The Role Of PRDX3 In Ischemia-Reperfusion Induced Neuronal Injury And Associated Mechanism

BackgroundStroke is a cerebrovascular disease accompanied with high incidence,high disability and high mortality rate.It is also the second leading cause of death in the world and is one of the main causes of disability.Moreover,the incidence of stroke is increasingly higher all over the world,particularly in developing countries.As one type of stroke,ischemic stroke（IS）is due to the lack of blood flow,which causes the dysfuction of brain.Every year,almost9.5 million people undergo IS and estimated 2.7 million people die of this disease.As the medical technology goes by,there is a rapid progress of various interventions in recent years.However,the prognosis of patients with ischemic stroke is still unsatisfactory.According to the GBD,ischemic stroke remains paramount of disability-adjusted life years among neurological diseases in many countries.It is estimated that the global cost of stroke exceeds721 billion dollors（0.66%of global GDP）,which places a huge burden on patients,their families,and the whole society.The high heterogeneity resulted from IS is an outgrowth of its pathogenesis,pathological alterations and the degree or location of injury.Most ischemic strokes are caused by emboli,which may arise from atherosclerotic plaques in the aortic arch,carotid artery or heart.Intracranial atherosclerosis with in situ thrombosis is also an important cause of IS.Some other subordinate causes include small vessel disease,carotid dissection and arteritis.Consequently,when intracranial feeding arteries are blocked for the above reasons,the clinical symptoms of patients will be continuing deterioration because of the restricting blood flow,which may contribute to the physiological dysfuction of neurons.Moreover,if cerebral blood flow is severely reduced,brain may suffer irreversible damage,resulting in the failure of respiratory and circulatory.Although the infarcted area can be reperfused to restore blood circulation through collateral circulation,reperfusion after ischemia can also bring secondary damage to brain,which may catalyze a series of neurochemical reactions and neurometabolic abnormalities,including glutamate excitotoxicity,mitochondrial dysfunction,oxidative stress injury,inflammatory response,Ca²+imbalance and blood-brain barrier dysfunction,etc.For decades,innumerable explorations have been carried out to elucidate the machenism of IS while the effective breakthrough of its mechanism and corresponding treatment strategies is still unfavourable.Some of potential preventions and related mechanisms are still needed to be explored in future researches.Mitochondria have multiple functions in eukaryotic cells.Besides producing ATP,it is involved in heme synthesis,amino acid metabolism and regulation of cellular redox state,which render it an integral subcellular organelle for cell survival.Additionally,studies have also shown that mitochondria can exert an indispensable impact in programmed cell death by regulating the release of pro-apoptotic factors.Meanwhile,it also has an association with necrosis and autophagy.Specifically,mitochondria perform these diverse functions by continuously communicating with other cells through signal transduction.Reactive oxygen species（ROS）generated from mitochondria,as a sort of secondary messengers,which plays an essential effect in cellular signal transduction,is one of the ways to carry out signal transduction.It is shown that brain IR injury is accompanied by massive production of ROS,including H₂O₂ and·OH.These ROS can modify intracellular molecules and disrupt cellular lipids,proteins and DNA.Consequently,cellular homeostasis is disrupted and apoptosis is ultimately induced.Most H₂O₂ is produced in mitochondria,but it also brings oxidative damage to mitochondria,thereby fracturing mitochondrial function and integrity,affecting redox signaling and oxidative stress,and ultimately inducing cellular necrosis and apoptosis.Mitochondrial oxidative damage involves in a wide range of pathological processes,such as liver fibrosis,atherosclerosis,diabetes,and IR damage.Many antioxidant enzymes（eg,superoxide dismutase,catalase,PRDXs）and other defense systems（eg,flavonoids,vitamin C,glutathione）are abundent in mitochondria to restrain cells from oxidative stress damage.Conclusively,identifying functional genes that can maintain ROS homeostasis to alleviate mitochondrial oxidative damage and exploring their mechanisms are crucial to defend cellular functions.Peroxiredoxins（PRDXs）are a family of potent thiol peroxidase enzymes in mammalian cells,consisting of at least six isoforms（PRDX1-6）.PRDX1,PRDX2 and PRDX6 are localized in the cytoplasm,PRDX4 is localized in the endoplasm,PRDX5 is localized to peroxisomes and mitochondria and PRDX3 is mainly present in mitochondria.Studies have shown that the sulfenic acid of PRDXs cysteine can form mixed disulfides with transcription factors after cells are treated with H₂O₂,thereby promoting cell survival.Furthermore,when the cell is suffering oxidative stress,PRDXs can form stable oxidative dimers which can be secreted into the extracellular space acting as extracellular"stress"signals.PRDX3 is the most abundant and efficient H₂O₂ scavenging enzyme in mitochondria,and has abilities to remove about 90%of H₂O₂ in the matrix by being oxidized