The Effect Of AMP-activated Protein Kinase Activity Induced By Oxidative Stress In Isolated Pig Hepatocytes

The experiment was conducted to investigate the effect of the oxidative stress on the AMPK activity. Oxidative stress was produced by H₂O₂ or paraquat (PQ) in isolated perfused piglet hepatocytes in vitro. Vc was used to protect cells from oxidative stress.The experiment designed six treatments: control (basal culture), H₂O₂ (0.6mmol/L), PQ (1.00mmol/L), H₂O₂ (0.6mmol/L) and Vc (0.2mmol/L), PQ (1.00mmol/L) and Vc (0.2mmol/L), Vc (0.2mmol/L). The level of TC (total cholesterol), TG (triglycerides), ALT (alanine aminotransterase) and LDH(lactate dehydrogenase) in cells' cultural supernatant were measured. The activity of SOD (superoxide dismutase) and GSH-Px(glutathion peroxidase), the content of NO(nitric oxide) and MDA (malondialdehyde) in cells were determined. The activity of AMPK was also studied in piglet hepatocytes. The results were as follows:H₂O₂ can increase the activity of ALT by 30.2%(P<0.01) and LDH by 22.86%(P<0.01). ALT and LDH activities were increased by 24.03%(P<0.05) and 20.31%(P<0.01) respectively in cells treated with PQ. The activity of GSH-Px in cells after treated with H₂O₂ or PQ was decreased by 58.6% and 55.03% (P<0.01) respectively. The SOD activity was reduced by 47.01% (P<0.01) in H₂O₂ treatment, and reduced by 35.33% (P<0.01) in PQ treatment. The contents of NO and MDA were increased significantly after treat with H₂O₂ or PQ. NO content in PQ treatment was higher than that in H₂O₂ treatment.The results of these indexes showed that treatment with H₂O₂ or PQ would disrupt structure of hepatocyte membrane. H₂O₂ or PQ also reduced the activity of antioxidative enzymes, and these two oxidants increased the content of NO, MDA. From these facts, it was concluded that treatment with H₂O₂ or PQ resulted in oxidative stress in piglet hepatocytes.Pretreatment with Vc decreased ALT and LDH activity, and increased the activity ofGSH-Px and SOD compared to treatment with H2O2 or PQ. Vc added into cell culture supernatant reduced the content of NO and MDA in hepatocytes.These results suggested that Vc was an efficient antioxidant. It was able to prevent the decrease of oxidative enzymes and keep the content of NO, MDA at a normal level. It was concluded that Vc could protect the cells from oxidative stress induced by H2O2 and PQ.The activities of AMPK in six groups were different. After treated with H2O2 or PQ the activity of AMPK was increased by 55.11% and 45.97% respectively. Compared to H2O2 treatment, AMPK activity reduced 21.49% in cells pretreated with Vc. Vc also reduced AMPK activity by 27.21% compared to PQ treatment. There was a negative correlation between the activity of AMPK and the activity of GSH-Px, SOD. AMPK activity had a positive correlation with the contents of NO, MDA. These results suggested that AMPK would be activated by oxidative stress.The content of TG and TC were examined in the cultural supernatant. The result was not significantly different among treatments. Addition of Vc in hepatocytes could increase the content of TG.Taken together, our results demonstrated that H2O2 or PQ induced efficiently oxidative stress of piglet hepatocytes, and oxidative stress could activate AMPK activity.