The Role Of PINK1/Parkin Mediated Mitophagy In Cadmium-induced Steatosis In Primary Chicken Embryo Hepatocytes And Liver

According to National survey of soil pollution released by Chinese government in 2014,the use of cadmium(Cd)in production application was the top of the environmental inorganic pollutants’ list.Accompanied with layer scale breeding industry expanding,environmental Cd pollution caused a serious of problems,such as fat deposition,fat degeneration,and nonalcoholic fatty liver(NAFLD).Mitochondrial plays an important role in modulating the metabolism of sugar,amino acid and fat.And mitochondrial damage will disorder lipid metabolism and fat deposition.PINK1 and Parkin mediate in elimination of mitochondrial damage in the process of mitophagy,and this is a kind of mechanism in mitochondrial quantity and quality control.However,the role of mitophagy in Cd-induced fat deposition is unclear.Our study used primary embryonic hepatocytes and liver tissues as experimental models to study the role of PINK 1/Parkin-mediated mitophagy in Cd-induced fat deposition.1.Effects of cadmium on steatosis in chicken embryonic hepatocytes and liverIn order to study the effects of Cd on lipid metabolism of chicken embryo hepatocytes and chicken embryo liver,14-day-old chicken embryo hepatocytes and 7-day-old chicken embryo livers were used as the experimental models,and different concentrations of Cd(0,2.5,5,10 μM)were used to treat chicken embryo hepatocytes in vitro for 6 h.7-day-old chicken embryo yolk sacs were injected with different concentrations of Cd(0,10,100,200,500,1000 nM)for 7 days.TG kit was used to detect the content of TG in chicken embryo hepatocytes and livers;QRT-PCR was used to detect transcription levels of genes related to lipid metabolism in chicken embryo hepatocytes and livers;Nile red fluorescence staining was used to observe the accumulation of lipid droplets in chicken embryo hepatocytes;HE staining was used to observe the fat vacuolization in chicken embryo livers.The results showed that Cd treatment group compared to the control group,the content of TG in chicken embryonic hepatocytes and livers was significantly increased(P<0.05 or P<0.01);The transcription levels of fatty acid synthesis-related genes(FASN,SREBP,SCD,ACACA)were increased(P<0.05 or P<0.01).The transcription levels of fatty acid decomposition-related genes(ATGL,ACOX1,LPL,CPT1)were decreased in different degrees(P<0.05 or P<0.01).Accumulation of lipid droplets in chicken embryo hepatocytes and liver were aggravated.These data manifest that Cd exposure leads to the occurrence of steatosis in chicken embryonic hepatocytes and livers by disturbing normal lipid metabolism.2.PINK2/Parkin participates in Cd-induced mitophagy in chickenembryonic hepatocytes and liversIn order to study the effects of Cd on lipid metabolism of chicken embryo hepatocytes and chicken embryo livers,we used primary chicken embryo hepatocytes and chicken embryo livers as experimental models.Chicken embryo hepatocytes were exposed with Cd(0,2.5,5,10 μM)for 6 h.And 7-day-old chicken embryo yolk sacs were injected with different concentrations of Cd(0,10,100,200,500,1000 nM)for 7 days.Transmission electron microscopy(TEM)was used to detect the ultrastructure of mitochondria in chicken embryo hepatocytes and livers;immunofluorescence was used to observe the co-localization level of mitochondria and autophagosomes in chicken embryo hepatocytes;Western Blot was used to detect LC3-Ⅱ,PINK1 and Parkin protein expression levels.These data showed that Cd caused the membrane potential depolarization of mitochondria and the formation of mitochondrial-autophagosome in chicken embryonic hepatocytes and livers;the co-localization degree of mitochondria and autophagosomes in chicken embryonic hepatocytes was significantly increased;the expression of LC3-Ⅱ,PINK1 and Parkin in chicken embryonic hepatocytes and livers increased in different degrees(P<0.05 or P<0.01).It indicated that Cd caused mitochondrial damage in primary chicken embryonic hepatocytes and lives,and mitophagy occurred.The PINK1/Parkin pathway was involved in the regulation of the process.3.3.The role of PINK1/Parkin mediated mitophagy in Cd-induced steatosis in chicken embryonic hepatocytes and livers In order to investigate the effect of PINK1/Parkin mediated mitophagy in Cd-induced steatosis in chicken embryonic hepatocytes and livers,we used primary chicken embryo hepatocytes and chicken embryo livers as experimental models.Primary chicken embryo hepatocytes were treated with 10 μM Cd companied with or without 1 μM cyclosporine A(CsA)for 6 h.Chicken embryo yolk sacs were treated with 1 μM Cd with or without 1 μM CsA for 7 days.TEM was used to detect the ultrastructure of mitochondria in chicken embryo hepatocytes and liver.TG kit was used to detect the content of TG in chicken embryo hepatocytes and liver;Nile red fluorescence staining was used to observe the accumulation of lipid droplets in chicken embryo hepatocytes and HE staining was used to observe the fat vacuolization in chicken embryo livers;QRT-PCR was used to detect transcription levels of genes related to lipid metabolism in chicken embryo hepatocytes and livers;Western Blot was used to detect the level of LC3-Ⅱ,PINK1 and Parkin protein expression in chicken embryo hepatocytes and livers.Results showed that compared with the Cd group,1 μM CsA could inhibit the formation of Cd-induced mitochondrial autophagosomes in chicken embryonic hepatocytes and livers.The expression of autophagy-related proteins LC3-II,PINK1 and Parkin protein decreased significantly(P<0.05 or P<0.01);the content of TG in chicken embryonic hepatocytes increased significantly(P<0.01),the transcription level of lipid synthesis-related genes increased significantly(P<0.01),and the transcription level of lipid decomposition-related genes decreased significantly(P<0.05 or P<0.01);1μM CsA effectively inhibited the accumulation of Cd-induced fat droplets in chicken embryo livers,significantly reduced TG generation(P<0.01),and restored the transcription level of lipid metabolism-related genes to normal.It indicated that inhibiting mitochondrial membrane potential depolarization could effectively inhibit PINK1/Parkin-mediated mitochondrial autophagy in primary chicken embryonic hepatocytes and livers induced by Cd,aggravate Cd-induced steatosis in primary chicken embryonic hepatocytes,but effectively alleviate Cd-induced steatosis in chicken embryonic livers.In conclusion,Cd interferes with fatty acid metabolism leading to steatosis of chicken embryonic hepatocytes and livers,mainly manifesting mitochondrial swelling and formation of mitochondrial autophagosomes.PINK 1/Parkin is involved in the regulation of Cd-induced mitophagy in chicken embryonic hepatocytes and livers.