The Role Of TIPE2-Endoplasmic Rericulum Stress Signaling Pathway In Cerebral Ischemia Reperfusion Injury

OBJECTIVE:Ischemic cerebrovascular disease is seriously threating human health,affecting the quality of life,and has increasing trend.It is one of the most important diseases of death and disability in modern society.However,the pathogenesis is not clear,which brings difficulties to the prevention and treatment of the disease.Clinical and animal experiments showed that vascular recanalization after reperfusion injury(schemia-reperfusion,injury,IRI)play a key role in ischemic cerebrovascular disease in the process.Many factors were involved and more and more studies show that moderate inflammatory reaction in cerebral ischemia reperfusion can remove necrotic tissue,but severe inflammatory reaction can cause further damage to brain tissue.Therefore,finding the immune regulation mechanism of cerebral ischemia reperfusion injury is a new direction for treatment of ischemic cerebrovascular disease.Tumor necrosis factor alpha induced protein 8 molecule 2(tumor necrosis factor--induced protein 8-like alpha 2,TIPE2/TNFAIP8L2)is a new gene discovery in 2008,belonging to the TNFAIP8 family.Many research shows that the main function of TIPE2 is inhibited the apoptosis induced by inflammation.Our preliminary results identified that TIPE2 is an important immune negative regulation factor of cerebral ischemia reperfusion inflammation play an important protective role in cerebral ischemia by inhibiting the infiltration of inflammatory cells and inflammatory factors.So TIPE2 is expected to become a new target for treatment of ischemic cerebrovascular disease,but the specific mechanism of immune negative regulation the role of TIPE2 in cerebral ischemia reperfusion inflammation is unclear.The endoplasmic reticulum is an important cellular site of protein modification.when cells are subjected to ischemia and oxidative stress stimulation,activate the unfoldedprotein response(unfolded protein,response,UPR).Its primary goal is to clear unfolded protein,restore cellular homeostasis;but strong stimulation can cause endoplasmic reticulum stress(endoplasmic reticulum stress,ER Stress).UPR will start the process of cell death,so the endoplasmic reticulum stress involved in cancer,stroke and Alzheimer's disease and other diseases occurrence and development.Recent studies have found that endoplasmic reticulum stress plays an important role in the inflammatory response of the body.Whether TIPE2 has been involved in the regulation of ER Stress in cerebral ischemia-reperfusion has not yet been reported.Cerebral ischemia/reperfusion induced endoplasmic reticulum stress in cell by changing the transcription and translation of the elimination of excessive unfolded protein to restore cell homeostasis.On the other hand it is important factors causing inflammation,but the molecular mechanism is not clear.Innate immunity and adaptive immunity are involved in the development of inflammation.NOD like receptor(NLR)is a kind of cell in the induction of PRR[9],which NLRP3 plays an important role in inflammatory corpuscles as an important innate immune reaction in immunity and disease process.Our previous studies have shown that NLRP3 play an important role in cerebral ischemia reperfusion inflammation.Whether ER Stress has been involved in the regulation of NLRP3 in the inflammatory response in cerebral ischemia and reperfusion has not yet been reported.We has preliminarily explored the mechanism of ER Stress in the pathogenesis of cerebral ischemia reperfusion injury.Therefore,in the present study,we used an in vivo middle cerebral artery occlusion(MCAO)model and in vitro cell cultures by oxygen glucose deprivation(OGD)for the first time to investigate the expression and function of TIPE2 in ischemic brains.Find the key of CIR damage for the treatment of ischemic cerebrovascular disease.METHODS:1.To validate the regulation of TIPE2 on the expression of inflammatory factors in vitroMicroglia are important immune cells in the brain,which accounted for the brain cells about 10%-15%,and play an important role in cerebral ischemia reperfusion inflammation.Previous research results showed that TIPE2 was mainly expressed in microglia.Therefore,we selected the microglia cell line BV-2 for the OGD experiment to simulate the model of ischemia-reperfusion in vivo.1.1 To detect the changes of TIPE2 mRNA level and protein expression after glucose and oxygen deprivation,2h and reoxygenation,3h,6h,12h and 24h by real-time quantitative RT-PCR and Western blot.1.2 To detect the changes of TNF-alpha,IL-1 beta,IL-6,IL-18 and MCP-1 mRNA level after glucose and oxygen deprivation2h and reoxygenation 12h by real-time quantitative detection1.3 BV-2 cells were transfected with pRK5-TIPE2 