Autophagy Promotes Neuronal Ferroptosis In Early Brain Injury After Subarachnoid Hemorrhage

Objectives:To observe the effect of autophagy on the recovery of early brain injury after subarachnoid hemorrhage(SAH)in rats,and to reveal the mechanism how autophagy promotes neuronal ferroptosis and affects early brain injury.Methods:SD rats were randomly divided into control group,SAH group,SAH+LV-shATG5 group and SAH+LV-Scramble group,the number of rats in each group was 12.We constructed SAH model by intravascular puncture.We used ATG5(Autophagy-related 5)lentivirus(LV-shATG5)to inhibit autophagy,and immunofluorescence was used to detect the expression and location of ATG5,FTH1(ferritin heavy polypeptide 1)and GPX4(glutathione peroxidase 4)in neurons.The expression of ATG5 mRNA was detected by PCR.The expression of ATG5,LC3II/I,FTH1 and GPX4 were detected by Western blot.The changes of neuronal morphology and function were detected by Nissl staining,neurological score and brain water content were also detected.Intracellular iron deposition,iron content,and malonaldehyde(MDA)content,GPX4 activity were measured toevaluate the ferroptosis.Results:Immunofluorescence showed that ATG5,FTH1 and GPX4 were widely expressed in the cytoplasm of cortical neurons after SAH.The results of PCR and Western blot indicated that the expression of ATG5 mRNA and LC3II/I protein was significantly decreased(p<0.05),and autophagy was successfully inhibited.When autophagy was inhibited,Nissl staining showed improvements on neuronal morphology and function,neurological function score was improved(p<0.05),accompanied by brain water content decreased(p<0.05).To explore the specific pathways,Western blot showed that the expression of FTH1 and GPX4 decreased after SAH(p<0.05),however,the expression of FTH1 and GPX4 increased when autophagy was inhibited(p<0.05).Furthermore,autophagy inhibition also reduced iron deposition,cellular labile iron and MDA contents(p<0.05),also elevated GSH content and GPX4 activity(p<0.05).Conclusion : Autophagy may promotes neuronal ferroptosis by degrading ferritin,results in cellular labile iron increase,finally exaggerates early brain injury.