柚皮素通过激活mTOR/p70S6K信号通路减轻胰岛素抵抗 并改善H2O2诱导的SH-SY5Y细胞氧化应激损伤

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

摘要:

目的：观察柚皮素（naringenin，NAR）对H2O2诱导的SH-SY5Y细胞氧化应激损伤的影响，并探索其机制。方法：通过CCK-8检测细胞存活率，筛选出H2O2的半数致死浓度，用该浓度H2O2同时加入20、40、80 μmol/L柚皮素处理SH-SY5Y细胞4 h，通过CCK-8检测细胞存活率，选择适宜柚皮素浓度。按所选H2O2和柚皮素浓度进行后续实验。将实验分为3组。control组：细胞不进行任何干预处理；H2O2组：只加入600 μmol/L H2O2；H2O2+NAR组：同时加入600 μmol/L H2O2与40 μmol/L柚皮素。4 h后，通过ANNEXIN V-FITC/PI凋亡检测试剂盒检测细胞凋亡率，DCFH-DA荧光探针检测细胞内活性氧（reactive oxy－gen species，ROS），Western blot检测哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）、核糖体蛋白S6激酶（p70 ribosomal S6 kinase，p70S6K），以及各自磷酸化形式蛋白[p-mTOR（Ser2448）、p-p70S6K（Thr389）]，用胰岛素受体底物-1（insulin receptor substrate 1，IRS1）及其丝氨酸636+639位点磷酸化[p-IRS1（Ser636+Ser639）]蛋白的表达来观察胰岛素抵抗（insulin re－sistance，IR）。结果：H2O2降低SH-SY5Y细胞的存活率，呈剂量依赖性，600 μmol/L浓度为半数致死剂量。柚皮素明显降低了H2O2诱导的SH-SY5Y细胞死亡，最适剂量为40 μmol/L。与control组比，H2O2明显增加了细胞凋亡率、胞内ROS的产生，降低了p-mTOR（Ser2448）、p-p70S6K（Thr389）的表达，增加了p-IRS1（Ser636+Ser639）蛋白的表达（P<0.05）；加入柚皮素后，相对于H2O2组，上述作用明显减弱（P<0.05）。结论：在体外氧化应激模型中，柚皮素能明显降低细胞内ROS的积累，提高细胞的存活率。其机制可能与激活mTOR/p70S6K信号通路、改善IR有关。

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.