七叶皂苷HK-2细胞毒性的氧化应激机制

Oxidative mechanism for toxicity induced by sodium aescinate in HK-2 cells

目的进一步研究七叶皂苷肾细胞毒性的氧化应激机制及谷胱甘肽(GSH)对七叶皂苷毒性的保护作用。方法以人肾近曲小管上皮细胞(HK-2)为模型,荧光探针法检测七叶皂苷钠(SA)0,10,15,20和25μmol·L-1处理2h后细胞内活性氧(ROS)含量变化;分别用N-乙酰-L-半胱氨酸(NAC)1,5和10mmol·L-1,丁硫氨酸亚砜亚胺(BSO)0.5和2.5mmol·L-1预处理HK-2细胞4h,然后加入SA20μmol·L-1作用24h,DTNB法测定预处理后及加入SA后细胞内GSH的含量;MTT法测定经NAC5mmol·L-1或BSO2.5mmol·L-1预处理及未经预处理的HK-2细胞与SA10,15,20,25,30和40μmol·L-1作用24h后的细胞存活率,并计算IC50值。结果 SA15,20和25μmol·L-1作用2h后,HK-2细胞内ROS含量显著高于正常对照组(P<0.01)。与正常对照组1×106个细胞内的GSH含量(5.1±1.2)nmol相比,BSO2.5mmol·L-1及NAC10mmol·L-1预处理后1×106个细胞内的GSH含量2.8±0.8和(2.7±2.3)nmol均显著降低(P<0.05),其他预处理组GSH含量无显著变化。除NAC10mmol·L-1外,各预处理组与SA20μmol·L-1作用24h后GSH含量显著降低(P<0.01)。与未经预处理的SA20,25和30μmol·L-1细胞存活率〔(83±5)%,(69±5)%和(54±6)%〕相比,经BSO2.5mmol·L-1预处理后,对应的细胞存活率显著降低,分别为〔(69±6)%,(40±13)%和(25±15)%〕(P<0.05),NAC预处理对细胞存活率无显著影响;未经预处理及NAC和BSO预处理的SA的IC50分别为31.3±1.7,23.6±2.7和(34.2±1.5)μmol·L-1。结论七叶皂苷通过氧化应激途径发挥肾细胞毒性,细胞内谷胱甘肽可对其氧化损伤起到保护作用。

OBJECTIVE To explore the oxidative stress mechanism of aescin-induced nephrotoxicity and the protective role of glutathione(GSH) by using modulators of GSH.METHODSA fluorescent probe dichlorofluorescin diacetate(DCFH-DA) was used to determine the reactive oxygen species(ROS) changes in HK-2 cells following 2 h exposure to sodium aescinate(SA).HK-2 cells were pretreated with BSO 0.5 and 2.5 mmol·L-1 or NAC 1,5 and 10 mmol·L-1,respectively for 4 h,prior to 24 h exposure to SA 20 μmol·L-1.GSH concentrations were measured using GSH assay kit before and after SA treatment.HK-2 cells were pretreated with N-acetyl-L-cysteine(NAC) 5 mmol·L-1 or L-buthionine-sulfoximine(BSO) 2.5 mmol·L-1 for 4 h,and then exposed to SA 10,15,20,25,30 and 40 μmol·L-1 for 24 h.MTT assays were used to assess HK-2 cell survival.RESULTS SA 15,20 and 25 μmol·L-1 for 2 h enhanced significantly cellular ROS level in HK-2 cells(P<0.01).Compared with control group(5.1±1.2)nmol,pretreatment with BSO 2.5 mmol·L-1 or NAC 10 mmol·L-1 decrease GSH levels in cells 2.8±0.8 and(2.7±2.3)nmol,respectively.SA 20 μmol·L-1 caused GSH decrease in all the pretreated groups except that with NAC 10 mmol·L-1.Compared with SA group,SA 20,25 and 30 μmol·L-1 pretreated with BSO 2.5 mmol·L-1 lowered HK-2 cell survival,while pretreatment with NAC caused no significant change in cell viability at any concentration tested.IC50 of SA in control group,NAC and BSO pretreated group were 31.3±1.7,23.6±2.7 and(34.2±1.5)μmol·L-1,respectively.CONCLUSIONOxidative stress is the mechanism of aescin induced toxicity in renal epithelial cells.