The Mechanism Of P-glycoprotein On Glucocorticoid-induced Osteonecrosis Of Femoral Head

Objective:1.To observe the effects of glucocorticoids on bone marrow stromal cells(MSCs) adipogenesis when P-glycoprotein(P-gp) activity in vitro was down-regulated/up-regulated by verapamil/rifampicin.2.To study the physiopathologic mechanism of glucocorticoid-induced osteonecrosis of femoral head(ONF) when P-gp activity in vivo was down-regulated by verapamil.To predict ONF by assessing P-gp activity of peripheral blood monouclear cells3.To explore the feasibility of the prevention of ONF by rifampicin up-regulated P-gp activity.Method:1.MSCs were isolated from SD rats and cultures in vitro.10ug/ml verapamil or 5ug/ml rifampicin was used to regulate P-gp activity in vitro.On the basis,we observed 10^-6mol/l dexamethasone-induced MSCs adipogenesis for 7 days.At the end,Triglyceride(TG) and alkaline phosphatase(AKP) activity were qualitatively and quantitatively detected.2.25ug/kg/d verapamil was used to decrease P-gp activity in vivo.On the basis,we observed the effect of decreased P-gp activity on glucocorticoids-induced ONF by MRI,histological and P-gp immunohistochemical determination.3.10mg/kg/d rifampicin was used to enhance P-gp activity in vivo.On the basis,we observed the effect of enhanced P-gp activity on glucocorticoids-induced ONF by MRI and histological determination.Result:1.Verapamil/rifampicin down-regulated/up-regulated P-gp activity in vitro.In absence of DEX,regulated P-gp activity had no effects on MSCs differentiation. In presence of DEX,MSCs became larger and showed lipid droplets in the cytoplasma.When P-gp activity was decreased by verapamil,MSCs induced by DEX became much larger and showed bigger lipid droplets.When P-gp activity 2.Verapamil decreased P-gp activity in vivo.In absence of MPSL,TG level increased by verapamil.The MRI and histology of femoral head were similar to the saline-treated control.In presence of MPSL,MRI showed abnormal enhanced signals in the matching region of T₁ and T₂ images of femoral heads.The incidence of ONF was 80%.Epiphyseal ossification center and trabeculae volume decreased and adipocytes increased and became larger.When verapamil was included,mean area of MRI abnormal signal was the largest.The incidence of ONF was increased to 100%.Mean percentage area of epiphyseal ossification center and trabeculae volume were the smallest.The number and the size of adipocytes was the most and largest.3.Rifampicin enhanced P-gp activity in vivo.In absence of MPSL,TG level increased by rifampicin.The MRI and histology of femoral head were similar to the saline-treated control.In presence of MPSL,MRI showed abnormal enhanced signals in the matching region of T₁ and T₂ images of femoral heads.The incidence of ONF was 80%.Epiphyseal ossification center and trabeculae volume decreased and adipocytes increased and became larger.When rifampicin was included,mean area of MRI abnormal signal was the smallest.The incidence of ONF was decreased to 50%.Mean percentage area of epiphyseal ossification center and trabeculae volume were the largest.The number and the size of adipocytes was the least and smallest.Conclusion:1.Verapamil/rifampicin decreased/increased P-gp activity in vitro.DEX-induced MSCs adipogenesis was promoted by verapamil and inhibited by rifampicin.2.Verapamil decreased P-gp activity and increased the risk for steroid-induced ONF in vivo.P-gp activity of PBMC was closely related to the incidence of ONF.3.Rifampicin enhanced P-gp activity and decreased the risk for steroid-induced ONF in vivo.