摘要
目的探讨三氯乙烯(TCE)致人肝癌细胞系HepG2细胞氧化应激中核因子E2相关因子2(NRF2)信号通路的作用。方法取对数生长期HepG2细胞随机分为对照组和低、中、高剂量组,分别以终浓度为0、2、4、8 mmol/L的TCE刺激24 h后,收集细胞,采用2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H-四唑单钠盐法检测超氧化物歧化酶(SOD)活力,酶促法检测过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活力,硫代巴比妥酸法检测丙二醛(MDA)水平,酶联免疫吸附法检测8-羟基脱氧鸟苷(8-OHDG)水平,实时荧光聚合酶链式反应法检测NRF2、血红素加氧酶-1(HO1)、谷氨酸半胱氨酸连接酶催化亚基(GCLC)和醌氧化还原酶1(NQO1)的mRNA表达,蛋白免疫印迹法检测NRF2、HO1、GCLC和NQO1蛋白的表达。结果与对照组比较,3个剂量组HepG2细胞CAT活力均下降(P值均<0.05),GSH-Px活力均升高(P值均<0.05);低剂量组HepG2细胞MDA水平下降(P<0.05)。4组HepG2细胞SOD活力和8-OHDG水平分别比较,差异均无统计学意义(P值均>0.05)。与对照组比较,低、中剂量组HepG2细胞NRF2、GCLC的mRNA和蛋白相对表达水平均下降(P值均<0.05),HO1 mRNA相对表达水平均下降(P值均<0.05)。与对照组比较,低剂量组HepG2细胞HO1蛋白相对表达水平升高(P<0.05),NQO1的mRNA和蛋白相对表达水平均升高(P值均<0.05)。中剂量组HepG2细胞HO1蛋白相对表达水平低于对照组(P<0.05)。与其余3组比较,高剂量组HepG2细胞NRF2和NQO1的mRNA和蛋白相对表达水平均升高(P值均<0.05),HO1 mRNA相对表达水平均升高(P值均<0.05)。结论低、中剂量TCE刺激可导致HepG2细胞发生氧化应激,抗氧化物酶活力降低;高剂量TCE刺激HepG2细胞可激活NRF2信号通路,调控下游抗氧化物酶HO1、GCLC、NQO1的表达上调,缓解TCE所致的氧化损伤。
Objective To investigate the role of nuclear factor erythroid-2-related factor 2(NRF2) signaling pathway in trichloroethylene(TCE) induced oxidative stress in human liver cancer HepG2 cells. Methods HepG2 cells in the exponential growth phase were randomly divided into control group and low-, medium-and high-dose groups, and the cells were stimulated with TCE at the final concentrations of 0, 2, 4 or 8 mmol/L respectively for 24 hours. After TCE exposure, the cells were collected. The activity of superoxide dismutase(SOD) was measured by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphenyl)-2 h-tetrazole monosodium salt method.The activities of catalase(CAT) and glutathione peroxidase(GSH-Px) were detected by enzymatic method. The level of malondialdehyde(MDA) was detected by thiobarbituric acid method. The level of 8-hydroxydeoxyguanosine(8-OHDG) was detected by enzyme-linked immunosorbent assay. The mRNA expression of NRF2, heme oxygenase(HO1), glutamate-cysteine ligase catalytic subunit(GCLC), NAD(P) quinone oxidoreductase 1(NQO1) were detected by real-time polymerase chain reaction and the protein expression of NRF2, HO1, GCLC, NQO1 were detected by Western blotting. Results The activity of CAT in HepG2 cells decreased in the 3 doses drug exposure groups(all P<0.05). The activity of GSH-Px increased(all P<0.05), while the level of MDA in HepG2 cells decreased in low-dose group compared with the control group(P<0.05). There was no statistical significance in SOD activity and 8-OHDG level of HepG2 cells among these 4 groups(all P>0.05). The mRNA and protein relative expression of NRF2 and GCLC in HepG2 cells decreased in the low-and medium-dose groups(all P<0.05) compared with the control group. The mRNA relative expression of HO1 decreased(all P<0.05). The HO1 protein relative expression of HepG2 cells increased in low-dose group compared with the control group(P<0.05). The mRNA and protein relative expression of NQO1 in low-dose group increased(both P<0.05). The protein relative expression of HO1 in HepG2 cells in medium-dose group was lower than that in control group(P<0.05).The mRNA and protein relative expression levels of NRF2 and NQO1 increased in HepG2 cells of high-dose group(all P<0.05) and the mRNA relative expression of HO1 increased in HepG2 cells of high-dose group(all P<0.05) compared with the other 3 groups. Conclusion The low and medium dose TCE stimulation can cause oxidative stress in HepG2 cells and decrease the antioxidant enzyme activity. The high dose TCE stimulation to HepG2 cells can activate NRF2 signaling pathway, thus upregulating the expression of downstream antioxidant enzymes HO1, GCLC and NQO1, and relieving the oxidative damage caused by TCE.
作者
钟海
吴维权
唐飞
黄璐
邹志辉
余日安
ZHONG Hai;WU Weiquan;TANG Fei;HUANG Lu;ZOU Zhihui;YU Rian(Department of Occupational and Environmental Health,School of Public Health,Guangdong Pharmaceutical University,Guangzhou,Guangdong 510310,China;不详)
出处
《中国职业医学》
CAS
北大核心
2020年第6期660-665,共6页
China Occupational Medicine
基金
2018年宝安区医疗卫生基础研究项目(2018JD052)。
