| Objective:this is a research on the intervention of traditional Chinese medicine Bone Reconstruction Pill on early steroid-induced avascular necrosis of the femoral head,SANFH in rat model and bone marrow derived mesenchymal stem cells,BMSCs after osteogenic-adipogenic induction,,to explore the influence mechanism of Bone Remodeling Pill on SANFH and related bone factor in vitro osteogenic differentiation of BMSCs,and signaling pathway of Wnt/β-catenin;explain the mechanism of"activating Yang,promoting blood circulation,removing dampness"method in traditional Chinese medicine to prevent and treat SANFH,and provide a theoretical basis for the clinical application of Bone Reconstruction Pill.Methods:1.In vivo experiments:（1）Forty Wistar rats were randomly divided into 5 groups:Bone Reconstruction Pill high solution group,Bone Reconstruction Pill medium solution group,Bone Reconstruction Pill low solution group,positive control group（Xianlinggubao capsule solution[23]）,blank group（0.9%normal saline）.After accurate weighing,refering conversion formula:A（body surface area m2）=K（coefficient）x W（weight）2/3 x 10-4,we converted the dosage of human to rat for in tragastric administration.The blood samples were collected from the heart in aseptic environment after 5 days of intragastric administration,one time in the morning and one in the evening,and 1 hour after the total daily quantity adminis tration within the last 24 hours.The blood samples of each group were storedat-20℃.2.II.In vitro experiments:（1）BMSCs were isolated and identified.Osteogenic induction and lipogenic inducer were used to induce osteogenic fat-forming of BMSCs,and BMSCs osteogenic fat-forming was identified by oil red"O"staining and alizarin red staining.After induced successfully,the cells were intervened with dexamethasone for 72 hours,and then divided into blank group,model group,high,medium and Bone Reconstruction Pill low serum concentration group,blank serum group and Xianling Gubao capsule serum group.Morphological changes of cells were observed by inverted microscope.The activity of BMSCs and the content of alkaline phosphatase（AKP）were detected by MTT method to observe the osteogenic differentiation of BMSCs and to determine the best serum concentration of Bone Reconstruction Pill.（2）The test（1）cells were divided into Bone Reconstruction Pill best medicated serum group,blank serum group and Xianling Gubao capsule positive control serum group,select the appropriate serum to intervene,by using Western Blotting,WB,Polymerase Chain Reaction,PCR the detection of related osteog enic factor protein and gene in the Wnt/β-catenin signaling pathway was conducted.The inquiring into the mechanism of Bone Reconstruction Pill in the treatment of SANFH from the aspect of osteogenesis was carried out.Results:In vitro experimental results（1）Comparison of alizarin red staining:BMSCs alizarin red staining was negative before osteogenesis induction and BMSCs alizarin red staining was positive after osteogenesis induction.（2）Comparison of oil red "O" staining:before osteogenesis induction,BMSCs oil red"O"staining did not appear red fat particles,after osteogenesis induction BMSCs oil red"O"staining appeared a large number of fat particles.（3）The results of AKP showed that there was a difference between the low concentration Bone Reconstruction Pill medicated serum group and the positive control serum group comparing with the blank control serum group,and both of them were higher than those of the blank control serum group.Both Xianling Gubao capsule and Bone Reconstruction Pill promoted BMSCs osteogenic differentiation（P<0.05）,and the effect of them was similar（P>0.05）,and there was a difference between the low concentration group and the high and medium concentration group of Bone Reconstruction Pill,and with the decrease of serum concentration,the ability of promoting osteogenic differentiation of BMSCsc was enhanced（P<0.05）.（4）The results of BMSCs activity:there were differences between the low concentration group and the high and medium concentration group,and were higher than those in the medium and high concentration groups.The effect of Bone Reconstruction Pill on osteogenic differentiation of BMSCs was related to serum concentration（P<0.05）,and the cell activity of 72h was significantly different from that of 48h and 24h when serum concentration was diluted at 0%,2.5%,5%,10%,15%,respectively.The promoting effect of Bone Reconstruction Pill on osteogenic differentiation of BMSCs was enhenced with the prolongat ion of time（P<0.05）.The cell activity was different from that of 0%,10%an d 15%when the dilution ratio was 2.5%,and the different serum multiple dilution of Bone Reconstruction Pill had different promoting effects on BNSCs（P<0.05）.（5）The expression of osteogenic related factor mRNA in Wnt/β-catenin signaling pathways in BMSCs:mRNA of BMSCs osteogenic differentiation related promoting factorβ-catenin and Wnt showed a rising trend in the Wnt/β-catenin signaling pathway（P<0.05）,and the m RNA of BMSCs osteogenic differentiation inhibiting Dickkopf-1 showed the trend of decreasing（P<0.05）,and Bone Reconstruction Pill and Xianling Gubao capsule had a similar effect of promoting osteogenic differentiation（P>0.05）.β-catenin at 12,24,48h time point,there was a difference of Bone Reconstruction Pill low concentration serum group conmparing with the positive control group and the blank control group,and it was higher than that of the two groups,the difference was statistically significant.（P<0.05）.Wnt1 at 6,12,24h time point,there was a difference of the Bone Reconstruction Pill low concentration serum group comparing with the positive control group and the blank control group,and it was higher than that of the two groups,difference was statistically significant（P<0.05）.DKK1 at 6,12,48h time point,there was a difference of positive control serum group and blank control serum group comparing with the low concentration serum group and higher then it,the difference is statistically significant.（P<0.05）.（6）The expression of osteogenic related factor protein in the Wnt/β-catenin signaling pathway in BMSCs:BMSCs osteogenic differentiation related promoting factorβ-catenin and Wnt1 protein showed a rising trend in the Wnt/β-caten in signaling pathway（P<0.05）,and the osteogenic differentiation of BMSCs inhibiting DKK1 protein showed the decreasing trend（P<0.05）,β-cateni protein in Bone Reconstruction Pill medicated serum group and Xianling Gubao capsule serum group were highly expressed（P<0.05）;Wnt1 protein in Bone Reconstruction Pill medicated serum low concentration group and positive control serum group had a high expression（P<0.05）;DKK1 protein in the Bone Reconstruction Pill medicated serum low concentration group were persistently inhibited（P<0.05）.Conclusion:1.Bone Reconstruction Pill has good curative effect on steroid-induced avascular necrosis of the femoral head and can promote osteogenic differentiation of bone marrow mesenchymal stem cells.2.Bone Reconstruction Pill can promote BMSCs osteogenesis differentiation by regulating the expression of Wnt/β-catenin signaling pathway,high expression of β-catenin and Wnt1 and inhibiting the low expression of DKK-1,soas to achieve the effect of curing steroid-induced necrosis of the femoral head. |
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