| ObjectiveTrichloroethylene is an important productive toxicant,which can induce occupational medicamentosa-like dermatitis.Clinical studies have shown that occupational trichloroethylene poisoning is accompanied by multiple organ injury,especially liver injury.However,the mechanism of acute liver injury has not been fully elucidated.Studies have shown that oxidative stress is an important mechanism of liver injury induced by trichloroethylene.Nrf2 signaling pathway is the central regulator of cellular antioxidant stress,which is responsible for regulating the expression of antioxidant proteins.However,there are few reports about the oxidative stress effect of trichloroethylene on liver and its effect on Nrf2 signaling pathway.In this study,HepG2 cells were treated with different doses of trichloroethylene to establish trichloroethylene exposure model,to explore the role of Nrf2 signaling pathway in oxidative stress in HepG2 cells induced by trichloroethylene.Furthermore,Nrf2 agonist tBHQ and inhibitor luteolin were selected to up-regulate or down-regulate Nrf2 signaling factors,respectively,to explore the regulatory role of Nrf2 signaling pathway in trichloroethylene induced oxidative stress in HepG2 cells,and to further reveal the toxic mechanism of trichloroethylene.MethodHepG2 cells were cultured in vitro.The specific operation is as follows:HepG2 cells in logarithmic growth period were divided into the following groups: control group and low-,medium-and high-dose TCE groups: treated with 0,2,4 and 8 mmol/L trichloroethylene for 24 h;tBHQ control group and tBHQ+low-,medium-and high-dose TCE groups: after pretreated with 50 μmol/L tBHQ for 12 h,treated with 0,2,4 and 8 mmol/L trichloroethylene for 24 h;luteolin+low-,medium-and high-dose TCE groups: after pretreated with 10 μmol/L luteolin for 12 h,treated with complete medium containing 0,2,4 and 8 mmol/L trichloroethylene for 24 h.After treatment,the cells were harvested.The activity of super oxidedismutase(SOD)was detected by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphenyl)-2h-tetrazolemonosodium 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 and the level of 8-hydroxydeoxyguanosine(8-OHDG)was detected by enzyme-linked immunosorbent assay.The mRNA expression of Nrf2,hemeoxygenase(HO-1),glutamate-cysteine ligase catalytic subunit(GCLC),NAD(P)Hquinone oxidoreductase 1(NQO1)was detected by real-time polymerase chain reaction and the protein expression of Nrf2,HO-1,GCLC,NQO1 was detected by Western blot.Results(1)Effect of trichloroethylene on oxidative stress in HepG2 cells: after TCE exposure for 24 h,compared with control group,the level of MDA in low-dose group decreased(P<0.05),the activity of SOD in high-dose group increased(P<0.05),the activity of CAT in low-,medium-and high-dose groups decreased(P<0.05),but the activity of GSH-PX increased(P<0.05).Under the condition of medium dose of TCE(4 mmol/L),the activity of CAT was significantly lower than that of low dose group(P<0.05),the level of MDA was significantly higher than that of low dose group(P<0.05);compared with medium-dose group,the activities of SOD and CAT of high dose group were significantly lower(P<0.05),the levels of MDA and 8-OHDG were lower(P<0.05).(2)Effect of TCE on Nrf2 signaling pathway in HepG2 cells: after TCE exposure for 24 h,compared with control group,the relative expression levels of Nrf2 and GCLC mRNA and protein in low-and medium-dose groups were significantly decreased(P<0.05),the relative expression levels of HO-1 mRNA were significantly decreased(P<0.05),the relative expression levels of HO-1 protein in medium-dose group were significantly decreased(P<0.05).In high-dose group,the relative expression levels of Nrf2 and HO-1 protein were significantly decreased(P<0.05),the relative expression levels of GCLC mRNA and protein were significantly decreased(P<0.05),the relative level of NQO1 mRNA was significantly increased(P<0.05).The expression levels of NQO1 protein were significantly increased in low-,medium-and high-dose groups(P<0.05).In the high-dose group,the relative expression levels of Nrf2 and NQO1 mRNA and protein,and the relative expression levels of HO-1 mRNA were significantly higher than those in low-and medium-dose groups(P<0.05),and the relative expression levels of HO-1 and GCLC protein in high-dose group were significantly higher than those in medium-dose group(P<0.05).