高糖抑制HK-2细胞NRF2信号轴相关蛋白表达并诱导线粒体损伤
High Glucose Inhibits the Expression of NRF2 Signaling Axis-related Proteins and Induces Mitochondrial Damage in HK-2 Cells
温海滨 1谭宁 2覃宇奇 3陈意 4王梦玲 5李鸿文 2魏兵6
作者信息
- 1. 广西壮族自治区江滨医院肾内科(南宁 530021)
- 2. 桂林医学院基础医学院(桂林 541199)
- 3. 广西壮族自治区江滨医院病理科(南宁 530021)
- 4. 桂林医学院临床医学院(桂林 541199)
- 5. 桂林医学院药学院(桂林 541199)
- 6. 桂林医学院国际教育学院(桂林 541199)
- 折叠
摘要
目的:探讨高糖培养对人肾小管上皮细胞HK-2 氧化应激的影响及其相关机制.方法:传代培养HK-2 细胞,并将细胞分为 4 组:对照组(5.5 mmol/L 葡萄糖)、甘露醇组(5.5 mmol/L 葡萄糖+24.5 mmol/L 甘露醇)、高糖组(30 mmol/L葡萄糖)和高糖+Ferrostatin-1 组(5.5 mmol/L葡萄糖+1 mmol/L Ferrostatin-1).采用CCK-8 法检测细胞的增殖活性;使用流式细胞术检测细胞凋亡情况;利用ROS和GSH试剂盒测定细胞内ROS和GSH水平;通过蛋白免疫印迹实验(Western blot)检测NRF2、SLC7A11、GPX4 蛋白表达水平;进行细胞线粒体荧光染色,观察并检测各组细胞的线粒体荧光密度.结果:与对照组相比,高糖可以抑制HK-2 细胞的增殖活性(P<0.05),促进细胞凋亡(P<0.05),升高细胞内ROS水平(P<0.05),降低GSH水平(P<0.05),下调NRF2、SLC7A11(System X(c))、GPX4 蛋白表达(P<0.01),降低线粒体荧光光密度(P<0.01).Ferrostatin-1 干预后,高糖介导的细胞增殖抑制、凋亡促进、升高 ROS、降低 GSH 等效应均被削弱(P<0.05),同时NRF2、GPX4 蛋白表达水平上调(P<0.01),线粒体荧光光密度增加(P<0.01).结论:高糖可显著抑制人肾小管上皮细胞HK-2 的增殖活性,其机制可能与抑制NRF2 信号轴相关蛋白的表达,促进细胞发生氧化应激,诱导线粒体损伤有关;而铁凋亡抑制剂Ferrostatin-1 能削弱高糖对细胞产生的上述氧化损伤作用,提示铁凋亡参与了高糖诱导HK-2 细胞的损伤.
Abstract
Objective:Explore the effect of high glucose culture conditions on the oxidative stress in human renal tubular ep-ithelial cells HK-2 and its related mechanisms.Methods:HK-2 cells were divided into four groups:normal group(5.5 mmol/L glucose),mannitol group(5.5 mmol/L glucose+24.5 mmol/L mannitol),high glucose group(30 mmol/L glucose),and high glu-cose+Ferrostatin-1 group(5.5 mmol/L glucose+1 mmol/L Ferrostatin-1).The cell proliferation activity of cells was detected by the CCK-8 method.The cell apoptosis was detected by flow cytometry.The intracellular ROS and GSH levels were measured by ROS and GSH kits.Western blot was used to assay the protein expression levels of NRF2,SLC7A11,and GPX4.Perform mitochondrial fluorescence staining to observe and detect the mitochondrial fluorescence density of cells.Results:Compared with the control group,high glucose can inhibit the proliferation activity of HK-2 cells(P<0.05),promote apoptosis(P<0.05),increase intracellular ROS levels(P<0.05),reduce GSH levels(P<0.05),down-regulate the proteins expression of NRF2,SLC7A11(System X(c)),GPX4(P<0.01),and reduce mitochondrial fluorescence intensity(P<0.01).After the intervention of Ferrostatin-1,the cell proliferation inhibition,apoptosis promotion,ROS increase,and GSH decrease mediated by high glucose were all attenuated(P<0.05),while the proteins expression levels of NRF2 and GPX4 were up-regulated(P<0.01),and mitochondrial fluorescence in-tensity increased(P<0.01).Conclusion:High glucose can significantly inhibit the proliferative activity of human renal tubular epi-thelial cells HK-2,which may be related to inhibiting the expression of NRF2 signaling axis-related proteins,promoting oxidative stress,and inducing mitochondrial damage in cells.Ferrostatin-1,an inhibitor of ferroptosis,can mitiqate the oxidative damage in-duced by high glucose in cells,suggesting that ferroptosis contributes to the damage in HK-2 cells,induced by high glucose.
关键词
糖尿病肾病/氧化应激/铁凋亡/核因子E2/相关因子2(NRF2)System/X©Key words
Diabetic nephropathy/Oxidative stress/Ferroptosis/Nuclear factor erythroid 2-related factor 2(NRF2)/System X©引用本文复制引用
基金项目
国家自然科学基金资助项目(82160699)
广西壮族自治区自然科学基金资助项目(2020JJA140128)
广西壮族自治区自然科学基金资助项目(2022JJA140859)
广西壮族自治区自然科学基金资助项目(2022JJA140918)
广西壮族自治区卫生健康委员会自筹经费科研项目(Z20201164)
出版年
2024
NETL
NSTL
万方数据