饮用水有机提取物对正常人肝细胞中活性氧及细胞凋亡因子表达的影响

Effects of organic extracts from drinking water on ROS and apoptosis factors in L02 cells

目的观察饮用水有机提取物染毒对正常人肝细胞(L02)中活性氧(reactive oxygen species,ROS)及细胞凋亡因子表达水平的影响。方法利用固相萃取法提取饮用水中有机污染物。将L02细胞分别暴露于培养液(空白对照)、0.1%DMSO(溶剂对照)和0.3125、0.6250、1.2500、2.5000、5.0000 L/ml饮用水有机提取物中72 h。采用流式细胞术检测ROS水平,分别采用实时荧光定量PCR(qRT-PCR)法和Western blot法检测细胞色素C(cytochrome C,Cyt C)mRNA、蛋白的表达水平,采用酶联免疫吸附试验(Elisa)检测天冬氨酸特异性半胱氨酸蛋白酶-9(Caspase-9)、天冬氨酸特异性半胱氨酸蛋白酶-3(Caspase-3)酶的活力。结果与溶剂对照组比较,2.5000、5.0000 L/ml有机提取物染毒组L02细胞内的ROS水平较高,差异均有统计学意义(P<0.05);且随着有机提取物染毒剂量的升高,L02细胞内的ROS水平呈上升趋势。与溶剂对照组比较,1.2500、2.5000、5.0000 L/ml有机提取物染毒组L02细胞内的Cyt C mRNA和蛋白表达水平均较高,差异均有统计学意义(P<0.05);且随着有机提取物染毒剂量的升高,L02细胞内的Cyt C mRNA和蛋白表达水平均呈上升趋势。与溶剂对照组比较,各剂量有机提取物染毒组L02细胞内的Caspase-9及Caspase-3酶活力均较高,差异均有统计学意义(P<0.05);且随着有机提取物染毒剂量的升高,L02细胞内的Caspase-9及Caspase-3酶活力均呈上升趋势。结论饮用水有机提取物可引起L02细胞ROS水平增加,导致Cyt C表达水平上调,进而诱导Caspase-9和Caspase-3酶活力水平升高。氧化损伤调控肝细胞凋亡因子变化可能是饮用水有机提取物致肝细胞损伤的重要环节。

Objective To observe the effects of ROS and apoptosis factors levels in human hepatocytes(L02)cells exposed to organic extracts from drinking water.Methods Organic pollutants in water samples were extracted by solid phase extraction(SPE)methods.L02 cells were exposed to culture medium(blank control),0.1%DMSO(solvent control),organic extracts from drinking water at the dose of 0.3125,0.6250,1.2500,2.5000,5.0000 L/ml for 72 h respectively.The level of ROS was measured by flow cytometry.mRNA and protein expression levels of cytochrome C(Cyt C)gene were determined by real-time fluorescence quantitative PCR(qRT-PCR)and Western blot.The levels of Caspase-9 and Caspase-3 were detected by ELISA.Results Compared with the solvent control group,the ROS levels were increased in 2.5000 and 5.0000 L/ml organic extract groups in L02 cells,the difference was statistically significant(P<0.05);And the level of ROS in the exposure groups presented a gradually increasing trend with the increasing organic extract dose in L02 cells.Compared with the solvent control group,mRNA and protein expression levels of Cyt C were increased in 1.2500,2.5000 and 5.0000 L/ml organic extract groups in L02 cells,the difference was statistically significant(P<0.05);mRNA and protein expression levels of Cyt C gene in the exposure groups presented a gradually increasing trend with the increasing organic extract dose in L02 cells.Compared with the solvent control group,the activities of Caspase-9 and Caspase-3 were increased in organic extract groups in L02 cells,the difference was statistically significant(P<0.05);And the activities of caspase-9 and caspase-3 in the exposure groups presented a gradually increasing trend with the increasing organic extract dose in L02 cells.Conclusion Organic extracts from drinking water may induce the increase of ROS level in L02 cells,up-regulate the expression level of Cyt C gene,and cause the high levels of Caspase-9 and Caspase-3 at last.Oxidative damage,apoptosis might be the mechanisms for L02 cells damage caused by organic extracts from drinking water.