Study On Mechanism Of Nrf2 In L02 Cell Injury Induced By Alcohol

Objective:To explore the injury of alcohol on human liver L02 cells and the mechanism of Nrf2 in the process of liver cell injury induced by alcohol.Methods:RNA interference was used to down-regulate the expression of Nrf2 in L02 cells.The sequence of Nrf2-si RNA used to transfect L02 cells was: sense: 5'-CCAGAACACUCAGUGGAAUTT-3';antisense:5'-AUUCCACUGAGUGUUCUGGTT-3'.The expression of Nrf2 m RNA in L02 cells was assayed by RT-PCR.The expression of Nrf2 in L02 cells was tested by Western blotting.MTT assay was used to detect the change of survival rate of L02 cells.The activity of glutathione peroxidase in L02 cells was determined by colorimetric method.The activity of superoxide dismutase in L02 cells was determined by hydroxylamine method.The content and distribution of ROS was observed by DCFH-DA staining,and flow cytometry(FCM)was used to detect the ROS content in L02 cells.The change of cell nucleus morphology was observed by DAPI staining and to judge the change of apoptosis from this.The rate of cell apoptosis was detected with Annexin V-FITC/PI dual staining.Results:1.L02 cells were treated with different concentrations of alcohol,and the cell survival rate was determined by MTT assay.Ethanol at the concerntration of 100 mmol/L,200 mmol/L,400 mmol/L and800 mmol/L,the rates of different groups cells proliferation at 24 h were 72.30%,53.14%,40.79% and34.67%.The survival rate of L02 cells decreased significantly with the increase of alcohol concentration(P<0.01).2.L02 cells were treated with alcohol at concentrations of 100 mmol/L,200 mmol/L and 400mmol/L for 3 h,and expression level of Nrf2 in cytoplasm and nucleus was detected by Western blotting.The expression of Nrf2 in the cytoplasm was decreased and the expression of Nrf2 in the nucleus was increased with the increase of alcohol concentration.It showed that alcohol could promote the nucleartransfer of Nrf2.3.Nrf2-si RNA expression vector was constructed.The RT-PCR showed that Nrf2 m RNA was significantly down-regulated,and the Western blotting showed that Nrf2 protein expression was significantly down-regulated,which indicated that Nrf2-si RNA could effectively inhibit expression of Nrf2.4.Nrf2-si RNA was transfected into L02 cells,and cells were treated with the corresponding concentration of alcohol.Cell glutathione peroxidase activity was determined.The enzyme activities of 0mmol/L alcohol group,200 mmol/L alcohol group,0 mmol/L alcohol + Nrf2-si RNA group and 200mmol/L alcohol + Nrf2-sir RNA group were 1931.14 U,3569.90 U,1908.62 U and 2809.61 U.Compared with the 0 mmol/L alcohol group,the activity of glutathione peroxidase in the 200 mmol/L alcohol group was significantly increased(P<0.01).Compared with the 200 mmol/L alcohol group,the activity of glutathione peroxidase in the 200 mmol/L alcohol + Nrf2-si RNA group was significantly decreased(P<0.01).There was no significant difference in glutathione peroxidase activity between the 0 mmol/L alcohol group and the 0 mmol/L alcohol + Nrf2-si RNA group(P>0.05).The activity of superoxide dismutase in the 0 mmol/L alcohol group,200 mmol/L alcohol group,0 mmol/L alcohol + Nrf2-si RNA group and 200 mmol/L alcohol + Nrf2-si RNA group were 117.83 u/mg prot,195.26 u/mg prot,112.60u/mg prot and 162.05 u/mg prot.Compared with the 0 mmol/L alcohol group,the activity of superoxide dismutase in the 200 mmol/L alcohol group was significantly increased(P<0.01).Compared with the 200mmol/L alcohol group,the activity of superoxide dismutase in the 200 mmol/L alcohol + Nrf2-si RNA group was decreased(P<0.01).There was no significant difference in superoxide dismutase activity between the 0 mmol/L alcohol group and the 0 mmol/L alcohol + Nrf2-si RNA group(P>0.05).5.Nrf2-si RNA was transfected into L02 cells,and cells were