| Objective:To investigate the protective effect of gallic acid on the acute liver injury induced by dimethylnitrosamine and explore its molecular mechanism from a perspective of phaseâ…¡enzymes.Methods:Fifty KM male mice were randomly divided into five groups(n=10):control group; gallic acid group at a dose of 100mg/kg(H-GA group); DMN group; gallic acid pretreatment group at a relatively low dose of 50mg/kg plus dimethylnitrosamine injection(DMN+L-GA); gallic acid pretreatment group at a relatively high dose of 100mg/kg plus dimethy-lnitrosamine injection(DMN+H-GA). GA was administered orally to mice twice daily for three consecutive days while the control group and DMN group received vehicle. Mice were intraperitoneally injected with a single dose of dimethylnitrosamine twelve hours after the last treatment of gallic acid. Twenty four hours later, blood sample was collected. Mice were then sacrificed and liver was quickly excised, rinsed in cold saline and stored correspondingly at appropriate conditions for different means of measurement. Biochemical evaluation includes the activities of serum transaminase, liver SOD, GSH-px, GST and the contents of liver MDA, GSH. All the measurement was performed using commercially available kits and the operation was carried out according to manufacturer's instructions. Formalin-fixed liver samples were sliced into five-micrometer sections for H.E staining and immunohistochemical assessment of HO-1. Western blot analysis was employed to determine the expression level of HO-1, GSTA3 and nuclear Nrf2.Results:Toxicant caused severe liver damage, as was seen from significant increase of serum transaminase activities, submassive hemorrhagic necrosis, glutathione depletion and lipid peroxidation. The SOD and GSH-px activities decreased, which implied that DMN toxication reduced the antioxidant capacity of liver tissue. However, GA pretreatment provided hepatoprotection against DMN-induced liver injury in a dose-dependent manner, which could be observed from an improved liver histological structure, significant decrease of serum transaminase activities and inhibition of lipid peroxidation. GA also enhanced SOD, GSH-px, GST activities and attenuated glutathione depletion caused by DMN. No toxic effect was found when administered gallic acid orally to mice at a dose of 100mg/kg. Compared with the normal group, elevation of serum transaminase activities and MDA level was not observed. Mice also sustained a normal histological pattern. Difference between the normal group and H-GA group lies in the upregulated activities of SOD, GSH-px, GST and elevated level of GSH content. Western Blot analysis revealed that DMN increased the expression level of nuclear Nrf2 due to the ROS generated by DMN, which suggested the celluar defensive mechanism of mammalians. GA also facilitated the translocation of Nrf2 into the nucleus and this induction is more powerful than that of DMN. The DMN+H-GA group has the highest expression level of Nrf2, possibly due to the dual induction effect afforded by DMN and GA. The expression level of cytoplasm HO-1 and GSTA3 corresponds to the nuclear level of Nrf2, as these two enzymes were mainly regulated by the transcriptional factor Nrf2. Immunohistochemisty also provided the same result of HO-1 expression.Conclusions:Short-term administration of GA could enhance the antioxidant capacity of liver tissue in a dose-dependent manner. The upregulation of HO-1 and GSTA3 via Nrf2-ARE mediated by GA contributes to cytoprotection against oxidative stress caused by DMN. |
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