Effect Mechanism Of Copper Sulfate On Hepatic Apoptosis,Autophagy And Inflammation In Mice

It has been reported that excessive copper can induce oxidative stress,apoptosis and inflammation,which in turn causes damage to tissue structure and function.Liver is the main organ of copper metabolism in the body and one of the main target organs of copper poisoning.However,there have no systematic studies on the liver damage of humans and animals caused by excessive copper and its mechanism of action.Therefore,the present study was aimed to explore the potential molecular mechanisms of CuSO₄-damaged liver by observing the changes of the histological structure,function,oxidative stress,apoptosis,autophagy and inflammatory responses,which provide a new theoretical basis for the prevention and treatment of copper poisoning.The results are as follows:1.Effects of CuSO₄ on liver histological structure,function and Antioxidant ability in miceA total of 240 healthy ICR mice aged four weeks（half male and half female）were acclimated for a week,followed by random division into four groups treated with 0,10,20,and 40 mg/kg CuSO₄ for 42 days,respectively.Clinical signs were observed daily during the experiment.The results showed that CuSO₄ in excess of 10 mg/kg decreased the liver volume and viscera index.Histopathological lesions induced by CuSO₄ varied with dose and time,including granular degeneration,vacuolar degeneration and necrosis.Meanwhile,the serum activities of liver functional parameters,such as ALT and AST,were increased in the CuSO₄-treated groups.The liver function was impaired.CuSO₄ in excess of 10 mg/kg also induced oxidative stress by increasing the levels of ROS and PC,and suppressing the abilities of ASA and AHR,as well as activities and mRNA expression levels of Antioxidant indexes.2.Effects of CuSO₄ on liver apoptosis in miceGrouping and treatment of experimental animals were the same as in 1.On days 21 and 42 during the experiment,the results of flow cytometry showed that the percentages of hepatic apoptosis and the proportions of hepatocytes depolarized with MMP collapse were increased in the CuSO₄-treated groups.The TEM observations found that mitochondria with blurred or broken cristae were present in liver cells.Concurrently,CuSO₄ increased the protein and mRNA expression levels of Cytc,caspase-3/9/8,PARP,Bax,TNF-R1,FADD and TRADD,and decreased the protein and mRNA expression levels of Bcl-2和Bcl-xl.Abovementioned results demonstrated that CuSO₄ in excess of 10 mg/kg induced hepatic apoptosis via activating both mitochondrial apoptotic pathway and TNF-R1 signaling pathway.3.Effects of CuSO4 on liver autophagy in miceGrouping and treatment of experimental animals were the same as in 1.At days 21 and 42 of the experiment,the immunochemistry results showed that the protein expression levels of LC3B were increased in the CuSO₄ treatment groups.The TEM observations showed that CuSO₄ strengthened autophagosome formation in hepatocytes.CuSO₄ also caused a significant decrease in mTOR and p62 protein and mRNA expression levels,and increase in ULK1,Atg12/5,Atg16L1,LC3 protein and mRNA expression levels.Abovementioned results demonstrated that CuSO₄ in excess of 10 mg/kg induced hepatocyte autophagy by inhibiting the m TOR signaling pathway and up-regulating expression levels of autophagy-promoted factors.4.Effects of CuSO4 on liver inflammatory responses in miceGrouping and treatment of experimental animals were the same as in 1.At day 42 of the experiment,inflammatory cells were observed in the liver of the CuSO₄ treatment groups.At days 21 and 42 of the experiment,CuSO₄ in excess of 10 mg/kg activated the MAPK/NF-κB signaling pathway through increasing protein and mRNA expression levels of p38,JNK,Erk1/2,NF-κB and pro-inflammatory mediators including iNOS,COX-2,IL-1β,IL-8,and decreasing protein and mRNA expression levels of IκB.Meanwhile,the protein and mRNA expression levels of anti-inflammatory mediators including IL-4 and TGF-βwere suppressed in the CuSO₄-treated groups.The imbalance between the levels of pro-inflammatory and anti-inflammatory mediators induced hepatic inflammatory responses.Also,the up-regulated protein expression levels of caspase-1p12 and cleaved-IL-1β suggested that CuSO₄ can also promote hepatic inflammation by inducing the formation of inflammasomes.5.The role of apoptosis and autophagy in liver injury induced by CuSO4A total of eight-week 84 ICR male mice were randomly allocated to six groups treated with distilled water,CuSO₄,Z-VAD-FMK,CuSO₄+Z-VAD-FMK,3-MA and CuSO₄+3-MA for 3 days,respectively.Clinical signs were observed daily during the experiment.The results showed that the mice in the CuSO₄ group were depressed.Also,2 mice died in total by the third day of the experiment in the CuSO₄ group,and the mortality rate was 14%.CuSO₄ impaired liver function by increasing serum activities of ALT and AST.The percentage of hepatic apoptosis was increased in CuSO₄ group.Meanwhile,CuSO₄ up-regulated the protein expression levels of Bax,cleaved-caspase-3/9,cleaved-PARP and LC3B,and decreased the protein expression levels of Bcl-2 and p62.When compared to CuSO₄ group,the co-treatment of apoptosis inhibitor Z-VAD-FMK and CuSO4 reduced the mortality of mice and the activity of ALT and AST in serum,and significantly inhibited hepatocyte apoptosis.Meanwhile,Z-VAD-FMK decreased the protein expression levels of Bax,cleaved-caspase-3/9,cleaved-PARP and LC3B,and increased the protein expression levels of Bcl-2 and p62.In comparison to CuSO₄ group,the co-treatment of autophagy inhibitor 3-MA and CuSO₄ raised the mortality of mice,and promoted hepatocyte apoptosis.Also,3-MA decreased the protein expression levels of LC3B,and increased the protein expression levels of p62,cleaved-caspase-3/9 and cleaved-PARP.Abovementioned results demonstrated that Z-VAD-FMK reduced CuSO₄-induced liver injure by inhibiting apoptosis,and inhibited autophagy while inhibiting apoptosis.The autophagy inhibited by 3-MA promoted hepatocyte apoptosis and also enhanced the liver injure induced by CuSO₄.In conclusion,CuSO₄ in excess of 10 mg/kg induced histological lesions and dysfunctions,oxidative damage,apoptosis,autophagy and inflammatory responses in the mouse liver.Oxidative damage was the pathological basis of CuSO₄-induced hepatic damage.Apoptosis,autophagy and inflammatory responses were the pathways of CuSO₄-induced hepatic damage.At the same time,studies discovered that CuSO₄ mainly impaired liver damage by causing apoptosis,and apoptosis also caused autophagy,which in turn inhibited apoptosis.This study was firstly to systematically illustrate the toxic effects,effect pathway and molecular mechanism of CuSO₄ on liver by observing lesions,dysfunction and anti-oxidation,apoptosis,autophagy and inflammatory responses from cellular and molecular levels.