Naringenin alleviated the SH-SY5Y cells death and insulin resistance by activating mTOR/p70S6K signalling pathway in oxidative stress induced by H2O2

Abstract:

Objective：To investigate the effect of naringenin on SH-SY5Y cells of neuroblastoma in oxidative stress induced by H2O2 and the possible mechanism. Methods：Firstly，a concentration of H2O2 that caused half cells death was chosen by CCK-8 assay. Three concentrations of naringenin（20，40，80 μmol/L） were added into H2O2-treated media，and cells survival rates were tested by CCK-8 assay after 4 hours. An optimal concentration of naringenin was chosen for the next experment in naringenin group. Next，three groups were chosen for the next experiment（control group：cells without any treatment；H2O2 group：cells with 600 μmol/L H2O2；H2O2+NAR group：cells with 600 μmol/L H2O2 and 40 μmol/L NAR simultaneously）. The apoptosis rate was measured by Annexin V-FITC/PI Apoptosis Detection Kit，intercellular reactive oxygen species（ROS） was measured by fluorescent probe through flow cytometry，and mTOR/p70S6K signalling pathway-related protein[mTOR，p-mTOR（Ser2448），p70S6K，p-p70S6K（Thr389）] were tested by Western blot. Finally，insulin resistance-related protein insulin receptor substrate 1（IRS1） and phosphorylated IRS1 in serine 636 and serine 639[p-IRS1（Ser636+Ser639）] were detected by Western blot as well. Results：H2O2 significantly increased the SH-SY5Y cells death，apoptosis，intercellular ROS and p-IRS1（Ser636+Ser639），and decreased the protein expression of p-mTOR（Ser2448） and p-p70S6K（Thr389） compared with control group（P<0.05）. These effects were attenuated after NAR treatment，compared with H2O2 group（P<0.05）. Conclusion：Naringenin reduced the ROS accumulation and the SH-SY5Y cells death. The activation of mTOR/p70S6K signalling pathway and decreasing of insulin resistance may be involved in naringenin-induced antioxidative effects.