The Role And Mechanism Of Ferroptosis In Early Brain Injury After Experimental Subarachnoid Hemorrhage

BackgroundSpontaneous subarachnoid hemorrhage(SAH)is an acute hemorrhagic stroke,the most common cause is ruptured aneurysm,in which more than 1/3 of the survivors will leave permanent disability,cognitive impairment,depression,anxiety and other mental symptoms,resulting in a significant decline in the quality of life of patients.Current studies have shown that timely intervention in the process of early brain injury(early brain injury,EBI)in patients with SAH can effectively improve the prognosis of patients.EBI refers to the pathophysiological process within 72 hours of SAH.Previous studies have confirmed that neuroinflammation is closely related to EBI,but the mechanism is not clear.Ferroptosis is an iron-dependent form of programmed cell death with elevated levels of lipid peroxidation that differs from other forms of cell death.Recently,studies have confirmed that ferroptosis is closely related to acute organ injury diseases such as acute lung injury,acute renal injury,traumatic brain injury,and may also involve the pathological process of neurodegenerative diseases,but the relationship between ferroptosis and EBI is not clear.Several studies have shown that EBI releases damage-related molecular model molecules(DAMPs)to aggravate neuroinflammation after SAH,which will further cause brain damage.Cells also release DAMPs after ferroptosis,so we speculate that ferroptosis may play an important role in a series of inflammatory responses after SAH.In this study,the rat model of SAH was established by arterial puncture.Through the detection of neurological function,brain edema and inflammation-related signal pathways in model rats,the role of ferroptosis in EBI and the key target factors of activating ferroptosis were deeply studied,to find a new method to reduce the risk of EBI and recover neurological function to the greatest extent.Objective1.To study the iron overload,ferroptosis and DAMPs in SAH rat model,and to determine the effect of ferroptosis on neuronal injury and neurological function in experimental rats after SAH.2.To explore whether ferroptosis inhibitor can reduce the damage of the blood-brain barrier,lighten the inflammatory reaction,and improve neurological dysfunction after SAH.3.To determine the regulatory effect of p53 on ferroptosis after SAH,and to study whether inhibition of p53 can reduce ferroptosis and improve neurological dysfunction after SAH.Methods:Part 11.The SAH model was established by the arterial puncture method.Male Sprague-Dawley rats 7-8 weeks old were randomly divided into Sham group and SAH group.The SAH group was divided into three subgroups of 6 hours,24 hours,and 48 hours according to the time point of taking materials after operation.2.The expression levels of ferroptosis related protein(p53,SLc7a11,GPx4),the level of lipid peroxide(4-HNE)and DAMPs related factor(HMGB1)in the cerebral cortex of rats in each group were detected by Western blot.3.Perls-DAB staining and iron content were used to detect the cerebral cortex of rat at 24 hours after operation.4.The morphological characteristics of ferroptosis(mitochondrial atrophy)were observed by transmission electron microscope(TEM)in the cerebral cortex of rats at 24 hours after operation.Part 21.Intervention with ferroptosis inhibitor Ferrostatin-1(Fer-1),rats were randomly divided into Sham group,SAH group,SAH+Vehicle group and SAH+Fer-1 group.DMSO or Fer-1 was injected intraperitoneally 30 minutes after operation in SAH+Vehicle group and SAH+Fer-1 group.2.The mortality of each group and the Garcia nerve score were evaluated at 24 hours after operation.3.The rats were sacrificed at 24 hours after operation,and the SAH grade and brain water content were measured.4.Brain tissue sections were taken for Perls-DAB staining,4-HNE