The Protective Role Of GPR30 In Inhibition Of Microglial Inflammation Via TLR4 In Cerebral Ischemia Injury

Stroke has become one of the major serious threat to human health that caused serious disability and high morbidity.In China,there are nearly 2 million people died from stroke each year,in other words,four people per minute on average,which brought heavy burden to family and society.So how to effectively prevent and reduce ischemic brain injury is the major problem urgently to be solved in neuroscience research at present.A number of studies have shown that estrogen have prominently nerve protective effect.Estrogen can not merely enhance cell survival signals,reduce glutamate excitatory toxic injury,improve local cerebral blood flow,reduce infarct volume and improve nerve function,but also inhibit neuroinflammation and relieve cerebral ischemia reperfusion injury.Large sample clinical trials obtain the opposite conclusion,leading to stagnation of clinical application of estrogen in prevention and treatment of ischemic brain injury.The above results suggest that estrogen exact role in central nervous system remains controversial,which makes it particularly important to explore new molecular mechanism and signal pathway to elucidate estrogen neural protection function,furthermore,to promote its clinical application.GPR30(G protein-coupled receptor 30)is a newly discovered membranous G protein coupled estrogen receptor in 2005 mediating the effect of rapid non-genome signals.GPR30 is highly expressed in the central nervous system and distribute in different brain regions,and participate in the various physiological function of the nervous system.Our team members found for the first time that GPR30 specificity agonists G1 can reduce ischemia-reperfusion injury of ovariectomized(OVX)mouse,suggest that GPR30 plays an important role in the nerve protective effect induced by estrogen.However its explicit molecular mechanism remains unclear.Our latest researches find that GPR30 expression is significantly increased after ischemic injury.What,s more,the agonist G1 can reduce the release of inflammatory factors TNF-α,IL-1β,IL-6 after ischemic injury,which implied that GPR30 is extremely likely to be an important molecular targets in the process of inflammatory reaction and neuron protection after ischemia.Previous studies have shown that both E2 and G1 could reduce the expression of TLR4(Toll-like receptor 4)on the surface of peripheral macrophages,thus relieved inflammation reaction.But this effect disappeared when GPR30 is knockout,suggest that TLR4 participates in the anti-inflammation reaction of GPR30.TLR4 is a key molecule mediating inflammatory reaction after cerebral ischemia-reperfusion injury and mainly expressed in microglia in the central nervous system.Our further study find that GPR30 and TLR4 have high colocalization in microglia and GPR30 agonist G1 can reduce the expression of TLR4 in microglia after ischemic or anoxic injury in vivo and in vitro experiment.Based on these findings,we finally confirmed that the activation of GPR30 in microglia can inhibit the expression of TLR4,thus relieve microglia activation and reduce the release of inflammatory cytokines,eventually alleviate ischemia injury.Our study provide a new molecular mechanism for estrogen neuroprotection pathway and offer a target in the treatment of acute ischemic stroke.Part 1: The influence of GPR30 on cerebral ischemia injuryObjective: To detect whether the activation of estrogen receptor GPR30 could reduce infarction volume and improve neurological deficit of ischemic injury.Methods: Forty-five female OVX mice were randomly divided into 5 groups: the Sham group,Vehicle group,E2 group,G1 group and ICI182780 + E2 group.E2,G1 and ICI182780 were administered immediately upon reperfusion,then infarct volume assessment,neurobehavioral evaluation,TUNEL staining,Nissol staining and immunofluorescence staining were performed at 24 h after reperfusion to evaluate brain function,neuronal necrosis and survival after ischemic injury.Results: Compared with Vehicle group,E2,G1 and ICI182780+E2 significantly reduced the infarct volume and neurological deficit score(P<0.05).TUNEL staining showed that E2,G1 and ICI182780+E2 significantly reduced the TUNEL positive cells(apoptosis)in penumbra compared with Vehicle group(P < 0.05).E2,G1 and ICI182780+E2 significantly reduced neuronal damage compared to Vehicle group(P<0.05).Neuronal nuclei staining showed that MCAO decreased Neu N positive cells(neurons)and E2,G1,ICI182780+E2 significantly increased Neu N positive cells in penumbra(P<0.05).Conclusion: The activation of estrogen receptor GPR30 could reduce infarction volume and improve neurological deficit of ischemic injury.Part 2: The influence of GPR30 on inflammatory response caused