Upregulation Of NOXA By 10-hydroxycamptothecin Plays A Key Role In Inducing Fibroblasts Apoptosis And Reducing Epidural Fibrosis

Objective:To investigate whether HCPT has positive effects on laminectomy-induced epidural fibrosis in rats,and also to explore the intrinsic mechanism of HCPT that underlies the induction of apoptosis in fibroblasts.Methods:Seventy-two mature male Sprague-Dawley rats were used for this study.All rats were randomly divided into 4 groups:0.2 mg/ml HCPT-treated group,0.1mg/ml HCPT-treated group,0.05 mg/ml HCPT-treated group and control group.We performed a laminectomy at the L1-L2 levels in rats and applied HCPT of different concentrations(0.2,0.1,0.05 mg/ml and saline)locally.4 weeks later,all rats were sacrificed.Macroscopic histological assessment, histological analysis,fibroblast counting,TUNEL assay and immunohistochemical staining of the scar tissue were performed to detect the effect of HCPT on preventing epidural scar adhesion following laminectomy.In vitro,fibroblasts were stimulated by HCPT for different concentrations(0,1,2,4?g/ml)and different durations(0,24,48,72 h),TUNEL staining was performed to detect fibroblasts apoptosis rate.Western-blot was performed to detect the expression of apoptosis marker proteins(NOXA,cleaved-PARP,Bax and Bcl-2).Results:In vivo studies,histological and immunohistochemical staining were used to evaluate the effect of HCPT and NOXA on reducing epidural fibrosis.Histological staining and TUNEL assay in epidural tissue shown that HCPT could obviously reduce epidural fibrosis and induce apoptosis of fibroblasts in a dose-dependent manner.Besides,immunohistochemical staining shown that the expression of NOXA was increased.The results in vitro showed that HCPT could induce apoptosis in fibroblasts and up-regulate the expressions of NOXA.Following knocking down NOXA in fibroblast,the results of westemblot shown that the apoptotic markers,such as cleaved-PARP and Bax,was decreased.The TUNEL assay also shown a decreasing apoptosis rate in fibroblast.Conclusion:HCPT could prevent laminectomy-induced epidural fibrosis,and the expression of NOXA regulated by HCPT plays a key role in inducing fibroblast apoptosis and in preventing epidural fibrosis...