咖啡酸对CCl_(4)诱导BRL大鼠肝细胞损伤的保护作用

Protective Effect of Caffeic Acid on CCl_(4)-Induced Injury of BRL Hepatocyte

采用体外四氯化碳(carbon tetrachloride,CCl_(4))诱导BRL大鼠肝细胞损伤模型,研究咖啡酸(caffeic acid,CA)对CCl;诱导BRL大鼠肝细胞损伤的保护作用。实验分为对照组、CCl;模型组(100 mmol/L CCl_(4)损伤3 h)和CA干预组(含0.2、0.4和0.8 mg/mL CA的DMEM溶液预处理4 h,再行CCl_(4)暴露3 h),采用全自动生化分析仪检测天冬氨酸氨基转移酶(aspartate aminotransferase,AST)、丙氨酸氨基转移酶(alanine aminotransferase,ALT)和乳酸脱氢酶(lactate dehydrogenase,LDH)活力,试剂盒测定胞内活性氧(reactive oxygen species,ROS)、细胞质中细胞色素C(cytochrome C,Cyt c)和8-羟基脱氧鸟苷(8-hydroxydeoxyguanosine,8-OHdG)水平,qRT-PCR检测核因子E2相关因子2(nuclearfactor erythroid-2-related factor 2,Nrf2)、谷胱甘肽还原酶(glutathione reductase,GSR)、醌氧化还原酶1(quinine oxidoreductase,NQO1)和超氧化物歧化酶(superoxide dismutase,SOD)的表达水平。结果显示,CA对CCl_(4)所致细胞培养液中AST、ALT和LDH活力以及胞内ROS、细胞质中Cyt c和8-OHdG水平的异常升高均具有显著的抑制作用(P<0.05);与对照组相比,模型组细胞中Nrf2、GSR、NQO1和SOD基因的mRNA表达水平无显著差异(P>0.05),而CA干预组中各检测基因的mRNA表达水平显著升高(P<0.05),且具有剂量依赖效应。结果表明,CCl_(4)暴露可使BRL大鼠肝细胞发生氧化应激,而CA可激活Nrf2/ARE信号通路、上调抗氧化基因的mRNA水平,从而有效抑制CCl_(4)对肝细胞的损伤。

This work aimed to investigate protective effect of caffeic acid on CCl_(4)-induced BRL hepatocyte injury. The experiment was divided into control group, CCl;model group(100 mmol/L CCl;injury for 3 hours) and CA pretreated group(pretreated with 0.2, 0.4 and 0.8 mg/mL CA in DMEM for 4 hours, followed by CCl_(4)exposure for 3 hours). The activities of aspartate aminotransferase(AST), alanine aminotransferase(ALT) and lactate dehydrogenase(LDH) were detected by automatic biochemical analyzer. The levels of reactive oxygen species(ROS), cytochrome C(Cyt c) and 8-hydroxydeoxyguanosine(8-OHdG) were measured by the kit. The expression levels of nuclear factor E2-related factor 2(Nrf2), glutathione reductase(GSR), quinine oxidoreductase(NQO1) and superoxide dismutase(SOD) were detected by qRT-PCR. Significant inhibitory effects of CA were observed on the CCl_(4)-induced elevation of AST, ALT, LDH, ROS, Cyt c and 8-OHdG in culture medium(P<0.05). Compared with the control group, no significant differences of the mRNA expression levels of Nrf2, GSR, NQO1 and SOD gene were found in the model group(P>0.05). However, the mRNA expression levels of all detected genes were significantly increased in CA-treated group compared with the control group and a dose-dependent manner was observed. In conclusion, CCl_(4)exposure induced oxidative stress in BRL hepatocytes and CA inhibited the CCl_(4)-induced BRL hepatocyte damage by activating the Nrf2/ARE signaling pathway and up-regulating the mRNA levels of antioxidant genes.