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

Effect of vaspin on high glucose and lipid induced oxidative stress in INS-1 cells via the Nrf2/ARE signaling pathway

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+80 ng/mL Vaspin组(HG+PA+V1组)、高糖高脂+160 ng/mL Vaspin组(HG+PA+V2组)、高糖高脂+320 ng/mL Vaspin组(HG+PA+V3组)、高糖高脂+640 ng/mL Vaspin组(HG+PA+V4组)。通过DCFH-DA荧光探针染色法检测细胞内活性氧(reactive oxygen species,ROS)水平;比色法、酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测氧化应激相关指标;葡萄糖(3.3 mmol/L和16.7 mmol/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;Western blot检测核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,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);④Western blot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±0.080 vs.0.579±0.065,P=0.039)。结论:Vaspin可以通过激活Nrf2/抗氧化反应原件(antioxidant response element,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。

Objective:To investigate the effect of vaspin on high glucose and lipid induced oxidative stress in INS-1 cells.Methods:INS-1 cells were cultured and divided into normal control group,high glucose(HG)+palmitic acid(PA)group,HG+PA+vaspin(80 ng/mL)group,HG+PA+vaspin(160 ng/mL)group,HG+PA+vaspin(320 ng/mL)group,HG+PA+vaspin(640 ng/mL)group.Colorimetry an d enzyme-linked immunosorbent assay were used to measure oxidative stress-related indicators;meanwhile,DCFH-DA fluorescent probe staining,glucose-stimulated insulin secretion test(3.3 and 16.7 mmol/L),flow cytometry,and Western blot were used to determine the levels of intracellular reactive oxygen species(ROS),insulin secretion,apoptosis,and protein expression of nuclear factor erythroid 2-related factor 2(Nrf2),respectively.Results:Compared with the HG+PA group,the HG+PA+vaspin(640 ng/mL)group had a significantly increased insulin secretion level after stimulation by both low-level and high-level glucose(both P<0.05)as well as significantly increased total antioxidant capacity and levels of superoxide dismutase and glutathione peroxidase(all P<0.05),but had significantly decreased levels of ROS,malondialdehyde,and 8-hydroxydeoxyguanosine(all P<0.05).With the increasing vaspin levels,the vaspin-treated groups had significantly reduced apoptosis in the INS-1 cells after intervention by high-level glucose and lipids compared with the HG+PA group,as revealed by flow cytometry(all P<0.05),and showed an increasing trend in intracytoplasmic Nrf2 expression as revealed by Western blot,while the intranuclear Nrf2 expression was significantly higher only in the HG+PA+vaspin(640 ng/mL)group compared with the HG+PA group(0.798±0.080 vs.0.579±0.065,P=0.039).Conclusion:Vaspin can suppress high glucose and palmitic acid induced oxidative stress injury in INS-1 cells,reduce apoptosis,and improve insulin secretion level by activating the Nrf2/ARE signaling pathway.