| Objective:To explore the possible mechanism of steroid-induced osteonecrosis of femoral head and the effects of Drynariae rhizoma on the proliferation and osteogenic-adipogenic differentiation of bone marrow mesenchymal stem cells based on the Wnt/?-Catenin signaling pathway,which provides a theoretical basis for clinical treatment.Methods:Part One:Clinical Experimental Research1.From 2015.05 to 2016.04,the bone marrow mesenchymal stem cells were taken from the proximalfemoral of 11 patients with SONFH undergoing arthroplasty(experimental group)and 7 patients with fracture of femoral neck andintertrochanter undergoing arthroplastyor interlock(control group).The experiment was approved by the hospital ethics committee,and each patient signed an informed consent form before surgery.The BMSCs were isolated,cultured and purified by whole bone marrow method,and P3 generation was used for experimental research.The surface antigen was identified by flow cytometry;the cell proliferation was detected by CCK-8 method and cell cycle assay;the adipogenic ability was identified by oil red O staining,and the osteogenic ability was identified by alizarin red staining.The m RNA and protein expressions of Wnt/?-Catenin signaling pathway-related factors(?-Catenin,Cyclin D1,Runx2,and PPAR-?)in bone marrow mesenchymal stem cells were detected by RT-PCR and Western-blot.To explore the possible mechanism of steroid-induced osteonecrosis of femoral head based on the Wnt/?-Catenin signaling pathway.2.Different concentrations of total flavonoids of Drynariae rhizoma were used to treat P3 bone marrow mesenchymal stem cells from patients with SONFH for 7 days,which were divided into 0g/L?10-1g/L?10-2g/L?10-3g/L?10-4g/L?10-5g/L?10-6g and 10-7g/L.The proliferation rate of each group was tested and the optimal concentration was obtained for the next experimental study.The best intervention concentration of total flavonoids and classical osteogenic inducer were used to intervene the P3 bone marrow mesenchymal stem cells of patients with SONFH.The test were divided into control group,total flavonoids treatment group and classical osteogenic inducer group.After 21 days of intervention,the ALP activity?alizarin red staining and quantitative determination of bone compensatory capacity of each group was detected,and the m RNA and protein expression of Wnt/?-Catenin signaling pathway ralated factors of each group of bone marrow mesenchymal stem cells was detected by RT-PCR and Western-blot.Differential m RNA and protein expression of Wnt/?-Catenin signaling pathway related factors(Wnt10b,LRP5,?-Catenin,Cyclin D1,Runx2,Osterix)explores the proliferation and osteogenic-adipogenic differentiation of BMSCs in patients with SONFH.Part Two:Animal Experimental Research:160 male Sprague-Dawley rats were randomly divided into control group,model group and treatment group I?II and III,and 32 rats were in each group.The animalmodel group was established by injecting dexamethasone sodium phosphate into gluteal muscles(30 mg/Kg,T.w.).On the basis of the model group,the treatment group I?IIand III were given the normal concentration of the Drynariae rhizomadecoction,thefive times concentration and the 10 timesconcentration,and the control group was treated with normal saline.After 12 weeks,all rats were sacrificed to obtain specimens-femoral head tissue and bone marrow mesenchymal stem cells for test.The trabecular area,fat vacuole area and cartilage thickness of each group were detected by HE staining and Image J software.The mechanical changes of femoral head were measured by axial pressure test of femoral head.The whole bone marrowmethod was used to separate and culture the r BMSCs of above five groups.Five groups of bone marrow mesenchymal stem cells were passaged and purified,and P3 generationwasused forexperimental research.The proliferation and osteogenic-adipogenic differentiation of P3 bone marrow mesenchymal stem cells of five groups of rats were detected by CCK-8,type I collagen SP staining,alizarin red staining,oil red O staining and quantitative,ALP quantitative after induction,and the m RNA and protein expression levels of Wnt/?-Catenin signaling pathways ralated factors(Wnt10b,?-Catenin,Cyclin D1,Runx2,Osterix,PPAR-?)were detected by RT-PCR and Western Blot.The possible pathogenesis of SONFH based on Wnt/?