Regulatory Effect Of Activin A In The Polarization Phenotype Of Microglia In Ischemic Brain Injury

Ischemic brain injury has a complex pathophysiological process involving multiple injury and repair mechanisms.Microglia in the central nervous system respond differently to the microenvironment and different stimulation signals,showing different functional phenotypes including M1 phenotype and M2 phenotype.This polarization behavior of microglia plays a "double-edged sword" role in cerebral ischemia injury,and the polarity of the phenotype may aggravate the injury or promote repair.Activin A(activin A,A)act as transforming growth factor-?(transforming growth factor beta,TGF-?)over one of the members of this family,and its receptor binding protein is widely distributed in the brain tissue,ischemic brain injury can cause Act A expression,by activating the different signaling pathways,neuroprotective activity,involved in neuron damage after repair.Act A may be an important factor in regulating microglia behavior,but its mechanism is still unclear.In this study we aim to explore the rule of ischemic brain damage microglia polarization and mechanisms that Act A involved in regulation of microglia polarization.We establish the rat model of focal cerebral ischemia injury and in vitro experiments BV2 OGD/R(oxygen and glucose deprivation/repoxygenation)model was made.According to different time points of ischemia/hypoxia,the Act A/Smads signaling pathway and the expression changes of microglia phenotype in ischemic brain injury were detected,and the change trend of exogenous activin A interfering microglia polarization was studied.This study includes the following contents:1.Expression of Act A/Smads signaling pathway in focal ischemic brain injuryObjective:To investigate the expression of Act A/Smads signaling pathway in focal ischemic brain injury.Methods:The middle cerebral artery occlusion model(MCAO)was established by Longa method.Different ischemic time points(0 d,1 d,3 d,5 d 7 d and 14 d)were selected to randomly group.Modified neurological severity score(mNSS)was used to evaluate the neurological damage,TTC staining was used to analyze the area of ischemic cerebral infarction,and HE staining was used to evaluate the pathological changes after infarction.Western blot assay was used to detect the proteins expression levels of ActR?and downstream factors Smad2 associated with Act A/Smads pathway in brain tissue.Results:After the establishment of MCAO focal cerebral ischemia model,the neurofunctional score indicated obvious functional defect,and the score gradually decreased with the prolongation of ischemic time.Cerebral infarction lesions were observed with TTC staining,and the infarct tissue volume gradually decreased with the extension of ischemia time.HE staining of coronal section of brain tissue showed a large number of neuronal cell necrosis in the infarcted core area of cortex,with unclear nuclear contraction and nucleoli.In the Act A/Smads signaling pathway,Act A,Act All receptor,receptor complex Smad2 were respectively expressed in higher gene and protein levels.With the extension of ischemia time,the expression peak reached 3-5 days after ischemia,and the difference was statistically significant(P<0.05).Conclusion:Ischemic brain injury can induce the expression of Act A and up-regulate genes and proteins expression of transmemmal receptor and downstream receptor factor in the Act A/Smads signaling pathway.Act A/Smads signaling pathway is involved in the process of ischemic brain injury,and its expression reaches a peak in 3-5 days after ischemia,laying an experimental foundation for the subsequent study of the relationship between Act A/Smads signaling pathway and microglia cells in ischemic brain injury.2.The phenotypic change of microglia during focal ischemic brain injuryObjective:To study the phenotypic changes of microglia at different ischemic time points of ischemic brain injury.Methods:The MCAO model was established at different time points of ischemia(1 d,3 d,5 d,7 d and 14 d).The expression of M1 and M2 microglia marker gene was detected by Realtime PCR,and the expression of M1 and M2 microglia marker protein in situ was detected by immunohistochemistry.Results:Real-time PCR showed that the expression level of M1-phenotypic markers(iNOS,CD86)and M2-phenotypic markers(CD206,Arg-1)in the 1st day ischemia group was at a low level,and the expression level of iNOS gradually increased from the 3rd day of MCAO and maintained an increasing trend for at least 14 days after ischemia.The expression level of CD86 gene decreased gradually after reaching the peak expression level on Day 5 of MCAO,and the expression level of CD86 gene on day 14 of MCAO was close to the initial expression level of ischemic injury.The gene expression trends of CD206 and ARG-1 were the same,both of which were induced to express in MCAO at 1-3 d,and reached the peak expression at 3 D after injury.With the extension of ischemia time,the expression levels of CD206 and Arg-1 gradually decreased.Immunohistochemical results and the change trend of real-time fluorescent quantitative PCR results are basically identical.The difference was statistically significant(P<0.05).Conclusion:brain injury.Ischemic brain injury can induce the activation of microglia.The M2 phenotype dominates the early ischemic stage and the M1 phenotype dominates the later