to an inactive dimer.It has been reported that PRDX3 can effectively ameliorate oxidative stress and apoptosis,and thereby reducing cell damage.In traumatic brain injury models,the PRDX3gene can reduce trauma-induced cell damage by protecting mitochondrial function.Furthermore,compared to normal mice,transgenic mice overexpressing PRDX3 can reduce mitochondrial H₂O₂ production and thus reduce oxidative damage.Additionally,in thymoma cells,PRDX3 overexpression can significantly reduce the level of intracellular H₂O₂ and inhibit tumor cell proliferation.Since PRDX3 serves as an essential role in oxidative injury,considering the scarce studies of this gene in cerebral ischemia-reperfusion injury,we focus PRDX3 on the further study in terms of cerebral ischemia-reperfusion injury and explore its regulatory mechanism on mitochondrial oxidative damage in IS.ObjectiveThis study aims to elucidate the function of PRDX3 on oxidative damage of neurons after IR injury and reveal the relating mechanism.Furthermore,we explore the effect of PRDX3 on mitochondrial function during IR injury.Conclusively,we explore the profound mechanism of PRDX3 to provide a new view of neuronal oxidative stress injury and mitochondrial dysfunction for improving the prognosis of IS.Methods1)Clinical case information of patients prevented in hospital was collected,and univariate analysis was performed using chi-square test.2)The OGD injury models of mouse hippocampal neuron cell line HT-22 and mouse primary cortical neuron were cultured respectively.The expression of PRDX3 at the RNA level in the two cells after OGD treatment was detected by real-time quantitative PCR.In addition,PRDX3 at the protein level was verified by Western-Blot.3)Lentivirus transfection overexpressed PRDX3 in HT-22 and mouse primary cortical neurons respectively.TUNEL assay was used to detect the survival rate of two kinds of cells overexpressing PRDX3 after OGD injury,and Western-Blot was used to detect the expression of apoptosis protein c-Caspase 3.Lactate dehydrogenase（LDH）was used to detect cell viability.4)The level of mitochondrial O₂^-was detected by using Mitochondrial superoxide indicator（Mito Sox）.Reactive oxygen species detection kit was used to detect ROS level of primary cortical neurons.Hydrogen peroxide kit was used to detect the concentration of intracellular H₂O₂.MDA and 4-HNE was used to analyze the level of intracellular lipid peroxidation.5)The level of mitochondrial energy metabolism was assessed by the content of adenosine triphosphate（ATP）.The changes in mitochondrial membrane potential（MMP）were detected by using the mitochondrial membrane potential detection kit（JC-1）.WB detected the expression of mitochondria-specific proteins COX IV and TOMM 20,which can reflect mitochondrial function.6)PCR was utilized to detect the RNA levels of D-loop,ATP 8,PGC-1,NRF-1 and TFAM,which can reflect the level of mitochondrial biosynthesisResults1)After clinical statistical analysis,it was found that the occurrence of IS events was highly associated with the patients’age,underlying diseases（hypertension and diabetes）,long-term smoking history,hypertriglyceridemia and hyperuricemia.The OGD model of HT-22 was successfully established.WB results showed that the expression of PRDX3gradually reduced and maintained a decreasing level for a long time.After successfully overexpressing PRDX3 in HT-22 by using lentivirus transfection,the results of TUNEL and LIVE or DEAD showed that,compared to untreated HT-22,the number of apoptosis of overexpressed HT-22 cells was significantly reduced during OGD injury.2)After successfully extracted mouse primary cortical neurons,the RT-PCR results showed that the expression of PRDX3 in primary cortical neurons decreased after OGD treatment,and maintained a low level for a long time.This result was further verified by WB at the protein level.Overexpression of PRDX3 in neurons by lentiviral transfection can effectively alleviate neuronal apoptosis after OGD treatment by TUNEL.Moreover,neurons overexpressed PRDX3 could significantly reduce the expression of LDH and c-Caspase 3.3)Overexpression of PRDX3 can efficaciously palliate the ROS content of neurons after OGD and alleviate the oxidative stress injury of cells.WB results showed that the expressions of COX IV and TOMM 20 were significantly increased in neurons overexpressed PRDX3.The fluorescence results showed that overexpression of PRDX3could effectively protect neuronal MMP and reduce the production of mitochondrial O₂^-through JC-1 and Mito Sox.The production of H₂O₂,MDA and 4-HNE was significantly reduced whereas the production of ATP was significantly increased in neurons overexpressed PRDX3 after OGD treatment.4)The results of q RT-PCR showed that overexpressed PRDX3 could increase the expression levels of D-loop,ATP 8,PGC-1,NRF-1 and TFAM and could also increase the content of mitochondrial DNA（mt DNA）.ConclusionsBased on the above results,PRDX3 can effectively promote mitochondrial biosynthesis and alleviate mitochondrial oxidative damage,which can protect mitochondrial function,alleviate the accumulation of cellular ROS,maintain cellular ATP function and further reduce cellular oxidative stress levels.Conclusively,PRDX3 serves as a protective role in neuronal oxidative stress damage induced by OGD and,furthermore,alleviates neuronal cell damage and apoptosis.