treated with the corresponding concentration of alcohol.The changes of ROS in cells were observed by DCFH-DA staining.Fluorescence intensity is positively correlated with ROS level.Compared with the 0 mmol/L alcohol group,the fluorescence intensity of the 200 mmol/L alcohol group was increased,which indicated that the ROS level of intracellular increased.Compared with the 200 mmol/L alcohol group,the ROS content of intracellular was significantly increased in the 200 mmol/L alcohol + Nrf2-si RNA group.Compared with the 0 mmol/L alcohol group,there was no significant change about ROS content in the 0 mmol/L alcohol + Nrf2-si RNAgroup.FCM was used to quantitatively detect changes of ROS.The fluorescence intensity of 0 mmol/L alcohol group,200 mmol/L alcohol group,0 mmol/L alcohol + Nrf2-si RNA group and 200 mmol/L alcohol + Nrf2-si RNA group were 102.50%,131.16%,104.43%,157.86 %.Compared with the 0 mmol/L alcohol group,the ROS level in the 200 mmol/L alcohol group was significantly higher(P<0.01).Compared with the 200 mmol/L alcohol group,the ROS content in the 200 mmol/L alcohol + Nrf2-si RNA group was increased significantly(P<0.01).Compared with the 0 mmol/L alcohol group,there was no significant change about ROS content in the 0 mmol/L alcohol + Nrf2-si RNA group(P>0.05).6.Nrf2-si RNA was transfected into L02 cells,and cells were treated with the corresponding concentration of alcohol.Survival rate was determined by MTT assay.Compared with the 0 mmol/L alcohol group,the survival rate of the 0 mmol/L alcohol + Nrf2-si RNA group was 97.20%,and there was no significant difference(P>0.05).The cell survival rate of the 200 mmol/L alcohol group was 57.31%,which was significantly lower than the 0 mmol/L alcohol group(P<0.01).The cell survival rate of the 200mmol/L alcohol + Nrf2-si RNA group was 48.10%,which was significantly lower than the 0 mmol/L alcohol group(P<0.01).The cell survival rate of the 200 mmol/L alcohol + Nrf2-si RNA group was lower than the 200 mmol/L alcohol group,and there was a significant difference(P<0.01).7.Nrf2-si RNA was transfected into L02 cells,and cells were treated with the corresponding concentration of alcohol.The change of cell nucleus morphology was observed by DAPI staining.Compared with the 0 mmol/L alcohol group,the 0 mmol/L alcohol + Nrf2-si RNA group showed no significant changes.There were apoptosis phenomena such as chromatin agglutination and shrinikage in the 200 mmol/L alcohol group and the 200 mmol/L alcohol + Nrf2-si RNA group.Compared with the 200mmol/L alcohol group,the apoptosis characteristics,such as chromatin agglutination,were more obvious in the 200 mmol/L alcohol + Nrf2-si RNA group.The rate of cell apoptosis was detected with Annexin V-FITC/PI dual staining.The apoptosis rates of 0 mmol/L alcohol group,200 mmol/L alcohol group,0mmol/L alcohol + Nrf2-si RNA group and 200 mmol/L alcohol + Nrf2-si RNA group were 2.29%,17.17%,3.15%,30.04%.Compared with the 0 mmol/L alcohol group,the apoptosis rate of the 200 mmol/L alcohol group was significantly increased(P<0.01).Compared with the 200 mmol/L alcohol group,the apoptosis rate of the 200 mmol/L alcohol + Nrf2-si RNA group was significantly increased(P<0.01).There was no significant difference in apoptosis rate between the 0 mmol/L alcohol group and the 0 mmol/L alcohol +Nrf2-si RNA group(P>0.05).Conclusion:1.When alcohol treated with L02 cells,the expression of Nrf2 was increases.The nuclear translocation of Nrf2 was obvious,which enhanced activity of transcriptional regulation.The activity of the targeted regulation of antioxidant enzymes was significantly improved.This reduced the content of ROS in cells.2.During the oxidative injury of liver L02 cells induced by alcohol,Nrf2 protein has a protective effect on cells.The cell survival rate was increases and the apoptosis rate was decreases..