The Role And Mechanism Of Endoplasmic Reticulum Stress In Early Brain Injury After Subarachnoid Hemorrhage

BackgroundAneurysmal subarachnoid hemorrhage(SAH)is a fatal cerebrovascular disease with unfavorable outcomes and a high rate of mortality.According to epidemiological survey,annual population prevalence rate is about 1‰.Past research efforts have traditionally focused on vasospasm,which was considered the most important determinant of brain injuries and outcomes after SAH.Nevertheless,the failure of anti-vasospastic treatment to improve the outcomes of SAH patients in clinical trials has shifted the research interest to SAH-induced early brain injury.Early brain injury refers to immediate injury that lasts within 72 h after SAH.EBI is the most important factor that determines the prognosis of SAH patients.The possible mechanisms of EBI after SAH involve the rapid rise of intracranial pressure and the reduction of cerebral perfusion pressure,Brain-Blood Barrier disruption,brain edema,oxidative and nitrosative stress,and neural apoptosis.However,the definitive mechanisms of early brain injury after SAH remain unclear.The endoplasmic reticulum(ER)is an essential intracellular organelle that is responsible for the synthesis and maturation of proteins.Disturbed ER functions result in the accumulation of unfolded proteins and induce endoplasmic reticulum stress.Subsequently,a signal transduction cascade,the unfolded protein response(UPR),is induced.UPR would lead to suspension of protein synthesis,up-regulation of chaperones and degradation of ER-associated protein,to deal with the stimuli.If the stimuli are severe and prolonged,ER stress is unable to compensate for these stimuli and cell apoptosis would be induced.Several studies have demonstrated that ER stress plays an important role in the process of neurodegeneration and neuroinflammation diseases.The potential contribution of ER stress to SAH-induced early brain injury is not completely understood.In the current study,we investigated the role of ER stress PERK-eIF2α-ATF4 pathway in early brain injury following SAH,and its influence on neural apoptosis and brain-blood barrier disruption.This study may provide a promising therapeutic strategy for SAH.MethodsPart 11.SAH model was performed using endovascular perforation technique.Adult male Sprague Dawley rats were randomly assigned into sham group,SAH 3 h group,SAH 6 h group,SAH 12 h group,SAH 24 h group,and SAH 72 h group.Neurological deficit scores and brain edema were evaluated.The expression of PERK,p-PERK,eIF2α,p-eIF2α and ATF4 were evaluated by Western blot analysis.Double immunohistochemistry staining was used to localize the expression of ER stress assioated proteins.Part 21.Adult male Sprague Dawley rats were treated with intracerebral ventricle infusion of PERK inhibitor GSK2606414(30 μg,90 μg)at 1 hour after SAH onset.Adult male Sprague Dawley rats were randomly assigned into sham group,SAH+vehicle group,SAH+GSK2606414(30 μg),SAH+GSK2606414(90 μg)group.2.SAH grade and neurological score were measured at 72 hours after SAH in all groups.Double immunohistochemistry staining of terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL)and NeuN were also performed in each group at 72 hours after SAH.Western blot analysis was performed to meature the expression of PERK,p-PERK,eIF2α,p-eIF2α,ATF,Akt,Bcl-2,Bax and Caspase-3.Akt inhibitor Wortmannin was used as an intervention to clarify the role of PERK inhibitor in SAH.Part 31.Adult male Sprague Dawley rats were administered with vehicle,GSK2606414 via I.C.V or Akt inhibitor Wortmannin via I.P.The rats were randomly assigned into sham group,SAH+vehicle group,SAH+GSK2606414 group,SAH+Wortmannin and SAH+GSK2606414+Wortmannin group.2.SAH grade,neurological score,and brain water content were measured at 72 hours after SAH in all groups.Brain-Blood Barrier disruption was quantitatively evaluated by Evans blue extravasation.Western blot analysis was performed to measure the expression of PERK,p-PERK,ZO-1,Occludin and Claudin5.Administration of Akt inhibitor Wortmannin was used as an intervention to clarify the role of PERK inhibitor on Brain-Blood Barrier in SAH.ResultsPart 11.Western blot analysis showed that the expression of p-PERK was significantly elevated three hours after SAH,and peaked at 72 hours after SAH.A similar tendency was observed in the expression of p-eIF2α and the downstream kinase ATF4.Both signals started to increase at 12 hours and peaked at 72 hours after SAH.Double immunostaining of PERK with NeuN or GFAP in both sham and vehicle groups suggested that PERK was expressed in neurons and astrocytes.Part 21.Post-SAH treatment of GSK2606414 significantly reduced the expression of p-PERK and p-eIF2α and ameliorated the neurological deficits after SAH.2.Administering GSK2606414 significantly lowered the expression of p-PERK and p-eIF2α compared to the SAH+vehicle group.The ratio of Bcl-2/Bax was significantly reduced,and the expression level of cleaved Caspase-3 was significantly increased in the SAH+vehicle group.The administration of GSK2606414 significantly reversed these processes,and there was a significant difference between the high dosage group and low dosage group.TUNEL staining localized with the neuronal marker NeuN.The total number of TUNEL and NeuN double-stained cells significantly increased at 72 hours after SAH.Treatment of GSK2606414 significantly reduced the number of TUNEL-positive neurons,and high dosage was more effective.The inhibition of PERK by GSK2606414 significantly promoted cell survival by increasing the Bcl-2/Bax ratio and reducing cleaved Caspase-3 expression.The anti-apoptotic effects of GSK2606414 were significantly blocked by the injection of Akt inhibitor Wortmannin.Part 31.Significant extravasation of Evans blue,an index of BBB disruption,was found in the left and right hemispheres in the SAH+vehicle group compared with the sham group at 72 hours after SAH.GSK2606414 treatment significantly reduced Evans blue dye extravasation and improved the neurological deficits compared with the SAH+vehicle group.Western blot analysis showed that GSK2606414 administration significantly increased the expression of ZO-1,Occludin and Claudin5,which were decreased after SAH.2.Preconditioning of Akt inhibitor Wortmannin injection aggravated brain edema,increased Evans blue dye extravasation,and reduced the expressions of ZO-1,Occludin and Claudin5 after GSK2606414 treatment in SAH rats.The increased expression of p-Akt by GSK2606414 treatment was significantly suppressed after Wortmannin administration.These results indicated that the neurovascular protective effects of GSK2606414 was significantly blocked by Wortmannin.Conclusion1.Endoplasmic reticulum stress PERK-eIF2a-ATF4 pathway is activated in early brain injury after SAH,and is widely distributed in neurons and astrocytes.These results indicate that ER stress may play a key role in SAH-induced early brain injury.2.Treatment of PERK inhibitor GSK2606414 can significantly reduce neuronal apoptosis,maintain BBB integrity,attenuate brain edema and improve neurological functions in early brain injury after SAH.3.Inhibition of PERK may have the potential to reduce early brain injury after SAH via Akt-related anti-apoptosis pathways.