Study On Ferroptosis In Neuronal Cell Induced By Arsenic Exposure

PART?FERROPTOSIS IN CEREBRAL CORTEX NEURONS OF MICE INDUCED BY ARSENITE EXPOSUREObjective:To investigate whether chronic exposure to arsenite could cause ferroptosis in cerebral cortical neurons of mice.Methods:The 32 C57BL/6J male mice,aged 7 weeks,were randomly divided into four groups as follows:control group,0.5,5,and 50 mg/L of arsenite.Mice were exposed to arsenite for 6 months via drinking water.The number of neurons in the cerebral cortex was observed by H&E staining.The relative protein level of NeuN and MAP2 were detected using immunofluorescence;The mitochondrial ultrastructure was observed using transmission electron microscope;Contents of SOD and GSH was detected by SOD and GSH assay kit.The total and mitochondrial MDA,ATP,iron contents were detected by MDA,ATP,iron assay kit,respectively;The expression levels of IREB2,SLC7A11,VDAC3 and GPX4 were detected by qPCR and Western blot.The expression levels of pJNK1/2,pERK1/2,GRP78 and CHOP were detected by Western blot.Results:?1?We observed that the number of nerve cells was significantly reduced and the fluorescence intensity of NeuN and MAP2were decreased in the cerebral cortex of mice after arsenite treatment,especially in 5 mg/L and 50 mg/L arsenite group?P<0.05?.?2?The ultrastructure of mitochondria showed that mitochondrial ridge decreased or disappeared,the mitochondrial membrane concentrated,and vacuoles appeared in cerebral cortex after arsenite treatment.?3?The contents of SOD and GSH were decreased gradually with the increasing dose of arsenite in cortex of mice.There were significant differences between arsenite groups and the control group?P<0.05?.The total and mitochondrial contents of MDA and ATP were decreased after arsenite treatment.The total and mitochondrial contents of Fe²⁺were increased after arsenite treatment.There were significant differences between exposed groups and the control group?P<0.05?;?4?The expression levels of IREB2,SLC7A11,GPX4 proteins and mRNA decreased gradually in arsenite groups,while the expression level of VDAC3 protein and mRNA increased gradually in arsenite groups.There were significant differences between arsenite groups and the control group?P<0.05?;?5?The protein levels of GRP78,CHOP,pJNK1/2 and pERK1/2 were increased after arsenite treatment,there were significant differences between arsenite groups and control group?P<0.05?.Conclusions:The neurons were lossed,the mitochondrial structure and function were destructed,the lipid peroxides and Fe²⁺were accumulated in response to chronic arsenite exposure in cerebral cortex.The endoplasmic reticulum and MAPK pathway were activated,and the expression levels of ferroptosis related genes were changed in cerebral cortex of mice.Our results show that ferroptosis is a newly characterized form of neuronal cell death in response to arsenite exposure.PART ?STUDY ON FERROPTOSIS IN PC-12 CELL INDUCED BY ARSENITE EXPOSUREObjective:To determine whether arsenite could cause ferroptosis in PC-12 cells.Methods:The cell viability of PC-12 was detected by CCK-8 assay;The mitochondrial ROS were detected using MitoSOX^TMM red probe by flow cytometry.Results:?1?The ferroptosis inhibitors DFO could effectively inhibit the cytotoxicity of PC-12 induced by arsenite.Moreover,ferroptosis inhibitors inhibited the accumulation of mitochondrial ROS in PC-12 cells effectively.There were significant differences between arsenite groups and the control group?P<0.05?.Conclusions:The cell viability of PC-12 cells was decreased,and the mitochondria ROS were increased after arsenite treatment.Ferroptosis inhibitors can inhibit the cytotoxicity induced by arsenite through inhibiting the mitochondria ROS.These results suggest that arsenite exposure may induce ferroptosis in PC-12 cells.