Western Blot.The results of r BMSCs osteogenic-adipogenic induction:Compared with CG,the results of osteogenic-adipogenic differentiation in MG were obviously difference(P<0.05)by alizarin red staining?oil red O staining and ALP quantitative detection.And the expression of gene and protain of the related factors and downstream specific factors(Wnt10??-Catenin ?Runx2?Osterix?PPAR-?)of Wnt/?-Catenin signaling pathway were obviously difference(P<0.05)by RT-PCR and Western Blot.Compared with MG,the results of osteogenic-adipogenic differentiation in TG were obviously difference(P<0.05)by alizarin red staining ? oil red O staining and ALP quantitative detection.And the expression of gene and protain of the related factors and downstream specific factors of Wnt/?-Catenin signaling pathway were obviously difference(P<0.05)by RT-PCR and Western Blot.2)In-vitro results:(1)The proliferation results by CCK-8:Without DG,the proliferation curve of other7 groups r BMSCs were "S" shapes.Compared with CG and DG,the proliferation of other groups were difference(P<0.05),and the difference was related with concentration.(2)The results of ARS and ALP:Compared with CG and DG,the results of ARS and ALP of other groups were difference(P<0.05),and the difference was related with concentration.(3)The results of RT-PCR and Western Blot:Compared with CG and DG,the expression of gene and protain of specific factors(?-Catenin ?Runx2?PPAR-?)of other groups were difference(P<0.05)by RT-PCR and Western Blot,and the difference was related with concentration.Conclusion:1.Clinical studies have shown that Bushenhuoxue capsule has the significant effect on SONFH.It can significantly relieve the clinical symptoms of patients with SONFH and delay necrosis and collapse of the femoral head,and no obvious liver and kidney dysfunction.2.The results of animal experiments in blood biochemistry and HE staining show that Bushenhuoxue capsule has a exactly therapeutic effect on SONFH.3.The mechanism of Bushenhuoxue capsule treating SONFH was possible achieved by regulating the balance of osteogenenic-adipogenic differentiation,and by regulating gene and protein expression of the related factors(Wnt10b,?-Catenin)and downstream specific transcription factors(Runx2,Osterix,VEGF,PPAR-?)of Wnt/?-Catenin signaling pathway.4.The results of r BMSCs in vitro and vivo showed that the Bushenhuoxue capsule could significantly promote the proliferation,osteogenic differentiation and inhibit adipogenic differentiation of r BMSCs,and the mechanism may be related to the gene and protein expression of the major Wnt signaling pathway(Wnts,?-Catenin)and downstream related specific transcription factors(Runx2?PPAR-??Cyclin D1). |
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