by cerebral ischemia injuryObjective: To detect whether the activation of estrogen receptor GPR30 could relieve inflammatory response caused by cerebral ischemia injury.Methods: Thirty female OVX mice were randomly divided into 5 groups: Sham group,Vehicle group,E2 group,G1 group and ICI + E2 group.E2,G1 and ICI were respectively administered immediately upon reperfusion,then ELISA was performed to measure the inflammatory cytokines TNF-α,IL-1β and IL-6 release levels in penumbra at 24 h after ischemia reperfusion.Primary microglia were randomly divided into 5 groups: Sham group,Vehicle group,E2 group,G1 group and ICI + E2 group.E2,G1 and ICI were respectively administered immediately upon the initiation of OGD,then ELISA was performed to measure the inflammatory cytokines TNF-α,IL-1β and IL-6 release levels in supernant and protein extracts at 12 h after reperfusion.Neuron-microglia co-culture model was established and then CCK8 and LDH was perforemed to detect cell viability and LDH release of neurons subjected to OGD/R injury.Results: ELISA results showed that E2,G1 and ICI+ E2 significantly reversed the MCAO/R or OGD/R induced upregulation of inflammatory cytokines TNF-α,IL-1β and IL-6 in penumbra or in microglia cells(P<0.05).CCK8 and LDH results showed that E2,G1 and ICI+ E2 significantly improved neuron cells viability(P<0.05)and reduced LDH release(P<0.05).Conclusion: The activation of estrogen receptor GPR30 could reduce the production and release of inflammatory cytokines TNF-α,IL-1β,IL-6 in microglia and alleviate neuron injury.Part 3: The influence of GPR30 on microglia activation state mediated via TLR4 and its potential molecular mechanismExperiment 1: The influence of GPR30 on microglia activation state mediated via TLR4Objective: To detect the influence of GPR30 activation on microglia activation state mediated by TLR4.Methods: Thirty female OVX mice were randomly divided into 5 groups:the Sham group,Vehicle group,E2 group,G1 group and ICI182780 + E2 group.E2,G1 and ICI182780 were administered immediately upon reperfusion,immunofluorescence staining was performed at 24 h after reperfusion to observe the expression of GPR30 and TLR4 in microglia and the state of its activation in penumbra.Western Blot was also performed to detect the expression of GPR30 and TLR4 protein.Immunofluorescence staining was performed to observe the expression of GPR30 and TLR4 in primary microglia.Primary microglia were randomly divided into 5 groups: Sham group,Vehicle group,E2 group,G1 group and ICI + E2 group.E2,G1 and ICI were respectively administered immediately upon the initiation of OGD,then Western Blot was performed to measure the expression of GPR30 and TLR4 protein.Results: GPR30 and TLR4 were both high expressed in microglia and displayed a strong colocalization in vivo and vitro experiments.Immunofluorescent staining of Iba1 showed that active microglias displayed larger somata and shorter coarser cytoplasmic processes than the resting ones in penumbra,E2,G1 and ICI+E2 treatment could relieve the activation state of microglia.Western blot analysis showed that compared with the sham group,Iba1,the microglia maker,and TLR4 expression were both reduced in penumbra or microglia after reperfusion(P<0.05).Conclusion: GPR30 activation on microglia could alleviate its activation state mediated via TLR4.Experiment 2: The role of TLR4 in neuroprotection effect of GPR30Objective: To detect the role of TLR4 in neuroprotection effect of GPR30 receptor.Methods: C57BL/6 OVX mice or tlr4-/-OVX mice were randomly divided into 5 groups: tlr4+/++Vehicle(WT)group,tlr4+/++G1(WT+G1)group,tlr4-/-+Vehicle(tlr4-/-)group,tlr4-/-+G1(tlr4-/-+G1)group,then MCAO was performed and Vehicle or G1 were administered immediately upon reperfusion.TTC staining were performed to measure infarct volume.Results: Compared with tlr4+/+ mice,the infarct volume of tlr4-/-mice was significantly reduced(P<0.05),however,the infarct volume of tlr4-/-mice accepted G1 administered was insignificantly changed when compared with tlr4-/-ones.Conclusion: After tlr4 gene was knocked out,the neuroprotection effect of GPR30 receptor abated,suggested that TLR4 participated in GPR30 signaling pathway.Experiment 3: The molecular mechanism of GPR30 regulating TLR4Objective: To investigate the molecular mechanism of GPR30 regulating TLR4.Methods: Primary microglia were randomly divided into 5 groups: Control group,Ig G group,OGD group,G1 group and Input group.Co-immunoprecipitation was performed to investigate whether GPR30 and TLR4 protein have direct interaction with each other.Results: There was no direct interaction between GPR30 and TLR4 protein.Conclusion: There was no direct interaction between GPR30 and TLR4 protein,GPR30 may modulate TLR4 protein via other moleculars or signaling pathways.