TIGAR Reduces Cerebral Ischemia Reperfusion Injury By Regulating Succinate Dehydrogenase (SDH) And Succinate

Background and purposeStroke has the characteristics of high morbidity,disability,and mortality.It is the second leading cause of death in the world.Stroke caused by cerebral ischemia is the most common type of stroke.The main treatments for ischemic stroke are thrombolysis and endovascular surgery to restore blood flow.However,these methods all have a short treatment time window and the risk of serious complications,so their clinical application is limited.Although the pathogenesis of ischemic stroke is complex,neuron survival is the focus of new methods for the prevention and treatment of cerebral ischemia-reperfusion injury at home and abroad.The survival of neurons and the maintenance of the function of the entire nervous system are extremely dependent on the integrity of mitochondrial functions.The dysfunction caused by mitochondrial metabolism disorder is the starting point and early stage of a series of events such as brain cell apoptosis and necrosis.Mitochondria are not only the hub of cellular calcium signal transduction,but also the enzyme complex on the inner membrane is also an essential component for maintaining cell energy requirements and metabolic homeostasis.Therefore,for the development of new anti-stroke drugs in the future,mitochondrial-targeted drugs are the most promising direction.Succinate dehydrogenase(SDH),also known as mitochondrial complex Ⅱ,is located in the inner mitochondrial membrane and couples two pathways necessary for oxidative phosphorylation:the tricarboxylic acid cycle and the respiratory chain.Hypoxia induces an increase in SDH activity,accelerates the rate of succinate oxidation,and drives the mitochondrial electron transport chain to produce a large amount of ROS through the reverse electron transport at complex I,resulting in oxidative stress damage.But succinate is not only a substrate of SDH,but also a downstream product of phosphorylation of substrates in the tricarboxylic acid cycle and α-ketoglutarate dehydrogenase complex,which is beneficial to maintaining the integrity of cell structure.Previous studies in our laboratory found that TIGAR can increase the flux of the pentose phosphate pathway by inhibiting the glycolytic pathway,thereby increasing the production of intracellular NADPH and generating reduced glutathione through glutathione reductase to eliminate ROS.Therefore,TIGAR shows strong antioxidant effect.Under hypoxic conditions,TIGAR translocates to mitochondria,which can reduce the production of mitochondrial ROS and increase ATP,enhance mitochondrial function,and reduce neuronal damage.Therefore,this project puts forward the hypothesis that "TIGAR protects neuronal mitochondrial function by regulating SDH and succinate to reduce cerebral ischemia-reperfusion injury".The tMCAO/R model and the OGD/R cell model were established through TIGAR genetically engineered mice and lentivirustransfected neuronal cells,respectively,and molecular biology techniques were used to reveal the effects of TIGAR on succinate metabolism of neuronal mitochondria induced by ischemia and hypoxia.The regulation mechanism and its effect on neuronal damage further clarify the new mechanism of TIGAR’s intracellular antioxidant effect.It provides new research directions and targets for TIGAR as the treatment of acute and chronic neurological diseases involving oxidative stress,especially ischemic stroke,and provides new ideas for future ischemic stroke prevention and treatment and new drug development.Method(1)Establish a transient middle cerebral artery occlusion/reperfusion(tMCAO/R)model,the succinate content and SDH activity in cerebral cortex of mice during cerebral ischemia-reperfusion were detected,and the protein expressions of SDH catalytic subunits SDH A and SDH B were detected by Western blot(WB).(2)Construct TIGAR knockout mice(TIGAR-KO)and TIGAR overexpression mice(TIGAR-TG),and evaluate the effects of TIGAR on cerebral ischemia-reperfusion injury by cerebral infarct volume,brain water content and neurological scores,and detect the effect of TIGAR on ischemia-induced succinate content and SDH activity.(3)Construct a stable transgenic HT22 strain that overexpresses and knocks down TIGAR by lentivirus infection,establish the model of oxygen glucose deprivation/reoxygenation(OGD/R).The effects of TIGAR on succinate content and SDH activity induced by OGD were detected.(4)Objective to evaluate the effects of dimethyl malonate(DMM),an inhibitor of SDH activity,and dimethyl succinate(DMS),an osmotic derivative of succinate,on cerebral ischemia-reperfusion injury in mice.(5)CCK8 was used to detect the protective effects of DMM,DMS and SDH ubiquinone binding site inhibitor atpenin A5(AA5)on OGD/R-induced neuronal death,and its effect on intracellular succinate content.(6)The effects of DMM,DMS and AA5 on mitochondrial function after OGD were investigated by measuring ATP,ROS,intracellular Ca2+concentration and mitochondrial membrane potential.(7)The cell co-localization of SDH and TIGAR was detected by immunofluorescence;the effect of TIGAR on the mRNA level and protein expression of SDH A and SDH B was detected by WB and qPCR;the effect of TIGAR on the acetylation level of SDH A was detected by immunoprecipitation.(8)Objective to evaluate the effect of inhibition of SDH activation by DMM or supplement of succinate by DMS on cerebral ischemia-reperfusion injury in TIGAR-KO mice.Results(1)During the period of cerebral ischemia-reperfusion,the succinate content in the brain of mice decreased continuously and stabilized at a low level after 1 h of reperfusion,while the activity of SDH only became active during the period of ischemia and the first 3 h after reperfusion.The protein expression of SDH A and SDH B increased in a timedependent manner and peaked at 12-24 h after reperfusion.(2)Overexpression of TIGAR can significantly reduce the cerebral infarct volume,brain water content and neurological scores of tMCAO mice;on the contrary,knocking out TIGAR significantly increased the cerebral infarction volume,brain water content and neurological scores of tMCAO mice.(3)Overexpression of TIAGR can significantly inhibit the intracellular SDH activation and the decrease of succinate content induced by ischemia or OGD,and up-regulate the intracellular succinate content;while interfering with TIAGR,SDH is over-activated and aggravates the decrease of succinate content.(4)Injection of DMM and DMS before ischemia can significantly reduce the cerebral infarction volume of tMCAO mice,and reduce the brain water content and neurological scores.(5)DMM,DMS,AA5 significantly reduced OGD/Rinduced cell death in a dose-dependent manner,and significantly inhibited the decrease in intracellular succinate content induced by OGD,and up-regulated the intracellular succinate content.(6)DMM,DMS,AA5 delay the depletion of intracellular ATP during OGD,increase intracellular ATP levels,inhibit intracellular ROS generation,inhibit Ca2+influx and increase mitochondrial membrane potential.(7)There is obvious mitochondrial colocalization between TIGAR and SDH A,SDH B.Overexpression of TIAGR significantly inhibits the up-regulation of the mRNA level and protein expression of SDH B induced by ischemia or OGD,and inhibits the decrease of SDH A acetylation level;interference with TIGAR reversed this phenomenon.(8)Both DMM and DMS significantly reduce the cerebral infarct volume after tMCAO/R in TIGAR-KO mice,reduce brain water content and the neurological scores.ConclusionDuring cerebral ischemia in mice,the content of succinate in the brain decreased,SDH was activated and the expression increased.Inhibiting SDH activity or increasing intracellular succinate content during ischemia can protect mitochondrial function.TIGAR has a protective effect on cerebral ischemia-reperfusion injury in mice,and its mechanism may be related to the regulation of SDH activity and the up-regulation of intracellular succinate content.This provides some new ideas for the future prevention and treatment of ischemic stroke and the development of new drugs.