Research On The Effect And Potential Mechanism Of Dioscin In Nucleus Pulposus Oxidative Stress Damage

Objective: To investigate the protective effect of Dioscin to suppress oxidative stress injury of nucleus pulposus and to explore the possible mechanism of drug action.Methods: The primary nucleus pulposus cells extracted from sprague-dawley rats were cultured to the second passage for experimental study.Hydrogen peroxide was used to induce oxidative stress in nucleus pulposus cells in vitro.The nucleus pulposus cells were treated with different concentrations of Dioscin and hydrogen peroxide,and the optimal concentration of Dioscin was determined by CCK8 cell activity assay.The apoptosis rate of nucleus pulposus cells was detected by flow cytometry of Annexin V-FITC/PI staining.The level of SOD,ROS and MDA was detected with different kits.Immunofluorescence was used to detect the distribution of intracellular ROS and NOX4.Meanwhile,Western Blot was used to analyze the expression of apoptotic regulatory proteins,inflammatory mediators,metalloproteinases,ECM anabolic factors and pathway proteins in the nucleus pulposus cells.Result: 200 u M H2O2 could reduce the vitality of nucleus pulposus cells by 51%,and thus was used to induce oxidative stress of rat nucleus pulposus cells in vitro.Dioscin with a concentration less than 600 n M showed no obvious cytotoxicity,and 200 n M Dioscin could significantly improve the vitality of rat nucleus pulposus cells down-regulated by hydrogen peroxide.Dioscin can down-regulate the expression levels of caspase3,Caspase9 and Bax,and inhibit the mitochondrial apoptosis induced by H₂0₂ in rat nucleus pulposus cells.Dioscin restores the redox balance in rat nucleus pulposus cells by reducing the production of ROS and MDA and enhancing the activity of SOD.Dioscin inhibits hydrogen peroxide-induced increase of INOS,COX2,and IL-6,and alleviates inflammatory response in rat nucleus pulposus cells.Diosicn can block the overexpression of matrix metalloproteinases such as ADAMTS-4,ADAMTS-5,MMP-3,and MMP-13 caused by hydrogen peroxide stimulation,and up-regulate the protein levels of synthetic factors including Collagen2 and SOX9.Hydrogen peroxide can induce high expression of NOX4,while Dioscin inhibits the activation of NF-?B and JNK pathway by down-regulating NOX4 expression and decreasing phosphorylation of NF-?B and JNK pathway proteins.Conclusion: Nucleus pulposus cells can promote the release of inflammatory mediators by activating the Nox4/NF-?B/JNK pathway under oxidative stress,up-regulating the catabolic factors of the intervertebral disc matrix and causing excessive matrix decomposition,and ultimately accelerate the degeneration of the intervertebral disc.Dioscin could inhibit ROS production and restore the oxidative and anti-oxidative balance,improve the inflammatory environment,and protect nucleus pulposus cells from oxidative stress damage.Therefore,Dioscin has potential therapeutic prospects in intervertebral disc degeneration.