The Effect And Mechanism Of Lipoxin A4 On The Microglia Polarization After Cerebral Ischemia-reperfusion Injury

BackgroundIschemic stroke refers to cerebral necrosis due to the stenosis or occlusion of the cerebral supplying artery?carotid artery or vertebral artery system?following insufficient blood supply of the brain.China is one of the countries with the highest incidence of stroke in the world.There are total 10×10⁶ cases of stroke patients,among them,85%are ischemic strokes,which brings great suffering to the patients and heavy medical and economic burden to the family and society.China is the largest populated country in the whole world.The incidence of ischemic stroke is increasing year by year,and the recurrence rate is higher than the global average,which seriously affects the national life and quality of life.The pathogenesis of ischemic stroke is complicated,involving various mechanisms such as inflammatory reaction,excitatory amino acid toxicity,Ca²⁺overload,oxidative stress damage and so on.More and more studies have shown that inflammatory reaction plays an essential role in the development of ischemic stroke.Inflammation can induce neuronal apoptosis,damage the integrity of the blood-brain barrier,and aggravate brain edema.Recently reducing the inflammatory reaction and preventing inflammatory injury have become the research direction and treatment strategy for improving the prognosis of ischemic stroke.Lipoxins?LXs?are the endogenous lipid mediators produced by the body in an inflammatory response,and have a strong anti-inflammatory and pro-inflammatory-reducing effect.In recent years,some studies have shown that LXA4 has protective effects on ischemic stroke,but the internal mechanism and exact link of its effects are still unclear.Recent studies have found that microglia can play an important role in the inflammatory response of ischemic stroke.Microglia are the resident macrophages of the central nervous system and it can be rapidly detected after ischemic stroke.The activated microglia mainly have two polarization states,M1 and M2,which play both pro-inflammatory and anti-inflammatory roles.Whether LXA4 can regulate the polarization of microglia and achieve anti-inflammatory and inflammatory damage in the pathogenesis of ischemic stroke is not clear,and no relevant research has been reported so far.The aim of this study was to elucidate the effects of LXA4 on microglial polarization after cerebral ischemia-reperfusion injury at animal and cell levels,and to explore its possible mechanism in order to provide a new way of thinking for the clinical treatment of ischemic stroke.To provide a new basis for the treatment of ischemic stroke with LXA4.Objective1.LXA4 can reduce the infarct volume and reduce the neurological damage after cerebral ischemia-reperfusion injury in rats.2.To elucidate the effect of LXA4 on the polarization of microglia after cerebral ischemia-reperfusion injury.3.To explore the molecular mechanism of LXA4 on the regulation of microglia polarization during cerebral ischemia-reperfusion injury.Methods1.The model of middle cerebral artery occlusion?MCAO?reperfusion injury was established in vivo by modified suture method.Immediately after the model was established,LXA4?5?L,0.2mmol/L?was injected into the lateral ventricle for drug intervention.After 2 hours of ischemia,the plug was pulled out to achieve reperfusion,and subsequent tests were performed at a given time point.2.BV-2 microglia were cultured in vitro to construct oxygen glucose deprivation/reoxygenation?OGD/R?model.BV2 microglia were treated with OGD for 6 hours and reoxygenation for 12 hours.3.The model of middle cerebral artery occlusion?MCAO?reperfusion injury was established in vivo by modified suture method.Immediately after the model was established,LXA4?5uL,0.2mmol/L?was injected into the lateral ventricle for drug intervention.After 2 hours of ischemia,the plug was pulled out to achieve reperfusion,and subsequent tests were performed at a given time point.4.Neurological scores and TTC staining were used to observe the size of cerebral infarction and edema by Longa method.The degree of infarction and pathological changes were observed by HE staining.The neuronal apoptosis around the infarcts was observed by TUNEL staining.5.RT-PCR detection of inflammatory factors?IL-1?,TNF-??,M1 microglia markers?iNOS,CD32?,M2 microglia markers(Arg-1,CD206 And mRNA expression of signaling pathways?Notch-1,Hes-1,Hes-5?;6.Western-blot was used to detect the protein expression of M1 microglial marker?iNOS?,M2 microglial marker?Arg-1?and signaling pathway?Notch-1,Hes-1,Hes-5?;7.ELISA detects the expression of cytokines of inflammatory factors IL-1?and TNF-?;8.The morphological changes of activated microglia were observed by immunofluorescence,and the protein expressions of M1 microglial marker?iNOS,CD32?and M2 microglial marker?Arg-1,CD206?were detected.Results1.In the rat model of focal cerebral ischemia-reperfusion injury,by behavioral score,TTC staining,HE staining and TUNEL staining,it was observed that intracerebroventricular injection of 1nmol LXA4 significantly reduced neurobehavioral score,infarct and edema volume,neuronal necrosis and apoptosis infarct and edema volume.2.Iba1 immunofluorescence results showed that the resting microglia showed small cell bodies,highly branched,and the activated microglia cell body became larger,the protrusion became shorter,and the spherical amoeba-like shape.LXA4 can slightly inhibit the activation of microglia after intervention,but most of the cell morphology is still"amebic".3.The mRNA and protein expression levels of IL-1?and TNF-?were significantly increased 24 h after MCAO and BV2 microglia cells in vitro?P<0.05?.After LXA4 intervention,the mRNA and protein expression levels of IL-1?and TNF-?were decreased?P<0.05?.4.The mRNA and protein expression levels of M1 type Marker?iNOS,CD32?and M2 type Marker?Arg-1,CD206?increased after 3 days of MCAO in vivo and BV2 microglia OGD/R12h in vitro.?P<0.05?;after LXA4 intervention,the mRNA and protein expression of M1 type Marker CD32 decreased?P<0.05?,the protein expression of M1 type Marker iNOS decreased?P<0.05?,and the expression of mRNA did not change significantly.?P>0.05?,mRNA and protein expression of M2type Marker?Arg-1,CD206?were increased?P<0.05?;The results of immunofluorescence double staining of iNOS,Arg-1,CD32 and CD206 after BV2microglia OGD/R12h in vitro were consistent with those mentioned above.5.The mRNA and protein expressions of Notch signaling pathway-related proteins Notch-1,Hes-1 and Hes-5 were increased on the 3rd day after MCAO in rats and in vitro.The expression of Notch-1,Hes-1 and Hes-5 in BV2 microglia cells was increased?P<0.05?.After LXA4 intervention,the mRNA and protein expression levels of Notch-1 and Hes-1 decreased?P<0.05?,and the expression of Hes-5 mRNA and protein increased?P<0.05?.Conclusions1.LXA4 could inhibit the inflammatory reaction and the expression of inflammatory factors in cultured microglia after cerebral ischemia-reperfusion and oxygen deprivation in vitro.2.LXA4 can reduce the infarct volume and decrease the nerve function injury after cerebral ischemia-reperfusion injury in rats.3.LXA4 can regulate the polarization of microglia and microglia in vitro after cerebral ischemia-reperfusion injury in rats,inhibiting its polarization into M1 type and promoting its polarization into M2 type.4.The regulation of LXA4 on the polarization of reoxygenated microglia after cerebral ischemia-reperfusion and oxygen deprivation may be achieved through the Notch signaling pathway.