The Effects Of N-acetylcysteine On Oxidative Stress And Autophagy-related Genes M Rna Expression Induced By High Copper In Chicken Hepatocyte

Copper is an essential trace element in all organs and cells,and plays an important role in biological function.Copper overload may lead to hepatotoxicity due to reactive oxygen species?ROS?induced by redox reaction and Haber–Weiss reaction.ROS cause a series of damages to cells,such as denaturation of nucleic acid and protein,degradation of polysaccharide and peroxidation of lipid.Studies shows that ROS can induce cell autophagy through oxidative damages of DNA,lipid and protein or act ing as a signal molecule.To study the effects of antioxidant N-acetylcysteine?NAC?on liver oxidative stress and autophagy induced by high copper in chicken,chicken primary hepatocytes were incubated in vitro with different concentrations of copper ions?0?M,10?M,50?M,100?M?and NAC?1 mM?,and the cells were collected at different time to determine cell activity by CCK-8 assay.The cells were divided into 4 group:contro l group,high copper group(100?M Cu²⁺),high copper with NAC group(100?M Cu²⁺+1 mM NAC)and NAC group?1 mM NAC?.After 24-hours incubation,the activities of lactate dehydrogenase?LDH?,aspartate transaminase?AST?,and alanine aminotransferase?ALT?in the culture medium was detected.Additionally,the activities or contents of superoxide dismutase?SOD?,catalasefrommicrococcuslysodeikticus?CAT?,glutathione?GSH?,malondialdehyde?MDA?and ROS in the cells were also determined to evaluate the degree of oxidative damage.Finally,the mRNA expression of autophagy-related genes?Beclin-1,ATG-5,LC3-I and LC3-II?were detected by fluorescence quantitative PCR.The results show that:there was no significant difference in the activity curve among low copper group(10?M Cu²⁺),1 mM NAC group and the control group?P>0.05?.While cell proliferation of the medium group(50?M Cu²⁺)and high copper group was significantly inhibited?P<0.05?.The growth of cells in the high copper with NAC group droped within 24 hours during incubation period,and was significantly inhibited after 24 hours?P<0.05?.?2?Compared with the control group,the activities of LDH,AST and ALT in the cell culture medium in high copper group was significantly increased?P<0.05?.And compared with high copper group,the activities of LDH,AST and ALT in the cell culture medium in was high copper with N AC group significantly decreased?P<0.05?.?3?Compared with the control group,the activities of SOD and CAT in high copper group were significantly increased,as well as the contents of MDA and ROS.The content of GSH was decreased.However,the high copper with NAC group showed significant inhibition of the above-mentioned trend?P<0.05?.The addition of NAC could alleviate oxidative damage caused by high copper in hepatocytes.?4?High copper can significantly increase the mRNA expression of autophagy-related genes?Beclin-1,ATG-5,LC3-I and LC3-II??P<0.05?,and NAC inhibited such expression significantly?P<0.05?.Conclusion:High copper cause oxidative damage of chicken hepatocytes and induce the mRNA expression of autophagy-related gene increased in the cells.NAC protect antioxidant function of hepatocytes and decrease the mRNA expression of autophagy-related gene.