plasmid 24h before OGD treatment,and the expression of inflammatory factors was detected by real-time quantitative RT-PCR.2.TIPE2 regulates the inflammatory response in cerebral ischemia reperfusion by inhibiting ER Stress.2.1 In the preliminary study of-the effects of TIPE2 on the expression of Bip:TIPE2 gene knockout rats were established to MCAO cerebral ischemia reperfusion injury model.By immunofluorescence and Western blot detected the expression of Bip,TIPE2.Preliminary study of-brain effect on ER stress in ischemia reperfusion injury.To verify TIPE2 negative regulation The expression of of Bip in vitro:inhibition of TIPE2 expression and overexpression TIPE2,Western blot detection the expression change of Bip.Overexpression of TIPE2 could inhibit the elevation of Bip:overexpression of pRK5-TIPE2 plasmid in BV-2 cells,OGD 2h reperfusion 12h and then the expression of Bip was detected by Western and Blot.Overexpression of TIPE2 could inhibit the elevation of Bip induced by ER Stress agonist TG:in vitro,BV-2 cells were overexpressed with pRK5-TIPE2 plasmids,and stimulated with TG(5uM)12h.The expression of Bip was detected by Western blot.Inhibition of TIPE2 expression could reverse the decrease of Bip expression induced by ER Stress inhibitor 4-PBA:in vitro,BV-2 cells were inhibited the expression of TIPE2 by siTIPE2,and the expression of Bip was detected by Western blot after 4-PBA(10mM)stimulation for 12h.2.2 TIPE2 regulate the expression of inflammatory factors caused by ER Stress:pRK5-TIPE2 plasmid was transfected into BV-2 cells and treated by endoplasmic reticulum stress agonist TG(5mM)to detect the expression of cytokines after 12h hours;at the same time,the application of siTIPE2 in BV-2 cells inhibits the expression of TIPE2 treated by ER Stress inhibitor 4-PBA(10mM)for 12h.2.3 TIPE2 inhibition of ER Stress pathway:in vitro BV-2 cells were treated with pRK5-TIPE2 plasmids and OGD2h followed by reperfusion.Western blot detected the expression of ER Stress three pathways:PERK,IRE1 and ATF6 changes.2.4 In vivo experiments to verify that TIPE2 down-regulate of ER Stress:8-10 weeks TIPE2-/-mice were injection of 4-PBA(100 mg/kg/)one day before ischemia reperfusion model.After ischemia and reperfusion,by HE,Nissl and TUNEL staining to observation the experimental mice s cortex and hippocampus changes.CBA method to detect the expression of inflammatory factor the serum in model mice.Western blot deteced the expression of Bip and endoplasmic reticulum stress related protein changes.3.To explore the mechanism of ER Stress in the regulation of cerebral ischemia reperfusion injury3.1 To explore the mechanism of the ER Stress regulation of inflammation:8-10 weeks 57BL/6J mice were injected of ER Stress inhibitor 4-PBA bef-ore ischemia reperfusion model.After ischemia reperfusion by Western blot detected the expression of-Bip,NLRP3.Application of ER inhibitor 4-PBA Stress(10mM)before OGD in BV-2cells.Western blot detected the expression of Bip,NLRP3.Real-Time quantitative RT-PCR detection of the mRNA levels of inflammatory cytokines.3.2 TIPE2 negative regulated the expression of NLRP3:BV-2 cells were transfected with pRK5-TIPE2 plasmid.Western blot detected the expression of TIPE2,NLRP3 expression changes.The expression and co-localization of TIPE2 and NLRP3 in microglia were detected by immunofluorescence double staining method.RESULTS:1.TIPE2 down-regulated the expression of inflammatory factors in vitro1.1 To detect the changes of TIPE2 mRNA level and protein expression after glucose and oxygen deprivation,2h and reoxygenation,3h,6h,12h and 24h by real-time quantitative RT-PCR and Western blot.The results showed that the mRNA and protein levels of TIPE2decreased in a time-dependent manner.1.2 To detect the changes of-TNF-alpha,IL-lbeta,IL-6,IL-18 and MCP-1 mRNA level after glucose and oxygen deprivation2h and reoxygenation 12h by real-time quantitative detection,The results showed that compared with the control group,the expression of cytokines TNF-alpha,IL-1 beta,IL-6,IL-18 and chemokine MCP-1 increased significantly.1.3 Real time quantitative RT-PCR showed that compared with the control group,the expression of cytokines after OGD reperfusion was significantly increased,but the expression of inflammatory factors after reperfusion could be inhibited by transfection of TIPE2.These results suggest that TIPE2 negatively regulates the expression of-inflammatory factors in cerebral ischemia reperfusion.2.TIPE2 regulates the inflammatory response by inhibiting ER Stress in cerebral ischemia and reperfusion2.1 The results showed that after ischemia reperfusion the number of microglia and the expression of Bip increased significantly.Compared with wild-type mice,microglia and the expression of Bip increased in TIPE2-/-mices.The Western