Arsenite Induces Ferroptosis In The Neuronal Cells Via Activation Of Ferritinophagy

Objective: To investigate whether arsenite-exposure can induce the ferroptosis in neuronal cells,and to verify whether ferritinophagy is involved in arsenite-induced ferroptosis.Methods: Healthy male C57BL/6J mice and weighted 20-24 g were subjected to experiment.After one week habituation to the environment,all32 mice were randomly divided into 4 groups: control(0 mg/L),low(0.5mg/L),medium(5 mg/L)and high(50 mg/L)(n=8).After 6 months of arsenite exposure via drinking,the hippocampus tissues were collected after sacrificed.H&E staining was used to detect the pathological changes of mice hippocampus.The ultrastructural changes of synapses and mitochondria in hippocampus were observed by transmission electron microscope.Then,the content of iron and MDA in hippocampus and PC-12 cells were measured by commercial kits,cell viability was measured by CCK-8.And western blot was used to detect the protein expression of SLC7A11,GPX4,NCOA4,Ferritin,and LC3 B in hippocampus and PC-12cells;q PCR was used to detect the genes expression of Slc11a2,Slc40a1,Tfrc and Iscu in hippocampus and PC-12 cells.Additionally,the expression level of neuron marker Neu N in hippocampus was detected by immunofluorescence.Results: Compared with the control: 1)As the concentrations of arsenite was increased,the number of neurons in the hippocampus was decreases,the cell body of some neuronal cells were remarkably shrunken or fractured with condensed nuclear chromatin,and the post-synaptic density thickness was increases,but no significant difference was observed on the width of the synaptic fissure or the length of the active area.2)Mitochondria in hippocampus exhibited vacuolar lesion,mitochondrial cristae disappeared,the content of iron and MDA were increased significantly,and the protein expression of SLC7A11 and GPX4 were decreased.3)The content of iron and MDA in PC-12 cells were significantly increased,and the protein expression of SLC7A11 and GPX4 were decreased.4)5 μM Ferrostatin-1 alleviated the cytotoxic effect of arsenite,reduced the elevated contents of iron and MDA induced by arsenite.The decreased protein expression of SLC7A11 and GPX4 were alleviated by Ferrostatin-1.5)Arsenite increased the m RNA expression of Slc11a2,Slc40a1 and Iscu in hippocampus and PC-12 cells,while decreased the Tfrc m RNA expression.It also significantly reduced the protein expression of NCOA4 and ferritin in hippocampus and PC-12 cells,whereas enhanced the protein expression of LC3 B.Besides,Arsenite was able to increase the co-localization of LC3 B and NCOA4.And the co-treatment of 10 m M 3-MA and 100 n M Baf A1 with arsenite significantly alleviated the cytotoxicity of arsenite.Conclusion: Arsenite triggers ferroptosis in the neuronal cells via activation ferrotinophagy.Ferrostatin-1 significantly rescued the phenotypes of ferroptosis induced by arsenite.