The
fibrosis that develops following
laminectomy or
discectomy often causes serious complications, and the proliferation of fibroblasts is thought to be the major cause of epidural
fibrosis.
10-Hydroxycamptothecin (
HCPT) has been proven to be efficient in preventing epidural
fibrosis, but the exact mechanism is still unclear. NOXA is a significant regulator of cell apoptosis, which has been reported to be beneficial in the treatment of
fibrosis. We performed a series of experiments, both in vitro and in vivo, to explore the intrinsic mechanism of
HCPT that underlies the induction of apoptosis in fibroblasts, and also to investigate whether
HCPT has positive effects on epidural
fibrosis following
laminectomy in rats. Fibroblasts were cultured in vitro and stimulated by varying concentrations of
HCPT (0, 1, 2, 4 µg/ml) for various durations (0, 24, 48, 72 h); the effect of
HCPT in inducing the apoptosis of fibroblasts was investigated via Western blots and TUNEL assay. Our results showed that
HCPT could induce apoptosis in fibroblasts and up-regulate the expression of NOXA. Following the knockdown of NOXA in fibroblasts, the results of Western blot analysis showed that the level of apoptotic markers, such as cleaved-PARP and Bax, was decreased. The results from the TUNEL assay also showed a decreased rate of apoptosis in NOXA-knocked down fibroblasts. For the in vivo studies, 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; the macroscopic histological assessment,
hydroxyproline content analysis and histological staining were performed to evaluate the effect of
HCPT on reducing epidural
fibrosis. The TUNEL assay in epidural tissues showed that
HCPT could obviously induce apoptosis in fibroblasts in a dose-dependent manner. Also, immunohistochemical staining showed that the expression of NOXA increased as the concentrations of
HCPT increased. Our findings are the first to demonstrate that upregulation of NOXA by
HCPT plays a key role in inducing fibroblast apoptosis and in reducing epidural
fibrosis. These findings might provide a potential therapeutic target for preventing epidural
fibrosis following
laminectomy.