Effect Of Endoplasmic Reticulum Stress-Autophagy On Energy Metabolism Disorder And Oxidative Stress During Cerebral Ischemia-Reperfusion

Energy metabolism disorder and oxidative stress are the major causes of cerebralischemia-reperfusion injury. Mitochondrial oxidative phosphorylation disorders aftercerebral ischemia-reperfusion can lead to decreased production of ATP andsimultaneously generate a large amount of reactive oxygen species (ROS), whichresults in oxidative stress. During cerebral ischemia-reperfusion, the decrease of ATPand the increase of free radicals can cause cell damaged, in addition, some adaptiveregulation mechanisms attract researchers’ attention.Endoplasmic reticulum stress and autophagy are two independent adaptiveresponses to cell stress injury. Endoplasmic reticulum stress-autophagy can maintaincell homeostasis, playing a protective role. The unfolded protein response uponendoplasmic reticulum stress can activate autophagy. Autophagy can alleviate theoverload of endoplasmic reticulum by degradation of misfolded or unfolded proteins,inhibiting overactivation of endoplasmic reticulum stress. In addition, autophagy canprovide raw materials for the synthesis of new proteins, reconstruction of cellstructure and ATP generation with the release of degradation products. However,excessive activation of endoplasmic reticulum stress-autophagy can increase celldamage or even cause cell death. Recent studies suggest that endoplasmic reticulumstress-autophagy plays a key role in energy metabolism disorder and oxidative stressinduced by cerebral ischemia-reperfusion, but the mechanism is not yet very clear.Keap1-Nrf2-ARE signaling pathway is a key cell defense mechanism underoxidative stress condition. As a multifunctional protein, autophagy-related protein p62not only can remove damaged cellular organelles and abnormal protein aggregation,but also can work as an adapter molecule in a variety of signal transduction pathways,such as cell stress and cell survival. Previous studies have shown that under oxidativestress condition, p62can form a positive feedback loop with Nrf2, promotes the xpression of antioxidant genes through the Keap1-Nrf2-ARE signaling pathway.Therefore, to explore the role of multifunctional protein p62in cerebral ischemia-reperfusion injury, may further clarify the regulation mechanism between autophagyand oxidative stress.The membrane fusion of autophagosome and lysosomes at the late stage ofautophagy pathway is one of the typical vesicle fusion events. Autophagy-relatedprotein NSF, as an ATPase, upon hydrolysis of ATP energy by itself, mediates therelease of SNARE complexes after the completion of vesicle fusion into the nextvesicle fusion process. NSF is a key protein indispensable for vesicle fusion process.In vitro studies show that only the the cytoplasmic soluble NSF can mediate theprocess of vesicle fusion through hydrolysis of ATP, NSF will lose its function ifaggregation and inactivation occurs or under ATP depletion condition. Thus, theremay exist some association between ATP generation and vesicle fusion. We observedchange of the vesicle fusion key protein NSF upon energy metabolism disorder by invitro experimental cerebral ischemia-reperfusion model, in order to further investigatethe possible regulation mechanisms between energy metabolism and autophagyduring cerebral-ischemic reperfusion injury.Objective:In this study, based on energy metabolism disorder/oxidative stress can inducecell injury，which may also activate some adaptive responses such as endoplasmicreticulum stress-autophagy, we investigated the role of endoplasmic reticulumstress-autophagy during energy metabolism disorder and oxidative stress in order toprovide new clues for the mechanism of cerebral ischemia-reperfusion injury.Methods:(1) In vivo:1) Rat transient middle cerebral artery occlusion (tMCAO) was induced by the suturemethod.2) TTC staining and HE staining were used to determine cortical injury after cerebralischemia-reperfusion.3) Immunohistochemistry, RT-PCR and western blot were used to detect theexpression of endoplasmic reticulum stress associated proteins, autophagy associattedproteins, proteins of Keap1-Nrf2-ARE pathway and expression of downstream genesin the cerebral cortex after ischemia-reperfusion. (2) In vitro:1) CHO cell culture ATP depletion and recovery model was induced by exposure to amixture of2-deoxy-D-glucose (2-DG,5mM) and oligomycin (2.5uM). CHO cellmedium LDH activities were determined to assess cell injury. Differentialcentrifugation and linear glycerol gradient centrifugation were used to get thedifferent protein fraction. Western blot was used to detect the expression of NSFprotein.1) PC12cell culture ATP depletion and recovery model was induced by exposure to amixture