摘要
目的探究依帕司他对高糖诱导的肾小球系膜细胞抑制氧化应激的分子机制。方法将106个肾小球系膜细胞转移至培养瓶中,分为正常生理浓度葡萄糖(5.5 mmol·L-1)组、高浓度葡萄糖(30 mmol·L-1)组、依帕司他高剂量浓度组(10μmol·L-1)、依帕司他中剂量浓度组(1μmol·L-1)、依帕司他低剂量浓度组(0.1μmol·L-1),在37℃,体积分数5%CO2培养箱内于无血清培养基培养48 h。分别测定细胞内活性氧(reactive oxygen species,ROS)、超氧化物歧化酶(superoxide dismutase,T-SOD)、铜锌-超氧化物歧化酶(Cu/Zn-SOD)、谷胱甘肽(glutathione,GSH)、一氧化氮(NO)含量。采用western-blot方法分别测定肾小球系膜细胞内硝化酪氨酸蛋白(NT)来反映肾小球细胞内过氧亚硝基阴离子(ONOO-)的生成量。结果与高浓度葡萄糖组对比,依帕司他各剂量组中的肾小球系膜细胞内ROS含量显著地降低;系膜细胞中T-SOD和Cu/Zn-SOD含量显著升高;系膜细胞中GSH的含量无显著变化。与高浓度葡萄糖组相比,依帕司他各剂量组的系膜细胞中NO含量显著地升高;高剂量(10μmol·L-1)和中剂量(1μmol·L-1)的依帕司他能够显著地降低NT的蛋白表达,表明ONOO-的生成被显著抑制。结论依帕司他能够升高由高糖诱导的肾小球系膜细胞中T-SOD和Cu/Zn-SOD含量,增强其对ROS的清除能力,降低由高糖诱导的系膜细胞中ROS含量。依帕司他虽能够升高NO生成,但仍能抑制高糖培养的系膜细胞中NT蛋白表达,抑制ONOO-生成。
Objective To investigate the mechanism of Epalrestat on oxidative stress induced by high glucose in glomerular mesangial cells.Methods The glomerular mesangial cells were were plated at 1×106 cells per flask,and were divided into five groups:control group,where cells were cultured in 5.5 m mol·L-1glucose(physiological level) dulbecco’s modified eagle medium(DMEM) without FBS;high glucose group,where cells were cultured in 30 mmol·L-1glucose DMEM without FBS;high glucose group treated with 10 μmol·L-1Epalrestat,1 μmol·L-1Epalrestat,or 0.1 μmol·L-1Epalrestat.After 48 h treatment,reactive oxygen species(ROS),total-superoxide dismutase(T-SOD),Copper/Zinc-superoxide dismutase(Cu/Zn-SOD),glutathione(GSH) and nitric oxide(NO) in glomerular mesangial cells were detected by microplate reader,respectively.The western-blot was used to qualify the nitrotyrosine(NT) to refleact the productuin of peroxynitrite(ONOO-).Results Compared with glomerular mesangial cells treated with high level of glucose alone,after cultured in different concentration of Epalrestat,the levels of ROS significantly reduced and the level of T-SOD and CuZn-SOD strikingly improved.Interestingly,the Epalrestat could not influence the level of GSH in the glomerular mesangial cells treated with high glucose.Compared with glomerular mesangial cells treated with high level of glucose alone,after cultured in different concentration of Epalrestat,their levels of NO significantly increased.Additionally,glomerular mesangial cells treated with Epalrestat(10 or 1 μmol·L-1) exhibited a lower expression of nitrotyrosine protein than they cultured in high level of glucose alone.Conclusion We found that through increased the level of T-SOD and Cu/Zn-SOD,Epalrestat could significantly enhance ROS clearance,thus reduce the high level of ROS in glomerular mesangial cells induced by high glucose.Epalrestat increase the production of NO in glomerular mesangial cells cultured in high level of glucose,however,Epalrestat could inhibit the expression of NT protein in glomerular mesangial cells cultured in high glucose,thus decreased the production of ONOO-.
作者
张瑞娇
崔皓月
匡亚飞
任舒
刘铮
ZHANG Ruijiao;CUI Haoyue;KUANG Yafei;REN Shu;LIU Zheng(School of Pharmacy,Shenyang Pharmaceutical University,Shenyang 110016,China)
出处
《沈阳药科大学学报》
CAS
CSCD
北大核心
2020年第2期157-161,共5页
Journal of Shenyang Pharmaceutical University
关键词
依帕司他
肾小球系膜细胞
氧化应激
糖尿病肾病
Epalrestat
glomerular mesangial cells
oxidative stress
diabetic nephropathy
