茶多酚激活Nrf2/HO-1通路减轻阿司匹林诱导的GES-1细胞损伤的机制研究

Mechanism Study of Tea Polyphenols Alleviating Aspirin-induced Injury in GES-1 Cells through Activating Nrf2/HO-1 Pathway

目的探讨茶多酚(tea polyphenols,TP)对阿司匹林诱导的人胃黏膜上皮细胞GES-1损伤的影响及分子机制。方法采用阿司匹林(19.27 mmol·L^(-1))和不同浓度的茶多酚(50、100、200μmol·L^(-1))处理GES-1细胞24 h,将GES-1细胞随机分为对照组、阿司匹林组(19.27 mmol·L^(-1))、阿司匹林19.27 mmol·L^(-1)+茶多酚50μmol·L^(-1)组、阿司匹林19.27 mmol·L^(-1)+茶多酚100μmol·L^(-1)组、阿司匹林19.27 mmol·L^(-1)+茶多酚200μmol·L^(-1)组。采用四甲基偶氮唑盐(MTT)检测GES-1细胞增殖水平,采用流式细胞术和TUNEL实验检测GES-1细胞凋亡水平。采用二氯荧光素双醋酸盐(DCFH-DA)荧光探针检测GES-1细胞活性氧(ROS)水平。采用试剂盒检测GES-1细胞超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、丙二醛(MDA)水平。采用蛋白免疫印迹(Western blot)检测GES-1细胞B淋巴细胞瘤-2(Bcl-2)、B淋巴细胞瘤-2相关X蛋白(Bax)、核因子E2相关因子2(Nrf2)、血红素单加氧酶1(HO-1)蛋白表达水平。结果与对照组比较,阿司匹林组GES-1细胞凋亡率、凋亡指数、ROS、MDA水平、细胞核Nrf2/细胞质Nrf2比值、Bax、HO-1蛋白表达水平显著增加,细胞存活率、Bcl-2蛋白表达水平、SOD、CAT水平显著降低(P<0.05);与阿司匹林组比较,阿司匹林19.27 mmol·L^(-1)+茶多酚(50、100、200μmol·L^(-1))组GES-1细胞亡率、凋亡指数、ROS、MDA水平、Bax蛋白表达水平显著降低,细胞存活率、细胞核Nrf2/细胞质Nrf2比值、Bcl-2、HO-1蛋白表达水平、SOD、CAT水平显著增加(P<0.05)。结论茶多酚可减轻阿司匹林诱导的GES-1细胞损伤,其机制可能与茶多酚激活Nrf2/HO-1抗氧化通路、抑制线粒体通路介导的细胞凋亡有关。

OBJECTIVE To investigate the effect of tea polyphenols(TP)on aspirin-induced injury of human gastric epithelial cells GES-1 and its molecular mechanism.METHODS GES-1 cells were treated with aspirin(19.27 mmol·L^(-1))and different concentrations of tea polyphenols(50,100,200μmol·L^(-1))for 24 h.GES-1 cells were randomly divided into control group,aspirin group(19.27 mmol·L^(-1)),aspirin 19.27 mmol·L^(-1)+TP 50μmol·L^(-1)group,aspirin 19.27 mmol·L^(-1)+TP 100μmol·L^(-1)group,aspirin 19.27 mmol·L^(-1)+TP 200μmol·L^(-1)group.Methyl thiazolyl tetrazolium(MTT)was used to detect the survival rate of GES-1 cells.Flow cytometry and TUNEL assay were used to detect apoptosis of GES-1 cells.Dichlorofluorescein diacetate(DCFH-DA)fluorescent probe was used to detect the levels of reactive oxygen species(ROS)of GES-1 cells.Corresponding kits were used to detect the levels of superoxide dismutase(SOD),catalase(CAT)and malondialdehyde(MDA)of GES-1 cells.Western blot was used to detect the expression levels of B-lymphocytoma-2(Bcl-2),B-lymphocytoma-2-associated X protein(Bax),nuclear factor E2 related factor 2(Nrf2)and heme monooxygenase 1(HO-1)of GES-1 cells.RESULTS Compared with the control group,the apoptosis rate,apoptosis index,ROS,MDA levels,nuclear Nrf2/cytoplasmic Nrf2 ratio,Bax,and HO-1 protein expression levels of GES-1 cells in 19.27 mmol·L^(-1)aspirin group were significantly increased,while the cell survival rate,Bcl-2 protein expression level,SOD and CAT levels were significantly decreased(P<0.05).Compared with 19.27 mmol·L^(-1)aspirin group,the apoptosis rate,apoptosis index,ROS,MDA levels and Bax protein expression levels in aspirin 19.27 mmol·L^(-1)+TP(50,100,200μmol·L^(-1))groups were significantly decreased,while the cell survival rate,nuclear Nrf2/cytoplasmic Nrf2 ratio,Bcl-2,Nrf2,HO-1 protein expression levels,SOD and CAT levels were significantly increased(P<0.05).CONCLUSION Tea polyphenols can alleviate aspirin-induced GES-1 cell damage,and the mechanism may be related to the activation of Nrf2/HO-1 antioxidant pathway and the inhibition of mitochondrial pathway mediated apoptosis.