The Role Of PERK-Mediated Endoplasmic Reticulum Stress In Ferroptosis Caused By Hexavalent Chromium In Chicken Hepatocytes

Chromium is widespread in nature.Due to the rapid development of chromium metal related industries,a large amount of chromium-containing waste is released beyond the selfhealing capacity of the environment,causing serious environmental pollution and also threatening human health.Cr（Ⅵ）is considered to be the most toxic chromium metal valence state owing to its high solubility and strong oxidizing properties.The liver is the primary organ affected by Cr（Ⅵ）poisoning.Ferroptosis is a type of programmed cell death characterized by lipid peroxidation.It is distinguished by changes in mitochondrial morphology,intracellular glutathione(GSH)depletion,decreased glutathione peroxidase 4(GPX4)activity,reduced cellular antioxidant capacity,and an increase in lipid reactive oxygen species(ROS).Early evidence showed that Cr（Ⅵ）could induce oxidative stress in liver tissue and lead to a large accumulation of ROS in DF-1 cells,as well as induce endoplasmic reticulum stress(ERS)in DF-1 cells.However,the link between Cr（Ⅵ）-induced ferroptosis and ERS remains unknown.Therefore,in this paper,we investigated whether Cr（Ⅵ）could induce ferroptosis in chicken hepatocytes by establishing a model of Cr（Ⅵ）toxicity in chicken hepatocytes,and the role of endoplasmic reticulum stress protein PERK in this process.In this experiment,the toxic effects of Cr（Ⅵ）on chicken hepatocytes,namely chicken primary hepatocytes and chicken hepatocellular carcinoma cell line(LMH),were investigated separately.To determine whether the hepatocytes were successfully extracted,the extracted chicken primary hepatocytes were stained with PAS.To determine the time and concentration of Cr（Ⅵ）,in vitro cultured chicken primary hepatocytes and LMH cells were treated with Cr（Ⅵ）,then the survival rate of chicken primary hepatocytes was examined by CCK-8 and the GPX4 protein expression was examined by Western Blot.To determine the induction effect of Cr（Ⅵ）on ferroptosis in chicken hepatocytes,the following experiments were performed.The expression of ferroptosis-related proteins COX2,HSP27 and GPX4 in chicken primary hepatocytes and LMH cells was detected by Western Blot.The contents of GSH,SOD and MDA in chicken primary hepatocytes and LMH cells were detected by assay kits.The lipid ROS in LMH cells were detected by flow cytometry and confocal laser microscopy.Next,chicken primary hepatocytes and LMH cells were treated using the same concentrations and times as described above.To verify the occurrence of ERS,the expression of ERS-related proteins GRP78 and PERK was observed by Western Blot and Immunofluorescence(IF),and in the meantime RT-q PCR was performed to detect the m RNA levels of GRP78 and PERK.Furthermore,the role of PERK in Cr（Ⅵ）-induced ferroptosis in chicken hepatocytes was clarified by utilizing si RNA to silence PERK and monitoring changes in the COX2,HSP27,and GPX4 proteins as well as the content of lipid ROS.The results of the experiment are as follows.The chicken primary hepatocytes were successfully extracted,as evidenced by the PAS glycogen staining results,which showed that the retrieved cells were stained with many purple-red dotted aggregates.After exposure to 40μM Cr（Ⅵ）for 6 h,the survival rate of chicken primary hepatocytes was around 50%.The down-regulation of GPX4 protein expression was significant after treatment of LMH cells with20 μM Cr（Ⅵ）for 20 h.In the Cr（Ⅵ）-treated group,protein expression of COX2,MDA content,and lipid ROS level were significantly up-regulated with the aforementioned time and concentration,whereas HSP27,GPX4,GSH,and SOD content were significantly downregulated,and all of these trends could be reversed by ferroptosis inhibitor(Ferrostatin-1,Fer-1).Subsequent Western Blot,IF and RT-q PCR results showed that protein and transcript levels of GRP78 as well as PERK were significantly increased in the Cr（Ⅵ）-treated group compared to the control group.In addition,silencing of PERK resulted in significant up-regulation of COX2 protein expression,a major down-regulation of GPX4 protein expression,and a significant increase in the accumulation of intracellular lipid ROS.In summary,this experiment demonstrated that Cr（Ⅵ）can disturb the antioxidant system,causing the accumulation of lipid peroxides,which eventually leads to ferroptosis in chicken hepatocytes.And PERK-mediated ERS plays an important role in this process,silencing PERK leads to the intensification of Cr（Ⅵ）-induced ferroptosis in chicken hepatocytes.