Regulation Of Parkin In Cr（Ⅵ）-induced Mitophagy In Chicken Hepatocytes

Chromium is one of the most common heavy metal contaminants.In the natural environment,the valence of chromium varies from-2 to +6.Trivalent chromium（Cr（III））and hexavalent chromium（Cr（VI））are the most common valence states,with Cr（VI）being the most toxic.Cr（VI）is a swallowed toxicant and is easily absorbed into the body through digestion,respiratory tract,skin and mucous membranes.It can react with reducing substances in the cells and prevent the timely removal of reactive oxygen species from the cells,causing symptoms such as mitochondrial damage and damage to cytogenetic material.In recent years,people have gradually recognized the harmful effects of chromium pollution,but the research on its pathogenic mechanism is not deep enough.Our group has previously conducted in vivo validation of Cr（VI）-induced mitophagy in chicken hepatocytes,but its regulatory mechanism is still unclear.Therefore,in this experiment,we established a model of Cr（VI）toxicity in chicken hepatocytes,determined the relationship between Cr（VI）-induced mitochondrial damage and mitophagy,and elucidated the role of Parkin in Cr（VI）-induced mitophagy.In this experiment,Primary cells of chicken liver were used to construct a Cr（VI）intoxication model,and a CCCP-treated positive control group,a Hank’ s-treated starvationtreated group,a blank control group and a Cr（VI）-treated group were set up to perform the following experiments.The optimal treatment time and concentration of Cr（VI）were screened by Western Blot to detect changes in the expression of autophagy-related proteins LC3 and p62.The intracellular MMP and ROS were measured qualitatively and quantitatively by laser confocal technique and flow cytometry,respectively,to verify that Cr（VI）could cause the occurrence of mitophagy in cells through damage to mitochondria.Subsequently,to further investigate the role of Parkin in Cr（VI）-induced mitophagy in chicken hepatocytes,this experiment used a chicken liver cancer cell line（LMH）to construct a Parkin silencing model with a blank control group,NC negative control group（si RNA empty carrier group）,Cr（VI）-treated group and si RNA + Cr（VI）co-treated Parkin silencing group.Parkin silencing was effectively verified by flow cytometry and Western Blot;changes in the content of mitophagy-related proteins PINK1,p62 and LC3 were detected;changes in mitochondrial morphology were observed using laser confocal technique,and changes in MMP and ROS were examined to further elucidate the effect of Parkin silencing on Cr（VI）-induced mitophagy production.The results showed that 40μM Cr（VI）-induced mitophagy was not affected.The results showed that the most significant changes in protein expression of autophagy-related proteins LC3 and p62 were observed in chicken liver primary cells treated with 40mM Cr（VI）for 6 h.At this concentration and time,the Cr（VI）-treated group caused a large accumulation of ROS and a significant decrease in MMP,and a significant increase in the expression of mitophagy-related proteins（PINK1 and Parkin）.In addition,knockdown of Parkin effectively blocked p62 degradation,LC3 lipidation and PINK1 expression in chicken liver cancer cells,and mitophagy was inhibited;in the Parkin-silenced group,mitochondrial morphology was drastically changed and fragmented;ROS accumulated heavily,MMP further decreased and mitochondrial damage increased.In conclusion,the present assay demonstrated that Cr（VI）can cause mitophagy through damage to mitochondria;and Parkin played a key role in Cr（VI）-induced mitophagy in chicken hepatocytes.This experiment further elucidated the toxicological effects of Cr（VI）on the liver and provided a theoretical basis for further studies on the toxic effects of Cr（VI）;in addition,understanding the role of Parkin in mitophagy is important for studying the pathogenesis and prevention of related diseases.