ischemic stage,suggesting that the M2 phenotype polarization of microglia induced by early ischemia may mediate phagocytosis and anti-inflammatory effects,while the Ml phenotype polarization may mediate chronic inflammatory injury.3.The effect of Act A on phenotypic changes of microglia in focal ischemic brain injury.Objective:To study the effect of Act A on microglia polarization in ischemic brain injury.yMethods:The middle cerebral artery occlusion(MCAO)model of rats was selected at the time point of 3 days of ischemia.After 6 hours of cerebral ischemia,exogenous Act A(2.5 ?g/kg,7.5 ?g/kg)was injected into the lateral ventricle.The expression of marker genes of M1 and M2 microglia was detected by realtime qPCR.In situ expression of M1 and M2 microglia markers was detected by immunohistochemistry.Results:Results of the evaluation of neurological functional defects after ischemic brain injury by mNSS showed that the scores of the exogenous Act A intervention group were significantly better than those of the MCAO 3d group,and Act A 7.5 ?g/kg group were better than Act A 2.5 ?g/kg group,with statistically significant differences between the groups(P<0.05).TTC staining results showed that the intervention group was significantly smaller than the MCAO 3d group,and Act A 7.5 ?g/kg group was smaller than Act A 2.5 ?g/kg group,and the difference was statistically significant(P<0.05).The real-time PCR results showed that the expression level of M1 phenotype marker iNOS was increased in the MCAO 3d group and decreased after the intervention of Act A.The levels of iNOS in the Act A 7.5 ?g/kg group and Act A 2.5 ?g/kg group were lower than those in the MCAO 3-d group.M2 phenotypic markers CD206 gene expression in MCAO 3 d group was higher than control group,the level in Act A 7.5 ?g/kg group and Act A 2.5 ?g/kg group were higher than MCAO 3 d.group.The expression of ActR ?gene showed that the level of the intervention group was higher than that of MCAO 3d group,and Act A 7.5 ?g/kg group was higher than that of Act A group 2.5 ?g/kg.The difference was statistically significant(P<0.05)..Conclusion:After focal ischemic brain injury,the intervention of exogenous Act A at different concentrations can reduce the degree of neurological injury and the volume of cerebral infarction,promote the expression of Act A/Smads signaling pathway,up-regulate the expression of the Phenotypic marker CD206,and downregulate the expression of the phenotypic marker iNOS of table Ml.The effect is correlated with the intervention concentration.The activation of Act A/Smads signaling pathway can induce the transformation of microglia phenotype from M1 phenotype to M2 phenotype,which may play an anti-ischemic injury role and provide potential transformation targets for the treatment of ischemic brain injury.4.The effect of Act A on the expression of Ml and M2 types of microglia during OGD/R of BV2 cells..Objective:Toinvestigate the expression of M1,M2 typing markers and key targets of signaling pathways in microglia at different time points during OGD/R in BV2 cells,and the effect of Act A on the expression of Ml and M2.Methods:BV2 microglia cells were cultured regularly(high-sugar DMEM medium,37?,5%CO2)and passed to the third generation.The OGD/R model of BV2 cells was established by changing sugar-free DMEM medium and three-gas incubator with low oxygen(94%N2,5%CO2,1%O2).The cells were divided into five groups:sham operation group,OGD 1 h/R 0 h,OGD 1 h/R 3 h,OGD 1 h/R 6 h,and OGD 1 h/R 12 h.The expression levels of iNOS,IL-10 and Smad2 were detected by Western blot.Select OGD 1 h/R6 h time point and culture BV2 cells with different concentrations of exogenous Act A.The rats were divided into control group,OGD 1 h/R 6 h,1 h/R 6 h+Act OGD A30nM group and OGD 1 h/R 6 h+Act A30nM group,further Western blot method to detect the phenotypic markers iNOS M1,M2 the phenotypic markers expression of IL-10 and Smad2 protein expression level of the situation.Results:BV2 cells were successfully cultured in vitro and the OGD/R model of BV2 cells was established.WesterBlot results showed that with the extension of reoxygenation time,the expression of Smad2 protein in Act A/Smads signaling pathway gradually increased,the expression of M1-phenotype marker iNOS protein gradually increased,and the expression of M2-phenotype marker IL-10 protein gradually increased,and the expression level decreased after reaching the peak with R6 h.After exogenous Act A pre-culture of B V2 microglia,Smad 2 protein expression was gradually increased in the intervention group compared with the OGD 1 h/R 6 h group,and the expression of Act A50nM group was higher than that of Act A30nM group.The M1 phenotype marker iNOS protein expression intervention group was lower than the OGD 1 h/R 6 h group,and the Act A50nM group was lower than the Act A30nM group.The expression level of M2 phenotype marker protein increased gradually,with the intervention group higher than the OGD 1 h/R 6 h group and the Act A50nM group higher than the Act A30nM group.Conclusion:Exogenous Act A can play a neuroprotective role by upregassing the expression level of the downstream factor Smad2 protein,which is also involved in the regulation of the polarization process of BV2 cells,suggesting that Act A/Smads signaling pathway may afect the outcome of neurons in ischemic injury by regulating the phenotype change of microglia cells.