immunohistochemistry,IgG immunofluorescence to detect the destruction of the bloodbrain barrier and FJB staining to detect neuronal death.5.The content of iron and GSH in brain tissue was measured.6.Ferroptosis related proteins(p53,SLc7a11,GPx4),the level of lipid peroxide(4-HNE),DAMPs related factor(HMGB1)and inflammatory factor(IL-1β,IL-6,TNF-α)were detected by Western blot at 24 hours after operation.6.Western blot to detect tight junction-associated protein(ZO-1)and matrix metalloproteinase family proteins(MMP9,MMP2)in rat cerebral cortex.7.The mitochondria of neurons were observed by TEM.Part 31.Intervention with p53 inhibitor Pifithrin-alpha(Pft-α),rats were randomly divided into Sham group,SAH group,SAH+Vehicle group and SAH+Pft-α group,in SAH+Vehicle and SAH+Pft-α were received intraperitoneal injection of DMSO or Pft-α 30 minutes after operation.2.The mortality and neurological score of each group were evaluated at 24 hours after operation.3.The rats were sacrificed at 24 hours after operation,and the SAH grade and brain water content were measured.4.Brain tissue sections were taken for p53 immunofluorescence staining.5.The content of GSH was measured in brain tissue.6.Ferroptosis related proteins(p53,SLc7a11,GPx4),lipid peroxide 4-HNE,DAMPs related factor(HMGB1)and inflammatory factor(IL-1β,IL-6,TNF-α)were detected by Western blot.7.The mitochondria of neurons were observed by TEM.Results:Part 11.Neurological dysfunction occurred after SAH in rats.2.Lipid peroxides increased significantly,accompanied by changes in ferroptosis related proteins and the release of HMGB1 in the cerebral cortex at 24 hours after SAH.3.The content of cortical iron increased and neuronal mitochondrial atrophy in the cerebral cortex at 24 hours after SAH.Part 21.Fer-1(ferroptosis inhibitor)could improve neurological function score and brain edema in rats at 24 hours after SAH.2.Fer-1 could reduce IgG extravasation,inhibit the decrease of ZO-1 level and the upregulation of MMP9 and MMP2 expression in the cerebral cortex at 24 hours after SAH.3.Fer-1 had no effect on iron content in the cerebral cortex after SAH.4.Fer1 could reduce the level of 4-HNE and neuronal mitochondrial atrophy.5.Fer-1 could restore the levels of SLc7a11,GPx4 and GSH,but had no effect on the expression of p53 in the cerebral cortex at 24 hours after SAH.6.Fer-1 could inhibit the expression of HMGB1,IL-1β,IL-6 and TNF-α after SAH.7.Fer-1 could reduce neuronal death in the cerebral cortex after SAH.Part 31.Pft-α(p53 inhibitor)could improve the neurological function score and brain edema of rats at 24 hours after SAH.2.Pft-α could inhibit the expression of p53 in the cerebral cortex after SAH.3.Pft-α could restore SLc7a11,GPx4 and GSH levels,reduced the expression of 4HNE and neuronal mitochondrial atrophy in the cerebral cortex at 24 hours after SAH.4.Pft-αcould inhibit the expression of HMGB1,IL-1β,IL-6 and TNF-α after SAH.Conclusion:Part 11.Ferroptosis occurred in cortical neurons at 24 hours after SAH.2.p53,SLc7a11 and GPx4 may be involved in the regulation of ferroptosis after SAH.3.Ferroptosis may promote DAMPs-related neuroinflammation.Part 21.Inhibition of ferroptosis could reduce the destruction of the cortical blood-brain barrier after SAH,the release of DAMPs,neuroinflammation,neuronal cell death,and improve neurological dysfunction,brain edema after SAH.Fer-1 plays a neuroprotective role in EBI.2.Fer-1 mainly mediated the increase the level of SLc7a11/GSH/GPx4 axis,reduced the level of lipid peroxidation and protected cortical neurons from ferroptosis after SAH.Part 31.Inhibition of p53 expression in cerebral cortex after SAH can mediate the increase of SLc7a11/GSH/GPx4 axis,reducing the occurrence of ferroptosis and EBI.2.Down-regulation of p53 expression after SAH could inhibit ferroptosis,thus reduce neuroinflammation and play a protective role in brain function.