Abstract
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.
Highlights
Laminectomy for treating lumbar disc herniation and other lumbar disorders often results in the formation of epidural fibrosis, and the development of epidural fibrosis is the major contributor to postoperative morbidities such as persistent low back pain and disability (Songer et al, 1995)
We showed that HCPT could induce fibroblast apoptosis and reduce epidural fibrosis by upregulating NOXA expression
Following HCPT treatment, the percentages of TdT-mediated dUTP-biotin nick-end labelling (TUNEL)-positive cells at1 μg/ml, 2 μg/ml and 4 μg/ml were 14.94% ± 1.40%, 20.06% ± 2.64% and 28.26% ± 2.64%, respectively (Fig. 1C). These results indicate that HCPT significantly induced apoptosis in fibroblasts
Summary
Laminectomy for treating lumbar disc herniation and other lumbar disorders often results in the formation of epidural fibrosis, and the development of epidural fibrosis is the major contributor to postoperative morbidities such as persistent low back pain and disability (Songer et al, 1995). The prevention of epidural fibrosis has been a subject of concern for many years. Many investigators have carried out studies to prevent epidural fibrosis via promoting fibroblast apoptosis, and some of them have achieved satisfactory results (Sun et al, 2015; Yang et al, 2016). Our previous study showed that local application of 0.1 mg/ml HCPT could conspicuously reduce postoperative epidural fibrosis formation in a rat laminectomy model (Sun et al, 2008). HCPT has shown its apoptosis-inducing character in some cell types (Yuan et al, 2016; Cheng et al, 2016), which implicated that it might be useful in the prevention of epidural fibrosis through inducing the apoptosis of fibroblasts
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