穿山龙多糖影响NADPH氧化酶/ROS信号通路抗H2O2诱导的HUVECs细胞损伤作用研究

摘要

目的 检测穿山龙多糖(RDNP)是否可以通过影响NADPH氧化酶/ROS信号通路对H2O2诱导的人脐静脉内皮细胞(HUVECs)氧化损伤产生保护作用.方法 H2O2作用HUVECs 4 h造成损伤,分别给予25、50、100、200、400、800 μg/mL RDNP进行干预,选取适当浓度进行后续实验.采用MTT法测定细胞活力;试剂盒检测 LDH、MDA、SOD、T-AOC、GSH-Px、ROS水平;Western Bolt及 qRT-PCR法检测Nox4、p22phox的蛋白及mRNA表达.结果 加入50、100和200 μg/mL浓度的RDNP与H2O2模型组间相比,活力增加,差异有统计学意义(P<0.05或P<0.01).因此选择50、100、200 μg/mL RDNP剂量组进行实验.不同浓度RDNP均可以显著改善H2O2损伤,使细胞形态逐渐趋于正常,以200 μg/mL RDNP给药组的效果最为显著.RDNP可以降低H2O2损伤HUVECs模型中LDH、MDA及ROS的水平,并增强细胞内SOD、 GSH-Px、T-AOC活力;且从mRNA水平及蛋白水平抑制Nox4、p22phox的表达.结论 RDNP可以通过干扰NADPH氧化酶/ROS信号通路抑制H2O2造成的HUVECs氧化损伤,保护内皮细胞.%Objective To investigate effect of Rhizoma Dioscoreae Nipponicae polysaccharides (RDNP) on protecting HUVECs from H2O2-induced injury by interfering NADPH oxidase/ROS pathway.Methods HUVECs were treated with RDNP in the presence/absence of H2O2 for 4 h.Then cell activity was detected by MTT method.LDH, MDA, SOD, T-AOC, GSH-Px and ROS were detected by reagent kits to evaluate the cell injuries and the antioxidant activity.Western blot and qRT-PCR was used to evaluate the protein expression and the mRNA expression of Nox4, p22phox.Results LDH, MDA, ROS levels decreased and intracellular SOD, T-AOC as well as GSH-Px activities enhanced in H2O2-treated HUVECs when RDNP was used, especially with 200 μg/mL.Simultaneously, Nox4, p22phox expression were both decreased in protein and mRNA levels in HUVECs.Conclusions RDNP could protect HUVECs from H2O2-induced injury by interfering NADPH oxidase/ROS pathway.