blot results also showed that,compared with the sham operated group,the expression of Bip inn the ischemia-reperfusion group was increased,and the increase of Bip in the TIPE2-/-group was more obvious than that in the WT group.These results suggest that TIPE2 can negatively regulate Bip and that TIPE2 may regulate the inflammatory response to cerebral ischemia and reperfusion via endoplasmic reticulum stress.BV-2 cell were intervention of TIPE2 expression.Western blot results showed that when inhibition of TIPE2,Bip expression significantly increased;when over expression of TIPE2,Bip expression decreased.These results indicate that TIPE2 can negatively regulate the expression of Bip.Overexpression of TIPE2 can inhibit the increase of Bip which induced by OGD:Western blot showed compared with control group,after reperfusion the protein level of Bip was significantly increased.Overexpression of TIPE2 can inhibit OGD induced Bip expression.Overexpression of TIPE2 could inhibit the expression of Bip by ER Stress agonist TG induced:the result showed that after TG actived the expression of Bip increased significantly,overexpression of TIPE2 can reverse the trendy.Inhibition the expression of TIPE2 can reverse ER Stress inhibitor 4PBA decreasing the expression of Bip.Results showed that the Bip protein expression level was significantly decreased after treatment with 4-PBA and TIPE2 can reversal of the Bip expression.2.2 Increase of TIPE2 can inhibit ER Stressinduced inflammatory factoroverexpression of TIPE2 in BV-2 cells,with endoplasmic reticulum stress agonist TG(5uM)12h hours,CBA results showed that the expression of cytokines,compared with the control group,TG stimulation group significantly increased the expression of inflammatory factors,while overexpression of TIPE2 can inhibit the inflammation caused by TG;while BV-2 cells inhibit the expression of TIPE2,4PBA(10mM)stimulate 12h.CBA results show that the expression of 4-PBA can inhibit the inflammatory cytokines,inhibiting the expression of TIPE2 can be reversed by 4-PBA treatment caused by decreased expression of inflammatory cytokines.2.3 In vitro BV-2 cells overexpressing TIPE2,after reperfusion.Western blot results showed that compared with the control group,the phosphorylation level of PERK,eIF2 alpha,IRE1 after reperfusion were significantly increased and the change of ATF6 is not obvious.Overexpression of TIPE2 can significantly inhibit the phosphorylation levels of PERK,eIF2,and IRE1.Suggesting that TIPE2 can regulate endoplasmic reticulum stress via the PERK-eIF2 alpha and Ire-1 pathways in the downstream pathway of ER Stress.2.4 TIPE2-/-mice were injected with 4PBA in advance,then establishing the model of ischemia-reperfusion was.By HE,Nissl and TUNEL staining,it was found that the cerebral cortex and hippocampus had obvious damage and apoptosis after ischemia-reperfusion.Compared with the WT mice group,the injury of TIPE2-/-mice was more serious,and the 4-PBA preconditioning group could obviously improve the damage caused by TIPE2 deletion.The results of in vivo CBA showed that the expression of inflammatory factors in TIPE2-/-mice was significantly higher than that in WT mice.Compared with the control group,the inflammatory expression in the 4PBA preconditioning group.The results of Western blot showed that the phosphorylation levels of PERK and IRE1 were significantly increased in the TIPE2-/-mice group compared with the WT mice.After pretreatment with 4PBA,the phosphorylation levels of PERK and IRE1 were reduced.3.Preliminarily explored the mechanism of ER Stress in promoting cerebral ischemia-reperfusion injury3.1 Establish the model of ischemia reperfusion mice after 4PBA injection.Western blot results showed that compared with sham operation group,the expression of Bip and NLRP3 were increased significantly after ischemia reperfusion,4-PBA can inhibition the expression of NLRP3 and Bip.After inhibiting TIPE2 and treatment with 4-PBA,Western blot showed that the protein level of Bip and NLRP3 increased after OGD reperfusion,and the expression of Bip and NLRP3 was inhibited by the addition of ER-stress inhibitor 4-PBA.The in vitro and in vivo results suggest that the proinflammatory action of ER Stress is closely related to the regulation of NLRP3 expression.3.2 Overexpression of TIPE2 in BV2 cells,immunofluorescence showed that expression of TIPE2 can significantly inhibit NLRP3;TIPE2 expression decreased and NLRP3 expression increased after reperfusion;overexpression of TIPE2 plasmid can inhibit the elevation expression of NLRP3.TIPE2 and NLRP3 were localized in the cytoplasm.CONCLUSION:1.TIPE2 modulates the inflammatory response by inhibiting endoplasmic reticulum stress.2.Endoplasmic reticulum stress partly promots proinflammatory action by regulating NLRP3.