Study On The Mechanism Of Glucocorticoid Induced Avascular Necrosis Of Femoral Head In Juvenile Rabbits

Objective To detect the expressions of R-spondin1,DKK-1 and beta-catenin in the tissue of avascular necrosis femoral head of steroid-induced juvenile rabbit model,compare the expression of R-spondin1 and the rate of apoptosis of human osteoblast treated with different doses of methylprednisolone,to discuss related mechanism of avascular necrosis of juvenile rabbit induced by glucocorticoid.Methods1.The group of avascular necrosis of juvenile rabbit(n=12): New Zealand juvenile rabbit with steroid-induced avascular necrosis of femoral head;the control group(n=12):normal New Zealand juvenile rabbit.The expression of R-spondin1,DKK-1 and ?-catenin in femoral head of each group were detected by Western Blot and RT-q PCR,the correlation between R-spondin1 and DKK-1,?-catenin were analyzed by Pearson's correlation test(Pearson).2.Human osteoblasts were cultured and passaged,the glucocorticoid intervention group were respectively added with methylprednisolone(10-8mol/L,10-7mol/L,10-6mol/L)and continued to culture 72 h,the control group were not added with methylprednisolone and continued to culture 72 h.The expression of R-spondin1 and the apoptosis rate of each group were detected by Western Blot and RT-q PCR.Results1.The expression of R-spondin1 in rabbit femoral head necrosis group and control group were 0.179±0.028 and 0.471±0.104 respectively,the difference was significant(t=9.38,P<0.01).The expression of ?-catenin in rabbit femoral head necrosis group and control were 0.195±0.041 and 0.632±0.129 respectively,the difference there was significant(t=11.15,P<0.01).The expression of Dkk1 in rabbit femoral head necrosis group and control group were 0.713±0.161 and 0.181±0.029 respectively,the difference was significant(t=-11.25,P<0.01).In rabbit femoral head necrosis group and control group,there was a positive correlation between the expression of R-spondin1 and that of ?-catenin(r=0.89,P<0.01),there was a negative correlation between R-spondin1 and Dkk1(r=-0.945,P<0.05).2.The expression of R-spondin1 in the gene of rabbit femoral head necrosis group and control group were 0.255±0.039 and 0.995±0.093,the difference was significant(t=25.33,P<0.01).The expression of beta-catenin in the gene of rabbit femoral head necrosis group and control group were 0.216±0.036 and 1.055±0.083,the difference was significant(t=32.49,P<0.01).The expression of Dkk1 in the gene of rabbit femoral head necrosis group and control group were 3.275±0.073 and 0.986±0.084,the difference was significant(t=-10.32,P<0.01).Compared the rabbit femoral head necrosis group with the control group,there was a positive correlation in the expression of R-spondin1 and that of beta-catenin(r=0.860,P<0.01),and a negative correlation between R-spondin1 and Dkk1(r=-0.945,P<0.01).3.The expression of R-spondin1 protein in control group and these three different doses of methylprednisolone(10-8mol/L,10-7mol/L and 10-6mol/L)in cultured cells were0.622±0.105,0.441±0.125,0.194±0.038 and 0.112±0.019 respectively,and the expression of R-spondin1 protein in geen decreased with increasing doses of methylprednisolone,and the difference was significant(F=76.37,P<0.01).The expression of R-spondin1 in gene were 0.999±0.168,0.897±0.131,0.624±0.101 and0.323±0.033,R-spondin1 gene expression decreased with increasing doses of methylprednisolone,and the difference was significant(F=64.79,P<0.01).4.The apoptosis rate(%)of control group and 10-8mol/L,10-7mol/L,10-6mol/L were7.41 + 1.11,13.14 + 1.92,25.01 + 5.21,31.99 + 9.48,with the intervention measurement increased the apoptosis rate of intervention is increased,the difference was significant(F=40.80,P<0.01).Conclusion1.Glucocorticoid can negatively regulate the expression of R-spondin1 in human osteoblasts,which may promote the apoptosis of human osteoblasts through negative regulation of R-spondin1 expression.2.In the mechanism of glucocorticoid induced avascular necrosis of femoral head in juvenile rabbits,the expression of R-spondin1 was positively correlated with beta-catenin,and negatively correlated with the expression of DKK-1.R-spondin1 may negatively regulate Wnt/ ?-catenin signaling pathway,promote apoptosis of osteoblasts,resulting in immature rabbit steroid induced avascular necrosis of femoral head.