The Mechanism Of Autophagy And Apoptosis Induced By High Levels Of Copper In Chicken Hepatocytes

Copper is one of the essential trace elements in most organisms,which involves in many physiological and biochemical activities.However,excessive copper can induce copper poisoning in the body,which cause damage to multiple tissues and organs.Studies have shown that the liver is the important target organ for copper toxicity.Domestic and foreign scholars have conducted many researches on the mechanism of hepatotoxicity induced by high levels of copper,but these studies mostly focus on the mechanism of apoptosis,and there are relatively few studies on the role and mechanism of autophagy and the regulatory relationship between autophagy and apoptosis in the process of hepatotoxicity induced by high levels of copper.Especially the study about autophagy induced by high levels of copper in poultry is rare.In this study,animal and cell culture models were established to explore the role of autophagy and apoptosis and their regulatory relationship in liver injury of chicken induced by high levels of copper in vivo and in vitro,and laying the foundation for the development of molecular pathology of copper toxicity and related research.To study the effects of high levels of copper on autophagy and apoptosis in chicken hepatocytes,the experiment was divided in two parts,in vivo and in vitro.In vivo experiments,192 one-day-old white-feathered broiler chickens were randomly divided into 4 groups with 48 birds in each group.Copper sulfate was selected as the experimental copper source,and 4 concentration groups were set.The concentration of copper of the basic diet in control group was 11 mg/kg,which was recommended by NRC(1994),and the concentrations of copper in the three experimental groups was 110 mg/kg,220 mg/kg,and 330 mg/kg,respectively.The experiments lasted 7 weeks,and livers were collected on weeks of 1,3,5,and 7.Histologic section and transmission electron microscopy were used for the observation of cytopathic effect.TUNEL assay was conducted to measure the apoptotic level.Additionally,the expression levels of autophagy-related and apoptosis-related genes and proteins were detected by real-time quantitative PCR(RT-qPCR)and western blotting,respectively.Moreover,the localization and expression of BECN1 and LC3? were determined by immunohistochemistry and immunofluorescence,respectively.Results showed that high dietary copper could induce necrosis,autophagosomes,karyopyknosis,and TUNEL-positive particles in hepatocytes;increase the mRNA expression of Beclin1,ATG5,LC3 I,LC3?,Dynein,P53,Bak1,Bax,Caspase3 and the protein expression of BECN1,LC3?/LC3?,P53,Bak1,Bax,Cyt C,Caspase3;but decrease the m RNA expression of P62,mTOR,Bcl2 and the protein expression of P62,Bcl2.In vitro experiments,a culture model of chicken embryo primary hepatocyte was carried out by enzymatic digestion.Chicken hepatocytes were treated with different concentrations of copper sulfate(0,10,50,100 ?M)for 24 h and/or copper sulfate(100 ?M)at different times(0,6,12,24 h).Transmission electron microscopy was used to observe the autophagosomes,and the autophagic level was detected by monodansylcadaverine(MDC)fluorescence probe.Additionally,flow cytometry was used to detect the cell cycle,apoptotic rate was determined by fluorescent staining,and the localization and expression of LC3? were measured by immunofluorescence.Moreover,the expression levels of autophagyrelated and apoptosis-related genes and proteins were detected by RT-qPCR and western blotting,respectively.Results showed that copper ion could increase the number of autophagosomes,the autophagic level,and LC3 puncta;induce S phase arrest and apoptotic rate;up-regulate the m RNA expression of Beclin1,ATG5,LC3 I,LC3?,P62,mTOR,P53,Bak1,Bax,Cyt C,Caspase3 and the protein expression of BECN1,LC3?/LC3 I,Bak1,Bax,Cyt C,Caspase3,but down-regulate Bcl2 mRNA and protein expression.In vivo and in vitro experiments suggest that high levels of copper could induce autophagy and apoptosis in chicken hepatocytes.To explore the role of autophagy on injury and apoptosis induced by high levels of copper in chicken hepatocytes,copper sulfate and autophagic disruptors were used to treat chicken hepatocytes in this study.Cellular morphology was observed by microscopy;the cell viability was measured by CCK-8 method and the activities of lactic dehydrogenase(LDH),aspartate aminotransferase(AST),alanine aminotransferase(ALT) and content of albumin(ALB) in the cell supernatant were detected by biochemical analyzer;additionally,flow cytometry was used to determine reactive oxygen species(ROS) level,the number of mitochondria,mitochondrial membrane potential(MMP) and cell cycle;moreover,the levels of intracellular antioxidant indexes and antioxidant genes were detected by spectrophotometry and RT-q PCR,respectively;the cellular adenosine triphosphate(ATP) level was measured by luciferase;apoptotic rate was determined by fluorescent staining;the expression levels of apoptosis-related genes and proteins were detected by RT-qPCR and western blotting,respectively.Results showed that compared to the copper sulfate group,the combined group of copper sulfate and Rapa(rapamycin) could reduce cell shedding;increase cell viability;decrease levels of ROS,hydrogen peroxide,superoxide dismutase(SOD),malondialdehyde(MDA),catalase(CAT),HO-1 mRNA expression,and apoptotic rate;upregulate the number of mitochondria,MMP and ATP level;down-regulate the mRNA expression of P53,Bak1,Bax and Cyt C,Caspase3 proteins expression.But the treatment with combined group of 3-methyladenine(3-MA)and copper sulfate had the opposite effects on above factors compared to the treatment with copper.These suggest that the activation of autophagy might attenuate copper-induced damage and apoptosis in chicken hepatocyte,but the inhibition of autophagy had the opposite effect.