The Mechanism Of Mitochondrial Dysfunction Induced By Copper And Protection Of ALCAR In Primary Cultured Chicken Hepatocytes

Copper(Cu)is an essential trace element for organism.In aquaculture production,high concentration of copper is added to animal feed to improve animal production performance.The body can maintain copper ion balance through various regulatory mechanisms.However,the occurrence of copper poisoning can occur when the body has a copper metabolic disorder or excessive intake of copper.The liver is an important organ of copper accumulation,so copper poisoning is particularly damaging to the liver.Although copper toxicology studies have shown that copper causes oxidative stress,damage mitochondria and induce autophagy and apoptosis in cells,the exact mechanisms underline those effects still need to be verified.Mitochondria is an important organelle playing major roles in energy metabolism,regulate calcium ion homeostasis,autophagy and apoptosis.The function of mitochondria is closely related to the morphology of mitochondria,and the fusion and fission of mitochondria is crucial for maintaining normal physiological functions of mitochondria.Abnormal mitochondrial fusion and fission will result in mitochondrial dysfunction and even caused cell apoptosis or death.In present study,in order to investigate the effects of Cu on the cell morphology and viability,oxidative stress levels,mitochondrial structure and function and mitochondrial dynamics,the primary chicken hepatocytes were cultured by revised M199 medium in logarithmic growth phase and treated with different concentrations of copper(10-200 ?M)for 12 or 24 h.On this basis,the protect effect and mechanism of ALCAR on damaged hepatocytes induced by copper were investigated.As a result:In this experiment,we successfully cultured the primary cultured chicken hepatocytes with a purity of 95 % and a well viability in vitro.Furthermore,Compared with the control group,10 ?M copper treatment had no effect on the cell viability and morphology,and 100 ?M copper had a slight effect on cell morphology,while,200 ?M copper treated for 12 h can significantly inhibit the cell viability and cause the cell death(P < 0.05),and treated for 24 h can significantly inhibit cell viability(P < 0.01).The ROS content of hepatocytes increased with the increase of copper concentration.Compared with the control group,there was a significant increase in ROS content after treated with 100 and 200 ?M copper for 12 and 24 h.Similarly results were also showed in MDA content.We also found that there was no significantly effect on ATP content after hepatocytes treated with 10 ?M copper.but ATP content can be significantly decreased after treated 100 and 200 ?M copper for 12 and 24 h.The result of mitochondrial membrane potential was consistent with that.The expression of mitochondrial fusion(OPA1?MFN1?MFN2)and fission-related genes(DNM1?MTFP1?MTFR1)were increased but only the fission genes were significantly increased(P<0.05).Similar results also appeared in the protein expression,There was no significant effect on OPA1 protein expression,but compared with the control group,the DNM1 protein expression in the high concentration of copper group were significantly increased(P<0.01).In conclusion,medium and high concentration of copper can induce the oxidative stress,and destroy mitochondrial structure and function,and affect cell vitality.Mitochondrial dynamics may be involved in the process of copper-induced hepatocytes injury by increased the fission-related genes expression.ALCAR can improve the survival rate of hepatocytes by restoring mitochondrial function and lowering ROS level,however,it can only significantly inhibit MTFR1 gene,and its mechanism may not significantly related to mitochondrial dynamics.