Vaspin通过Nrf2/ARE信号通路对高糖高脂诱导的INS-1细胞氧化应激的影响

EffectofvaspinonhighglucoseandlipidinducedoxidativestressinINS-1cellsviatheNrf2/AREsignalingpathway

摘要:

目的：探讨Vaspin对高糖高脂诱导的INS-1细胞氧化应激的影响。方法：培养INS-1细胞，分为正常对照组（NC组）、高糖高脂组（HG+PA组）、高糖高脂+80ng/mLVaspin组（HG+PA+V1组）、高糖高脂+160ng/mLVaspin组（HG+PA+V2组）、高糖高脂+320ng/mLVaspin组（HG+PA+V3组）、高糖高脂+640ng/mLVaspin组（HG+PA+V4组）。通过DCFH-DA荧光探针染色法检测细胞内活性氧（reactiveoxygenspecies，ROS）水平；比色法、酶联免疫吸附试验（enzyme-linkedimmunosorbentassay，ELISA）检测氧化应激相关指标；葡萄糖（3.3mmol/L和16.7mmol/L）刺激胰岛素分泌实验检测各组胰岛素分泌水平；流式细胞仪检测细胞凋亡水平；Westernblot检测核因子E2相关因子2（nuclearfactorerythroid2-relatedfactor2，Nrf2）蛋白表达水平。结果：①与HG+PA组相比，HG+PA+V4组中低糖和高糖刺激后的胰岛素分泌水平均有明显升高（均P<0.05）；②与HG+PA组相比，HG+PA+V4组中高糖高脂诱导INS-1细胞中ROS、丙二醛、8-羟基脱氧鸟苷水平明显降低，总抗氧化能力、超氧化物歧化酶水平、谷胱甘肽过氧化物酶水平明显升高（均P<0.05）；③流式细胞实验发现，与HG+PA组相比，随着Vaspin浓度增加，高糖高脂干预后INS-1细胞的凋亡水平明显下降（均P<0.05）；④Westernblot结果表明，胞浆中Nrf2表达水平随着Vaspin浓度的增加呈上升趋势，胞核Nrf2表达水平仅在HG+PA+V4组较HG+PA组升高（0.798±0.080vs.0.579±0.065，P=0.039）。结论：Vaspin可以通过激活Nrf2/抗氧化反应原件（antioxidantresponseelement，ARE）信号通路，改善高糖高脂诱导的INS-1细胞氧化应激损伤，减少细胞凋亡，增加胰岛素分泌水平。

Abstract:

Objective：ToinvestigatetheeffectofvaspinonhighglucoseandlipidinducedoxidativestressinINS-1cells.Methods：INS-1cellswereculturedanddividedintonormalcontrolgroup，highglucose（HG）+palmiticacid（PA）group，HG+PA+vaspin（80ng/mL）group，HG+PA+vaspin（160ng/mL）group，HG+PA+vaspin（320ng/mL）group，HG+PA+vaspin（640ng/mL）group.Colorimetryandenzyme-linkedimmunosorbentassaywereusedtomeasureoxidativestress-relatedindicators；meanwhile，DCFH-DAfluorescentprobestaining，glucose-stimulatedinsulinsecretiontest（3.3and16.7mmol/L），flowcytometry，andWesternblotwereusedtodeterminethelevelsofintracellularreactiveoxygenspecies（ROS），insulinsecretion，apoptosis，andproteinexpressionofnuclearfactorerythroid2-relatedfactor2（Nrf2），respectively.Results：ComparedwiththeHG+PAgroup，theHG+PA+vaspin（640ng/mL）grouphadasignificantlyincreasedinsulinsecretionlevelafterstimulationbybothlow-levelandhigh-levelglucose（bothP<0.05）aswellassignificantlyincreasedtotalantioxidantcapacityandlevelsofsuperoxidedismutaseandglutathioneperoxidase（allP<0.05），buthadsignificantlydecreasedlevelsofROS，malondialdehyde，and8-hydroxydeox-yguanosine（allP<0.05）.Withtheincreasingvaspinlevels，thevaspin-treatedgroupshadsignificantlyreducedapoptosisintheINS-1cellsafterinterventionbyhigh-levelglucoseandlipidscomparedwiththeHG+PAgroup，asrevealedbyflowcytometry（allP<0.05），andshowedanincreasingtrendinintracytoplasmicNrf2expressionasrevealedbyWesternblot，whiletheintranuclearNrf2expressionwassignificantlyhigheronlyintheHG+PA+vaspin（640ng/mL）groupcomparedwiththeHG+PAgroup（0.798±0.080vs.0.579±0.065，P=0.039）.Conclusion：VaspincansuppresshighglucoseandpalmiticacidinducedoxidativestressinjuryinINS-1cells，reduceapoptosis，andimproveinsulinsecretionlevelbyactivatingtheNrf2/AREsignalingpathway.