(3)Effect of the up-regulation of Nrf2 signaling factor on oxidative stress and Nrf2 signaling pathway in HepG2 cells induced by trichloroethylene: Compared with the control group,in tBHQ control group the activities of SOD and GSH-Px increased significantly(P<0.05),the activity of CAT decreased significantly(P<0.05),and the level of MDA decreased significantly(P<0.05),the activity of GSH-PX increased significantly in tBHQ+low-,medium-and high-dose groups(P<0.05),and the activity of CAT increased significantly in tBHQ+medium-and high-dose groups(P<0.05);compared with the same dose TCE group without tBHQ pretreatment,the activity of SOD in tBHQ+medium-dose group increased significantly(P<0.05),the activities of GSH-Px and CAT in tBHQ+mediumand high-dose groups increased significantly(P<0.05),and the level of MDA decreased,but the change was not significant(P>0.05).Compared with the control group,in tBHQ control group the relative expression levels of Nrf2,NQO1 mRNA and protein were significantly increased(P<0.05),the relative expression levels of HO-1 protein were significantly increased(P<0.05);in tBHQ+low-,medium-and high-dose groups the relative expression levels of GCLC mRNA were significantly increased(P<0.05),the relative expression levels of NQO1 mRNA were significantly decreased(P<0.05),the relative expression levels of HO-1 protein were significantly decreased(P<0.05);in the tBHQ+medium-and high-dose groups the relative expression levels of Nrf2,HO-1 mRNA were significantly increased(P<0.05),the relative expression level of NQO1 mRNA was significantly decreased(P<0.05),the relative expression level of HO-1 protein was significantly decreased(P<0.05);in the tBHQ+high-dose group the relative expression levels of Nrf2,HO-1 and GCLC mRNA were significantly increased(P<0.05),the relative expression levels of Nrf2,HO-1,GCLC and NQO1 protein were significantly increased(P<0.05).Compared with the same dose TCE group without tBHQ pretreatment,in tBHQ+medium-dose group the relative expression levels of Nrf2,GCLC and NQO1 mRNA and protein were significantly increased(P<0.05),and the relative expression levels of HO-1protein were significantly increased(P<0.05);in the tBHQ+high-dose group the relative expression levels of Nrf2,GCLC mRNA and protein were significantly increased(P<0.05),and the relative expression levels of HO-1protein were significantly increased(P<0.05).(4)Effect of the down-regulation of Nrf2 signaling factor on oxidative stress and Nrf2 signaling pathway in HepG2 cells induced by trichloroethylene:Compared with the control group,the activities of CAT in the low-,medium-and high-dose groups were significantly decreased(P<0.05),and the activities of SOD in the luteolin+high-dose group was significantly decreased(P<0.05);compared with the same dose TCE group without luteolin pretreatment,in luteolin+low-,middle-and high-dose groups the activities of CAT decreased significantly(P<0.05)and the activities of GSH-Px increased significantly(P<0.05),the levels of MDA in luteolin+low-and high-dose groups was significantly increased(P<0.05),the levels of 8-OHDG in luteolin+medium-and high-dose groups increased significantly(P<0.05),the activity of SOD in luteolin+high-dose group decreased significantly(P<0.05).Compared with the control group,the relative expression levels of Nrf2,HO-1,GCLC and NQO1 mRNA and protein in luteolin control group were significantly decreased(P<0.05).Compared with the same dose TCE group without luteolin pretreatment,in the luteolin+low-dose group the relative expression level of Nrf2 protein was significantly decreased(P<0.05),and the expression levels of HO-1,GCLC and NQO1 mRNA and protein were significantly decreased(P<0.05),the the medium-dose group the expression levels of Nrf2,GCLC and NQO1 mRNA and protein were significantly decreased(P<0.05),the expression levels of HO-1 protein were significantly decreased,in the high-dose group the expression levels of Nrf2,HO-1,GCLC and NQO1 mRNA and protein were significantly decreased(P<0.05)Conclusion(1)A certain dose of trichloroethylene can induce oxidative stress in HepG2 cells,reduce the activity of antioxidant enzymes and cause oxidative damage.(2)A certain dose of trichloroethylene can activate Nrf2 signaling pathway,upregulate the gene and protein expression levels of the downstream HO-1,GCLC,NQO1,enhance the activity of antioxidant enzymes,and exert Nrf2 mediated antioxidant effect.(3)In this study,after Nrf2 signaling factor was up-regulated,in the process of trichloroethylene induced oxidative stress in HepG2 cells,the mediating speed of downstream phase Ⅱ detoxification enzyme and antioxidant enzyme was faster,the positive regulatory role was enhanced,and the cell antioxidant capacity was enhanced,so as to antagonize the oxidative damage caused by trichloroethylene.(4)In this study,after Nrf2 signaling factor was down-regulated the expression levels of downstream phase Ⅱ detoxification enzymes and antioxidant enzymes were significantly decreased.During trichloroethylene induced oxidative stress in HepG2 cells,the synthesis of genes and proteins was blocked,the antioxidant mechanism was unbalanced,and the oxidative damage of HepG2 cells was aggravated. |
PDF Full Text Request