Neuroprotective Effect Of ATRA Via TLR4/NF-κB Signaling Pathway On MCAO Rats And Its Mechanism

stroke is the second leading cause of death and the first leading cause of disability worldwide,and ischemic stroke(IS)accounts for the vast majority.Reperfusion therapy,with the aim of rapidly returning blood flow to the ischaemic brain tissue,has been a key part of the treatment of IS.Thrombolysis and endovascular reperfusion therapy,has been confirmed to be the most effective strategy to recanalize the occluded vessel and improve patient outcome after acute IS,but limited by time window and in the diotic condition,only a small subset of IS patients obtain above therapies in time.However,even patients undergoing vessel recanalization following endovascular therapy still face a host of adverse outcomes,such as ischemia-reperfusion injury,haemorrhagic transformation,cerebral oedema and inflammatory injury.patients will experience deterioration if those risks cannot be defused,while inflammation is the core of pathological reactions of above adverse outcomes.Inflammatory response of local cerebral ischemia plays an important role in the damage to the central nervous system in the early stages of IS,therefore inhibiting inflammation early might be an effective therapeutic strategy for treating IS.Inflammatory cells and the inflammatory mediators are primary constituents of the inflammatory response.Once IS occurs,Microglia,the resident immunological cells of the brain activated rapidly and migrate to the site of damage precede peripheral macrophages,cleanning dead cell debris through phagocytosis and proteolysis,also release inflammatory factors to damage nerve tissue,plays essential works in onset and resolution of inflammation.It has traditionally been thought that activated microglia can aggravate the damage of ischemic.More and more studies revealed that microglial activation after stroke has a dual role,its with both beneficial and detrimental actions,and its specific role mainly depends upon phenotypic transformation,pro-inflammatory M1 phenotype aggravates tissue damage while anti-inflammatory pro-healing M2 phenotype promote healing and tissue restoration.Thus,suppressing activation of M1 phenotype and promoting microglia polarization toward the M2 phenotype might be an new therapeutic targets to attenuate inflammatory injury after IS.Despite much research in recent years,the complex pathophysiological processes of inflammatory response has not been sufficiently elucidated.But we can certain that inflammation results in tissue hypoxia is an essential step in damaging brain tissues and promoting neuronal cell death.In recent years,studies found that toll-like receptor 4(TLR4)and nuclear factor-kappa B(NF-κB)pathway which closely associated with heart ischemic injuries and cerebral ischemia injuries,may be a critical signal pathway for regulation inflammation after IS.All transretinoic acid(ATRA),as a vital inducer of differentiation,has been successfully applied in clinic.Recent studies indicate that ATRA has protective effects on the ischemia reperfusion injuryof heart,kidney and brain,and its mechanism may involve the pathway in inflammation.The previous study of our team found that ATRA can downregulates the expression of pro-inflammatory cytokines and upregulated anti-inflammatory cytokines to ameliorates the stability of atherosclerotic plaques on carotid atherosclerotic plaques model in rabbit,but the mechanism of its action is unknown.The objective of the present study was to address the inflammatory impact and the possible mechanism of ATRA for middle cerebral artery occlusion(MCAO)in rat,finding new targets for the treatment of IS and providing experimental evidence for the development of new drugs.Objective: To investigate whether ATRA affects the inflammatory response of infarct brain tissue through the ATRA/NF-κB signaling pathway,thereby playing a neuroprotective effect on MCAO model rats.Methods:(1)Six-week-old male Sprague-Dawley(SD)rats were randomly divided into Sham group(Sham group),model group(MCAO group)and MCAO model + ATRA treatment group(ATRA group),with15 rats in each group.MCAO model was established to simulate IS in rats.(2)After MCAO model was established in rats,ATRA was intraperitoneally injected into the experimental group for drug intervention.Choosing the Sham group and the MCAO group as controls,the effects of ATRA on the symptoms of neurological defects,cerebral infarction volume and brain tissue morphology in post-IS rats were observed and compared to analyze its brain protection effect.(3)IL-6 levels of serum in each group were detected by ELISA to analyze the effects of ATRA on inflammation in IS rats.(4)IF staining method was used to detect the expression of Iba-1 and CD206 in the brain tissue of rats to observe the classification and morphological changes of microglia,analyzing whether ATRA played a neuroprotective role in rats after IS by regulating microglia.(5)The expression of TLR4,p65 NF-κB,Myd88 and IL-6 proteins was detected by WB method to analyze if ATRA inhibits the inflammatory response through regulating the TLR4/NF-κB signaling pathway.Results:1.Compared with the MCAO group,the neurobehavioral score of the ATRA group did not change significantly(P=0.999,P=0.535)during reperfusion for 2h and 24 h,and the neurobehaviorological score of rats in the ATRA group decreased compared with the MCAO group at 48 h and 72h(P<0.01).2.Compared with the MCAO group,the volume of cerebral infarction in the ATRA group was significantly reduced during reperfusion for 24 h and72h(P<0.001,P<0.01).3.Rats in the MCAO group developed edema and vacuoles in brain cells after ischemia reperfusion;after ATRA intervention,brain tissue edema decreased,vacuolar cells decreased.4.Compared with the Sham group,the serum IL-6 content in the MCAO group and the ATRA group was significantly increased(P<0.001),and compared with the MCAO group,ATRA intervention could reduce the serum IL-6 level(P<0.01).5.Compared with the Sham group,the MCAO group had an increase in Iba-1 positive cells,cell body enlargement,shortened protrusions,and the cells were round or "amoeba" like.After the ATRA intervention,may decrease in Iba-1 positive cells,the morphology was highly branched,and the expression of CD206-positive cells was significantly increased.6.Compared with the Sham group,the expression of TLR4,p65 NF-κB,Myd88 and IL-6 proteins in the brain tissue of MCAO group increased significantly(P<0.05),and the expression levels of TLR4,p65 NF-κB,Myd88 and IL-6 decreased after ATRA treatment(P<0.01).Conclusions:We defined the protection of ATRA on infarct brain tissue in MCAO model rats,and its mechanism is related to down-regulation of the protein expression of TLR4,p65 NF-κB,Myd88,regulating the TLR4/NF-κB signaling pathway,thereby reducing the release of downstream IL-6 and alleviating the inflammatory response of infarcted brain tissue.