-Catenin signaling pathway and the effects of Drynariae rhizoma on proliferation and osteogenic-adipogenic differentiation of BMSCs were explored further.Results:Part One:The Results of Clinical ExperimentsThe results of clinical experiment one:1.Results of cell surface antigen detection in two groups of hBMSCs:The surface antigens(CD29,CD44,CD45,CD54,CD90)in the experimental group were significantly different than the control group(P<0.05).2.The cell proliferation and cell cycle detection results of the two groups of hBMSCs:The experimental group was significantly lower than those of the control group(P<0.05).3.The osteogenic-adipogenic induction and quantitative detection results of the two groups of hBMSCs:The osteogenic differentiation ability of the experimental group was significantly weaker than that of the control group,and the adipogenic differentiation ability was enhanced.The difference of quantitative detection was statistically significant(P<0.05).4.RT-PCR results:Compared with the control group,the main related factors of Wnt/?-Catenin signaling pathway and the proliferation-promoting,osteogenic differentiation factors(Wnt10,LRP5,?-Catenin,Cyclin D1,Runx2 m RNA)were significantly down-regulated,and the m RNA expression of adipogenic-differentiation factor PPAR-?was up-regulated,the difference was statistically significant(P<0.05),and the m RNA expression of Osterix was not statistically significant(P>0.05).5.Western blot results:Compared with the control group,the main related factors of Wnt/?-Catenin signaling pathway and the proliferation-promoting,osteogenic differentiation factors(Wnt10,?-Catenin,Cyclin D1,Runx2)protein were down-regulated,and the adipogenic differentiation factor PPAR-?protein was significantly up-regulated,and the difference was statistically significant(P<0.05).The results of clinical experiment two:1.The effect of different concentrations of Drynariae rhizoma on the proliferation of hBMSCs in patients with SONFH showed that the cell proliferation rate of 10-5g/L total flavonoidswas the highest,which was significantly different than other groups(P<0.05),and it was the optimal intervention concentration.2.The results of ALP test:Compared with the control group,the total flavonoids group(10-5g/L)and the classic osteogenic inducer group after intervention 1 week?2 weeks ALP activity was significantly increased(P<0.05);The ALP activity of classic osteogenic inducer group was higher than that of the total flavonoids,but there was no significant difference between the two groups(P>0.05).3.The alizarin red staining and quantitative test results of hBMSCs intervention for 3weeks of three groups of SONFH patients:The control group calcium nodule staining results were negative;compared with the control group,the total flavonoids group and the classic osteogenic inducer group can be seen by alizarin red staining.There were significant differences in the quantitative detection of a large number of positive calcium nodules(P<0.05).There was a significant difference in the quantitative detection of calcium nodules between the classic osteogenic inducer group and the total flavonoids group(P<0.05).4.The relative expression of m RNA of hBMSCs in three groups of SONFH patients was detected by RT-PCR.Compared with the control group,the m RNA expression of the main related factors(Wnt10,LRP5,?-Catenin,Cyclin D1,Runx2 and Osterix)of Wnt/?-Catenin signaling pathway of hBMSCs of the total flavonoids treatment group and osteogenic inducer group were significantly different(P<0.05).There were significant differences in the expression of Wnt10??-Catenin?Runx2 and Osterix m RNA between the total flavonoids and osteogenic inducer groups(P<0.05).There was no significant difference in the expression of LRP5 and Cyclin D1 m RNA(P>0.05).5.The WB results of relative expression of each hBMSCs protein of three groups of SONFH patients:Compared with the control group,the main related factors of Wnt/?-Catenin signaling pathway and the promotion of proliferation,osteogenic differentiation factors?-Catenin,Cyclin D1,Runx2 and Osterix proteins were significantly increased in the total flavonoids treatment group and osteogenic inducer group(P<0.05).There were also significant differences in the expression of?