Mechanism Of Grape Seed Proanthocyanidins In Alleviating Intervertebral Disc Degeneration Induced By IL-1β

Background: Low back pain（LBP）is a common condition that approximately 84% of people will experience at some point in their lives.Many studies have shown that intervertebral disc degeneration（IDD）mediated by inflammatory response and oxidative pressure may be one of the main causes of LBP.The current treatment of IDD disease is mainly to relieve the clinical symptoms,but does not fundamentally delay the process of IDD.Therefore,it is necessary to study drugs to delay IDD.Purpose: procyanidins have been reported to have significant antiinflammatory and antioxidant effects,but whether they also have a protective effect on IDD remains unclear.The purpose of this study was to investigate the protective effects of grape seed proanthocyanidins（GSP）on the IDD induced by interleukin-1β（IL-1β）.And further study the anti-apoptotic and anti-inflammatory mechanism of GSP.In order to provide a new vision for the clinical development of drugs to delay the process of IDD.Methods: Primary nucleus pulposus（NP）cells from SD rat coccyx were obtained by type II collagenase and trypsin digestion.Rat NP cells were stimulated by IL-1β to establish IDD model in vitro.Cell counting kit 8（CCK-8）and ordinary light microscope were used to detect the effects of different concentrations of GSP on the activity,morphology and number of NP cells,and appropriate concentrations were selected for subsequent experiments.Western blotting,Hoechst 33258 staining and immunofluorescence were used to detect the protective effects and mechanism of GSP on IL-1β-induced IDD.EX527 was used to down-regulate SIRT1 expression in NP cells,and Western blotting was used to observe the effects of GSP on NF-κB signaling pathway and extracellular matrix（ECM）metabolism.Results: The primary NP cells of rat coccyx were successfully extracted and cultured,and were confirmed to be NP cells by red O staining and toluidine blue staining.In the model of NP cell degeneration induced by IL-1β,the activity of NP cells increased gradually and the number of NP cells increased when GSP concentration was2.5μM to 10μM by CCK-8 assay and ordinary light microscopy.Western blotting,immunofluorescence and Hoechst 33258 staining showed that GSP inhibited the expression of inflammatory cytokines（i NOs,COX-2）,pro-apoptotic protein（Bax,caspase-3）and matrix degrading enzyme（MMP13,ADAMTS4）in IL-1β-induced NP cells in a concentration-dependent manner,while promoting the expression of antiapoptotic protein（Bcl-2）,type II collagen,SIRT1 protein.In addition,GSP inhibited IL-1β-induced NF-κB signaling pathway activation.The protective effect of GSP on ECM and NF-κB pathway was significantly reduced after the expression of SIRT1 was specifically inhibited by EX527.Conclusion: In this study,we demonstrated that GSP can reduce inflammatory response,apoptosis of NP cells and ECM catabolism stimulated by IL-1β by regulating SIRT1/NF-κB signaling pathway in an in vitro IDD model.Therefore,GSP may be a potential drug for treating IDD.