Analysis Of GSTA1 Expression Regulated By HNF-1 On Acetaminophen Induced Acute Hepatic Injury In Mice

Drug-induced liver injury means that when using drug, itself or its metabolites will cause liver injury. Patients’ susceptibility to drugs can also lead to liver damage. Acetaminophen(APAP) becomes the liver injury model with ideal and practical application because of it exists in many kinds of antipyretic analgesics. Glutathione S-transferase A1(GSTA1) is the most important phase II metabolizing enzyme in vivo which can protect cells from oxidative damage and resist cancer reaction. It’s detoxification of foreign toxic substances and active oxygen has become an important cellular mechanism of oxidative stress. Hepatocyte nuclear factor-1(HNF-1) is a kind of transcription factor which can regulate specific gene expression in liver, it can be highly expressed in liver and contains the DNA binding region which is quite conservative in evolution. HNF-1 can regulate the target gene expression by combining with the cis acting elements in the regulatory regions of target genes to play an important role in the differentiation and metabolism of hepatocytes at the transcriptional level. This experiment would study on the regulation of GSTA1 expression in mice under the inhibitory effect of C2-ceramide and the stimulative function of oltipraz on HNF-1. The regulatory effect of HNF-1on the expression of GSTA1 was determined and analyzed. All the above have laid a theoretical and experimental basis for a comprehensive understanding of the important role of GSTA1 in acute drug induced liver injury and its mechanism of action. It is possible that GSTA1 expression regulated by HNF-1 will become an important new direction of drug development.In this experiment, the acute liver injury model induced by APAP in mice was first copied.According to the activities of ALT and AST, liver tissue homogenate indexes(SOD、MDA、GSH、GSH-Px) GSTA1 content and pathological sections in liver as the bases for judging liver injury. Then C2-ceramide and oltipraz were test for the optimum route of medication and concentration based on the activities of ALT and AST and used in liver injury model. Mice were randomly divided into six groups, control group, model group, C2 group, C2+AP group, OL group, OL+AP group. Activities of ALT and AST, liver tissue homogenate indexes(SOD 、MDA、GSH、GSH-Px) and pathological sections of liver tissue were used to analysis the effect of C2-ceramide and oltipraz on liver. The method of RT-PCR was used to measure the m RNA expression of GSTA1 and HNF-1, the protein expression level of GSTA1 and HNF-1 was determined by the method of western blot and GSTA1 content was determined by ELISA. The regulatory effect of HNF-1 on the expression of GSTA1 was studied under the inhibitory effect of C2-ceramide and the stimulative function of oltipraz on HNF-1.Results(1) In APAP induced acute liver injury model, there were damaged cells, nuclear condensation, vague liver cell cord, many inflammatory cell infiltrations and bleeding points.Compared with the control group, ALT and AST activities in the model group increased significantly(p<0.01), the levels of SOD, GSH and GSH-Px decreased significantly(p<0.01),MDA content increased significantly(p<0.01) and GSTA1 content in liver was significantly reduced(p<0.01), which indicated that the acute drug induced liver injury model would be successfully replicated in mice by perfusing 200 mg·kg-1 dose of APAP for 12 h.(2) The best route of administration is intragastric administration. The optimum concentration of C2-ceramide is 120 μmol·L-1 and oltipraz is 150 μmol·L-1.(3) C2-ceramide and oltipraz were applied into APAP induced acute liver injury model.Compared with the control group, the model group serum transaminase activities increased significantly(p<0.01), the indexes of liver tissue homogenate showed significant changes(p<0.01). However, all the indexes in the C2 group and OL group were not significantly changed,which proved that C2-ceramide and oltipraz can’t cause liver damage. Compared with the model group, serum transaminase activities in the C2+AP group increased significantly(p<0.01),OL+AP group decreased significantly(p<0.01). Liver tissue indexes changed significantly(p<0.01). Histological examination showed a large number of necrotic cells, fuzzy liver cell cords, inflammatory cell infiltration and bleeding points in C2+AP group. The severity level of liver injury in OL+AP group was almost close to the control group, but there was a small amount of inflammatory cell infiltration and nuclear condensation. All of the above results showed that C2-ceramide can increase the degree of liver injury, while oltipraz can reduce the liver damage.(4) The results showed that, compared with the control group, the HNF-1 expression level of the model group decreased significantly(p<0.01), the C2 group and OL group were not significantly changed. Compared with the model group, the expression level of HNF-1 in the C2+AP group decreased significantly(p<0.01), while the OL+AP group increased significantly(p<0.01). The above results showed that acute liver injury induced by APAP can reduce the expression level of HNF-1. When liver is damaged, C2-ceramide can down regulate the expression level of HNF-1, oltipraz can upregulate its expression level.(5) The results showed that, compared with the control group, the GSTA1 expression level and content of the model group decreased significantly(p<0.01), the C2 group and OL group were not significantly changed. Compared with the model group, the expression level and content of GSTA1 in the C2+AP group decreased significantly(p<0.01), while the OL+AP group increased significantly(p<0.01). The above results were consistent with the results of HNF-1 expression level.This study determined that the acute drug induced liver injury model was successfully replicated in mice with 200 mg·kg-1dose of APAP administered orally for 12 h. In the model, thecontent of GSTA1 in liver was significantly decreased, which indicated that GSTA1 plays an important role in antioxidant activities of bodies in the acute phase of drug induced liver injury.The optimal routes and optimum concentrations of C2-ceramide and oltipraz were determined. It is determined that C2-ceramide can increase the degree of liver injury, while oltipraz can reduce the liver damage. When liver is damaged, C2-ceramide can down regulate the expression level of HNF-1, oltipraz can upregulate its expression level. It is analyzed that during the acute drug-induced liver injury, HNF-1 can regulate GSTA1 expression level, and the expression level of m RNA was consistent with the trend of protein expression. It will play an indirect role in the protection of liver, that HNF-1 regulates the expression level of GSTA1 through some gene pathway. The mechanism needs to be studied further.