Buyang Huanwu Decoction Regulate Microglial Cell Polarization By FPR2 To Reduce Inflammatory After Cerebral Ischemia-reperfusion Injury In Rats

ObjectiveTo investigate the regulation of microglia M1/M2 polarization by FPR2.To study Whether Buyang huanwu Decoction(BYHWD)regulates microglia M1/M2 polarization through FPR2 and reduces the inflammatory injury after cerebral ischemia/reperfusionMethodsMale Sprague-Dawley rats weighing 250-300 g were selected,the middle cerebral artery occlusion(MCAO)was established by Longa method.According to neural function score ranging from 1 to 3,qualified rats was selected for experiment.1.Effect of FPR2 on the polarization of microglia after cerebral ischemia reperfusion.(1)Rat were randomly separated into two groups:Sham and MCAO(n=6 per group).The levels of endogenous FPR2 were evaluated at different time points by WB,and the levels of endogenous RvD1 were detected by ELISA.(2)Rat were randomly divided into four groups:Sham,MCAO,RVD1 and RVD1+Boc-2(n=6 per group).RVD 1(0.4 μg/kg)was given intraperitoneally(i.p.)after MCAO.Boc-2(0.4 mg/kg)was injected 30 min before MCAO.TTC staining were employed to determine infarct volume.The expressions of FPR2/Iba-1,MPO,CD16/Iba-1 and CD206/Iba-1 was determined by double immune-fluorescence labeling.The expression of microglia M1/M2 markers were detected by RT-qPCR.2.Buyang Huanwu Decoction Inhibits Inflammation via Regulating Microglia Polarization by FPR2 after Cerebral Ischemia reperfusion.(1)Rat were randomly separated into two groups:Sham,MCAO and BYHWD(n=6 per group),the dose of BYHWD was 16 g/kg each time and given twice a day.Time course expressions of FPR2,LXA4 and RvD1 were detected by Western blot and ELISA respectively at 6 h,24 h,2 d,3 d,7d after ischemia-reperfusion.(2)The rats after surgery were randomly divided into:sham,MCAO,BYHWD,BYHWD+Boc-2(FPR2 antagonist);The neurological performance was evaluated by beam-walking test and grip strength experiment after 24 h.TTC staining were employed to determine infarct volume.The expressions of FPR2/Iba-1,MPO,CD16/Iba-1 and CD206/Iba-1 was determined by double immune-fluorescence labeling.The expression of microglia M1/M2 markers were detected by RT-qPCR.Results1.The experimental results in chapter 1 showed that:(1)The data showed that FPR2 was increased after MCAO,reaching significance at 24 h post MCAO(P<0.05).Compared with Sham,the expression of RvD1was decreased at different time points(P<0.05).(2)RvD1 significantly reduced cerebral infarct volume and the expression of MPO(P<0.05),and its receptor FPR2 was expressed in microglia.RvD1 down-regulated M1 markers CD16+/Iba-1+ cells and the mRNA expression of TNF-α,IL-1β,iNOS,and up-regulated M2 markers CD206+/Iba-1+cells and the mRNA expression of TGF-β1,IL-10,Arg-1(/P<0.05).However,RVD1+BOC-2 group increased the inflammatory of M1-type microglia and inhibited the anti-inflammatory of M2-type microglia(P<0.05).2.The experimental results in chapter 2 showed that:(1)Compared with MCAO group,BYHWD enhanced the expressions of FPR2 and RvD1,peaking at 24 h(P<0.05).Thus 24 h was chosen to be the observation time point for the following experiments.(2)Compared with MCAO group,it was found that BYHWD improved neurological behavior(indicated by beam-walking test and grip strength test)and decreased the cerebral infarct volume(P<0.05).Double immune-fluorescence labeling showed that BYHWD significantly reduced the expression of M1 marker CD16(P<0.05)and enhanced the expression of M2 marker CD206 in microglia(P<0.05).RT-qPCR results showed that BYHWD significantly down-regulated the mRNA expression of M1 microglia inflammatory cytokines TNF-α,IL-1β,iNOS(P<0.05).In contrast,BYHWD up-regulated the mRNA expression of M2 microglia Arg-1,IL-10 and TGF-β(P<0.05).However,BYHWD+BOC-2 group increased the inflammatory of Ml-type microglia and inhibited the anti-inflammatory of M2-type microglia(P<0.05).Conclusions1.The combination of FPR2 and RVD1 promote the polarization of the microglia from M1 to M2 phenotype,and alleviate the inflammatory injury after cerebral ischemia and reperfusion.2.BYHWD Inhibits Inflammation via Regulating the polarization of the microglia from M1 to M2 phenotype in the ischemic brain,the mechanism may be related to the increase the effect of FPR2.