Involvement Of Microglia Phenotype In Electroacupuncture-induced Neuroprotection Against Cerebral Ischemic Injury Through α7nAChR In Rat With Stroke

Stroke is the fifth leading cause of death worldwide with high mortality and disability,which seriously impairs people’s lives and is a common societal burden with substantial economic costs.Although great advances have been made in understanding the diverse mechanisms of neuronal cell death induced by ischemic stroke,clinically effective neuroprotective therapies are limited.It is a major imperative to gain an effective strategy for stroke intervention.Ischemic stroke constitutes more than 80% of strokes.Obstruction of main arterial leads to a depletion in energy and oxygen within the affected area,resulting in cell death and disruption.These dying cells secrete danger signals that stimulate an inflammatory response in the brain.Severe inflammatory response is an important cause of secondary brain injury.However,it has been suggested that long-term functional recovery could be worse when inflammation after stroke is inhibited.so,controlling the inflammation precisely during stroke will provide a new way on the treatment of stroke.Microglia is the resident immune cell in the central nervous system,activation of microglia is the first step in the inflammatory response of the brain.Microglia can be activated into two different phenotypes:classically activated(M1)and alternatively activated(M2).The M1 phenotype is a proinflammatory phenotype,releasing a large amount of inflammatory cytokines in the eraly stages of ischemic stroke,which exacerbates neuronal damage;However,M2 phenotype is a anti-inflammatory phenotype,facilitating damage repair by secreting anti-inflammatory cytokines and neurotrophic substances at later timepoints after ischemic stroke.Thus regulating microglia activation state to alleviate neuroinflammation may be the new target for the treatment of stroke.Cholinergic anti-inflammatory pathway inhibits inflammatory responses through communication between the nervous and immune systems.α7 nicotinic acetylcholine receptors(α7n ACh Rs)play a very important role in this process.α7n ACh Rs are widely expressed on the surface of neurons and non-neuronal cells,including astrocytes,microglia,oligodendrocytes,and endothelial cells.Numerous studies indicated that activating the α7n ACh Rs of microglia and macrophage can inhibit the inflammatory response,and play a neuroprotective effect.Our previous studies have found that the electroacupuncture pretreatment can effectively reduce cerebral ischemic injury,but the precise mechanisms of this phenomenon are unclear.Whether electroacupuncture pretreatment can regulate the activation of microglia through α7n ACh R,making transformation of microglia from proinflammatory phenotype to anti-inflammatory phenotype? We designed the following experiments to answer this question.Experiment 1.The effect of electroacupuncture pretreatment on the activation of microglia after ischemia and reperfusionObjective: To observe whether electroacupuncture pretreatment changes the activation state of microglia in cerebral ischemic penumbra after cerebral ischemia reperfusionMethods: Adult male Sprague-Dawley rats were randomly divided into 3 groups: Sham,MCAO and EA+MCAO.They were sacrificed at 3 days after reperfusion and the expression of specific microglial markers of M1/M2 phenotypes were analyzed by Western Blot.Results: Electroacupuncture pretreatment decreased the expression of i NOS and IL-1β and increased the expression of Arginase and TGF-β(P<0.05,vs.MCAO).Conclusion: Electroacupuncture pretreatment could change the activation state of microglia after cerebral ischemia and reperfusion,making microglia transformed from M1 to M2.Experiment 2.Electroacupuncture pretreatment regulates the activation of microglia after ischemia and reperfusion through α7n ACh RObjective: To observe whether electroacupuncture pretreatment changes the activation state of microglia in cerebral ischemic penumbra after cerebral ischemia reperfusion through α7n ACh R.Methods: 1.The effect of a7 n ACh R agonist PHA-543,613 on the activation of microglia after ischemia and reperfusion Adult male Sprague-Dawley rats were randomly divided into 5 groups: Sham,MCAO,PHA-543,613+MCAO,Vehicle+MCAO,EA+MCAO.They were sacrificed at 3 days after reperfusion and the expression of specific microglial markers of M1/M2 phenotypes were analyzed by Western Blot.2.The effect of α7n ACh R antagonist α-BGT on the activation of microglia after ischemia and reperfusion Adult male Sprague-Dawley rats were randomly divided into 5 groups: Sham,MCAO,EA+MCAO,α-BGT+EA+MCAO,Vehicle+EA+MCAO.They were sacrificed at 3 days after reperfusion and the expression of specific microglial markers of M1/M2 phenotypes were analyzed by Western Blot and Immunofluorescence staining.Results: 1.Compared with MCAO,the expression of i NOS and IL-1β were significantly reduced in PHA-543,613+MCAO and EA+MCAO,while the expression of Arginase and TGF-β were increased(P<0.05,vs.MCAO).There was no significant difference between MCAO and Vehicle+MCAO.2.Compared with EA+MCAO,the expression of iNOS and IL-1β were increased in α-BGT+EA+MCAO,while the expression of