-Catenin,Cyclin D1,Runx2 and Osterix between the total flavonoid group and the osteogenic inducer group(P<0.05).Part Two:The Results of Animal Experiments1.HE staining results of femoral head tissues of five groups of SD rats:Compared with the control group,the trabecular area and maximum cartilage thickness of the model group were significantly reduced,and the area offat vacuoles was significantly increased.The difference was statistically significant(P<0.05);Compared with the model group,the trabecular area and maximum cartilage thickness of the treatment group I,II,III were significantly increased,and the area offat vacuole was significantly reduced.The difference was statistically significant,especially in the treatment group II(P<0.05).2.Biomechanical test results of femoral head of five groups of SD rats:Compared with the control group,the maximum axial load,stress intensity and axial stiffness of the femoral head subchondral bone in the model group and the treatment group I,II and III were significantly weaker.The maximum displacement of the subchondral bone of the femoral head was significantly increased(P<0.05).Compared with the model group,the maximum axial load,stress intensity and axial stiffness of the subchondral bone of the femoral head in group I,II and III were obviously stronger,the maximum displacement of the subchondral bone of the femoral head was significantly reduced,there was a significant difference(P<0.05),especially in the treatment of group II and III,there was no significant difference between the two groups.3.The proliferation of r BMSCs of five groups of SD rats showed that the proliferation curves of r BMSCsof five groups of SD rats were"S"type.Compared with the control group,the cell proliferation curve of the model group wasobviously down-regulated,andwere significant difference(P<0.05).Compared with the model group,the cell proliferation curves of the treatment group I,II and III were up-regulated,and the difference was statistically significant(P<0.05),especially in the treatment group II,III up-regulation was obviously.4.The results of osteogenic function test of r BMSCs of five groups of SD rats:Compared with the control group,the model group whose SP staining of type I collagen,staining and quantification of alizarin red,and detection of ALP activity were significantly lower.Compared with the model group,the indexes of treatment group I,II and III were significantly increased,and the difference of quantitative analysis was statistically significant(P<0.05),especially in the treatment of group II and III,but the difference between the two groups was not statistically significant(P>0.05).The results of RT-PCR and Western blot showed that the main factors of Wnt/?-Catenin signaling pathway and the expression of osteogenic differentiation factors(Wnt10,?-Catenin,Runx2 and Osterix)m RNA and protein were significantly down-regulated in the model group;compared with the model group,the m RNA and protein expression of the factors in the treatment group I,II,III were significantly up-regulated,the difference was statistically significant(P<0.05),especially in the treatment group II,III.5.The results of adipogenic function of r BMSCs of five groups of SD rats:Compared with the control group,the oil red O staining of the model group was significantly enhanced;compared with the model group,the treatment groups I,II and III were significantly weakened,and the differences in quantitative detection and analysis were statistically significant(P<0.05),especially in the treatment of group II,III,but the difference between group II,III were not statistically significant(P>0.05).The results of RT-PCR and Western blot showed that the expressions of m RNA and protein of Wnt10and?-Catenin were down-regulated in the model group,and the expression of m RNA and protein of PPAR-?was up-regulated,and the results werestatistical significance(P<0.05).Compared with the model group,the expression ofm RNA and protein of Wnt10and?-Catenin of treatment group I,II and III were significantly up-regulated,and the expression m RNA and proteinof PPAR-?were down-regulated,and the difference was statistically significant.(P<0.05),especially in the treatment of group II and III.Conclusion:1.The proliferation and osteogenic-differentiation of BMSCs of SONFH are significantly reduced,and the adipogenic-differentiation is enhanced,which is consistent with the theory of“Medullary blight causes atrophic debility of bones”and its mechanism may be related to the inhibition of Wnt/?-Catenin signaling pathway,and the expression of Wnt10,LRP5,?-Catenin,Cyclin D1,Runx2 is down-regulation,and the expression of PPAR-?is up-regulation.2.Drynariae rhizoma can play a therapeutic role by promoting the proliferation and osteogenic-differentiationof BMSCs of SONFH,andattenuating adipogenic-differentiation.It is in line with the theory of“Strong kidney can strong marrow and bone”.And its mechanism may be related to the activation of Wnt/?-Catenin signaling pathway,witch promotes expression of proliferative and osteogenic-differentiation factors(Wnt10,?-Catenin,Cyclin D1,Runx2,Osterix)and inhibits expression of adipogenic-differentiation factor(PPAR-?). |
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