of2-deoxy-D-glucose (2-DG,5mM) and oligomycin (2.5uM). MTT methodwas used to detect cell viability. Western blot was used to detect the expression ofautophagy and endoplasmic reticulum stress associated proteins.Results:(1) In vivo:1) The results of neurological deficit scores, TTC and H&E staining revealed thatcerebral cortex injury was aggravated with prolongation of ischaemia afterischemia-reperfusion.2) After1hour of tMCAO and24hours of reperfusion, the expression ofubiquitinated proteins in the cortex increased, the expression of Grp78and Bcl-2alsoincreased, but the expression of Bax decreased. With prolongation of ischaemia, at3hours of ischaemia and24hours of reperfusion, the formation of ubiquitinated proteinaggregates and CHOP/GADD153expression notablely increased. Meanwhile, theexpression of Bax increased and expression of Bcl-2decreased.3) The expression of ubiquitinated proteins in the cortex increases after1hour and1.5hours of ischaemia and24hours of reperfusion, indicative of the accumulation ofprotein aggregates.Meanwhile, the expression of the autophagy conjugate proteinsAtg12-Atg5and LC3-PE, two factors essential for autophagy, also increased.However, at3hours of ischaemia and24hours of reperfusion, the formation ofubiquitinated protein aggregates notablely increased, while the expression ofAtg12-Atg5and LC3-PE decreased.3) After1hour and1.5hours of ischaemia and24hours of reperfusion, the expressionof the autophagy conjugate proteins Atg12-Atg5and LC3-PE, two factors essentialfor autophagy, increased. However, at3hours of ischaemia and24hours ofreperfusion, the expression of Atg12-Atg5and LC3-PE decreased. 4）The nuclear localization of Nrf2increases after1hour or1.5hours of ischaemiaand24hours of reperfusion. This RT-PCR analysis confirmed a marked increase inthe expression of the Nrf2down-stream target antioxidant genes, NQO1, GCLM andHO1. Prolongation to3hours of ischaemia followed by24hours of reperfusion,resulted in a decrease in the nucleic localization of Nrf2and inactivation of the Nrf2down-stream antioxidant target genes.5) The expression of p62increased dramatically after3hours of tMCAO and24hoursof reperfusion, while p62mRNA expression firstly increased and then decreased withprolongation of ischaemic time.(2) In vitro:1) Mild ATP depletion following by24hour recovery under glucose-free condition,the viability of PC12cell slightly elevated, while the protein levels of LC3-II and p62did not have significant changes; Mild ATP depletion following by24hour recoverywith the existence of glucose, the viability of PC12cell was higher than that in severeATP depletion group, Grp78protein significantly increased and p62proteinsignificantly decreased; Severe ATP depletion following by24hour recovery, theviability of PC12cell significant decreased, the protein levels of LC3-II and p62dramatically increased. Inhibition of autophagy by3-MA reduced the viability ofPC12cells upon ATP depletion.2) ATP depletion causes the aggregation of cytoplasmic soluble protein NSF in CHOcell culture and cell injury. Overexpression of NSF mitigates ATP depletion/recovery-induced cell injury.Conclusion:1. Endoplasmic reticulum stress-autophagy pathway is involved in cerebral ischemia-reperfusion injury. Upon short-time cerebral ischemia-reperfusion, the activation ofendoplasmic reticulum stress-autophagy plays a protective role; Upon long-timecerebral ischemia-reperfusion, the excessive activation of endoplasmic reticulumstress response and the decrease of autophagic degradation can aggravate braindamage.2. During cerebral ischaemia-reperfusion, autophagy may be involved in theKeap1-Nrf2-ARE signalling pathway through p62, which collectively induces theexpression of ARE downstream antioxidant proteins, playing a role in alleviating cellinjury induced by oxidative stress and overactivation of endoplasmic reticulum stress. 3. The inhibition of autophagy aggravates cell death, which suggest that endoplasmicreticulum stress-autophagy pathway play a protective role on energy metabolismdisorder.4. Energy metabolism disorder leads to aggregation and inactivation of cytoplasmicsoluble NSF protein, losing its function in mediating vesicle fusion, which results inthe malfunction of autophagic degradation and causes cell damage. Overexpression ofNSF can reduce cell damage induced by ATP depletion by supplying somecytoplasmic soluble NSF.Overall, we think that the cell death induced by brain ischemia-reperfusion isclosely related with energy metabolism disorder/oxidative stress, while mildendoplasmic reticulum stress-autophagy have some protective effect in this process.The study about endoplasmic reticulum stress-autophagy in brain cells probablyprovide new clue for the prevention and therapy of brain ischemia-reperfusion.