Experimental Study On The Nrf2-ARE Pathway During The Isoniazid-induced Liver Injury In Mice

Objectives To observe the expressions of NF-E2-related factor-2(Nrf2), superoxidedismutase(SOD) and glutathione-S-transferase(GST) which induces by isonicotinic acidhydrazide(INH) in mice liver. To explore the effect of Nrf2-ARE pathways in developmentand progression in detoxification and antioxidation.Methods A total of112clean Kunming mice were randomly divided into14groups.The experimental mice were given isoniazid orally at90mg/kg body weigh at1d,3d,5d,7d,2w,3w,4w and the control group was given distilled water. Serum ALT, AST, ALPand ALB levels were determined using an automatic biochemical analyzer, andGST/SOD activities and GSH/MDA contents were detected by biochemical method.Enzyme-Linked immunosorbant assay was used to test the protein contents of Nrf2, GSTand SOD. SYBR Green Real-time RT-PCR was used to test the expressions of Nrf2,GST and SOD mRNA. Immunohistopathology was used to observe the membranetransport of Nrf2.Results The general situation in the process of experimental were observed firstly, themice in control group were in good station and in the treatment groups became dispirited.With the increasing of time, the weights of mice in every group were increased. Theweight of the mice in experimental group and control group was significantly lower at4wthan the control group(t=3.780, P=0.002). Then the pathological changes were observedand the general liver function index for clinical diagnosis of liver disease was checked.Hepatocytes showed swelling at3d, dual-core cells increased and liver cell regeneratedat4w. The liver index was higher than the control group in the group of5d and7d. Thelevels of ALT were significantly higher at2w (F=13.425, P=0.000) than the control ones;and AST were significantly higher at4w (F=3.498, P=0.004) than the control ones.Theresults prompted liver function index can not reflect the damage of liver cell timely.Thirdly, the indicators of the damage of liver tissue, the ability of Ⅱdrug metabolismand the capacity of oxidative stress resistance showed the concentration of MDA at7d,2w,4w groups were higher (F=3.816, P=0.002); and it was highest in the group of7d.The content of GSH in the group of7d was significantly lower than those in control group (F=11.869, P<0.001). The total activity of GST and SOD at5d,7d,2w weresignificantly lower than control group(F=3.365, P=0.005; F=3.769, P=0.002), then wererecovered to normal levers at3w and4w gradually. Fourthly, the results of the situationof across nuclear membrane transport showed that in the normal control group mice livertissue Nrf2protein mainly expressed in cytoplasm, a visible expression in nucleus in thegroup of7d (F=161.477, P<0.001). The characteristic was synchronous with activity ofGST/SOD and the content changes of MDA/GSH in the first2weeks. Prompted thatNrf2protein appeared across the nuclear membrane transport with the occurrence of livercell injury and both were positively correlated. Fifthly, mRNA and protein expression ofdownstream related indicators of Nrf2-ARE pathways were analyzed. With the increaseof GSTA1, GSTM1, Cu-ZnSOD and MnSOD mRNA expression, the correspondingprotein expression also showed a trend of increase accordingly. Nrf2protein began toacross the nuclear membrane at7d, then GSTA1mRNA expression ratio in2w/3w and4w were increased (F=9.375, P<0.001), the content of protein was significantly lowest at2w (F=10.157, P<0.001) than the control ones and in3w/4w when the protein contentwas higher than2w. The GSTM1mRNA expression ratio in3w and4w were increased(F=3.259, P=0.005), the content of GSTM1were significantly lower at2w (F=6.374,P<0.001) than the control ones. At the same time, the activity of GST rose to normallevel at3w and4w. Prompted that Nrf2protein appeared across the nuclear membranetransport and up-regulated the expression of GST, then adjusted the ability of phaseⅡ drug metabolism. Cu-ZnSOD and MnSOD mRNA expression ratio were increased at3w (F=8.261, P<0.001; F=4.026, P=0.002); the content of protein were significantlylowest at2w (F=2.303, P=0.039; F=10.928, P<0.001) than the control ones and thecontent rose to normal level at3w and4w. Prompted that Nrf2protein appeared acrossthe nuclear membrane transport and up-regulated the expression of SOD, then adjustedthe ability of antioxidation.Conclusions In the development of liver injury by INH, the Nrf2-ARE access isactivated, GST and SOD gene expression up-regulated, then adjusts the ability of phaseⅡdrug metabolism and antioxidation.