Arginase and TGF-β were decreased(P<0.05,vs.EA+MCAO).There was no significant difference between EA+MCAO and Vehicle+EA+MCAO.The results of immunofluorescence staining were consistent with Western Blot.Conclusion: Electroacupuncture pretreatment regulated the activation of microglia through α7n ACh R.Experiment 3.Electroacupuncture pretreatment alleviates cerebral ischemia injury through α7n ACh RObjective: To verify the neuroprotective effects of regulating α7nAChR after cerebral ischemia-reperfusionMethods: Adult male Sprague-Dawley rats were randomly divided into 6 groups: Sham,MCAO,PHA-543,613+MCAO,Vehicle+MCAO,EA+MCAO,α-BGT+EA+MCAO.They were sacrificed at 3 days after reperfusion.We tested inflammatory factors by ELISA Kits and assessed brain function by TTC staining,neurobehavioral scores(Garcia score).Results: ELISA: the level of TNF-α was reduced in PHA-543,613+MCAO and EA+MCAO compared with MCAO,while the level of IL-10 was increased(P<0.05,vs.MCAO);the level of TNF-α was increased in α-BGT+EA+MCAO compared with EA+MCAO,while the level of IL-10 was decreased(P<0.05,vs.EA+MCAO).TTC: compared with MCAO,the infarct size was decreased in PHA-543,613+MCAO and EA+MCAO(P<0.05,vs.MCAO);compared with EA+MCAO,the infarct size was increased in α-BGT+EA+MCAO(P<0.05,vs.EA+MCAO).neurological behavior score: compared with MCAO,the neurological behavior score was increased in PHA-543,613+MCAO and EA+MCAO(P<0.05,vs.MCAO);compared with EA+MCAO,the neurological behavior score was decreased in α-BGT+EA+MCAO(P<0.05,vs.EA+MCAO).Conclusion: a7 n ACh R agonist PHA-543,613 could simulate the effect of electroacupuncture pretreatment and improve neurological function after ischemia-reperfusion injury;α7n ACh R antagonist α-BGT reversed it.Experiment 4.The effects of regulating a7 n ACh R of microglia to microglia activation and neuronal survival after oxygen and glucose deprivationObjective: To further verify the role of a7 n ACh R in altering the microglia activation state and improving neurological function in vitro.Methods: 1.The best effective concentration of α7n ACh R agonist PHA-543,613 in cell protection Building oxygen and glucose deprivation(OGD)model simulated cerebral ischemia injury.The purified primary microglia were randomly divided into 6 groups: Control,OGD,OGD+ PHA-543,613 0.1μM,OGD+ PHA-543,613 1μM,OGD+ PHA-543,613 10μM,OGD+ PHA-543,613 100μM.The expression of Arginase was detected by Western Blot after regaining oxygen and glucose for 24 h.2.The effects of regulating a7 n ACh R of microglia to microglia activation after oxygen and glucose deprivation The purified primary microglia were randomly divided into 5 groups: Control,OGD,PHA-543,613+OGD,PHA-543,613+α-BGT+OGD,α-BGT+OGD.Drugs pretreatment for 24 hours and treated by OGD model for 2h,then regained the oxygen and glucose for 24 h.the expression of IL-1β and Arginase were analyzed by Western Blot and Immunofluorescence staining.3.The effects of regulating a7 n ACh R of microglia to neuronal survival after oxygen and glucose deprivation Neuron-microglia model was established and divided into 5 groups: Control,OGD,PHA-543,613+OGD,PHA-543,613+α-BGT+OGD,α-BGT+OGD.Then CCK8 and LDH was perforemed to detect cell viability and LDH release of neurons subjected to OGD/R injury after different treatment.Results: 1.Western Blot showed that the expression of Arginase was significantly increased when the concentration of PHA-543,613 was 100μM(P<0.05,vs.OGD),and there was no significant difference between the other concentrations.Thus,the concentration of 100 μM was chosen as the best effective concentration for the follow-up experiments.2.The expression of IL-1β in PHA-543,613 + OGD group was significantly lower than that in OGD group,while the expression of Arginase was higher(P <0.05,vs.OGD);There was no significant difference of PHA-543,613+α-BGT+OGD group,α-BGT+OGD group and OGD group.The results of Immunofluorescence staining were consistent with those of Western Blot.3.Compared with OGD group,neuron cell viability increased and LHD release decreased in PHA-543,613 + OGD(P <0.05,vs.OGD);There was no significant difference of PHA-543,613+α-BGT+OGD group,α-BGT+OGD group and OGD group.Conclusion: Up-regulation α7nAChR of microglia could promote microglia switching from M1 to M2,which was beneficial to neuronal survival;and inhibition of α7n ACh R inhibited this effect,which was harmful to neuronal survival.Summary In current research,we found that electroacupuncture pretreatment provided neuroprotective effect by changing the activation state of microglia,making transformation of microglia from M1 to M2.The expression of proinflammatory cytokines were reduced following electroacupuncture preconditioning after ischemia and reperfusion,while the expression of anti-inflammatory factors were increased.EA also decreased the infarct size and increased the neurological behavior score.The a7 n ACh R agonist PHA-543,613 could simulate the effect of electroacupuncture preconditioning,while inhibition of α7n ACh R inhibited this effect.Therefore,we concluded that the electroacupuncture pretreatment can alleviate cerebral ischemia injury by making transformation of microglia from M1 to M2,and α7n ACh R was involved in this process.