Mechanism Of Corticotropin-Releasing Factor Aggravating Ischemic Stroke Injury By The Inflammatory Activation Of Microglia

Ischemic stroke is the second leading cause of death worldwide.Therefore,exploring effective and emerging molecular targets for ischemic stroke is a primary task of basic and clinical research.The aim of the present study was to investigate the function of corticotropin-releasing factor(CRF)in ischemic stroke and its related mechanisms,to provide a reference for the treatment of ischemic stroke.CRF,antalarmin,or astressin-2B were used to activate or block the CRF1(CRF receptor 1)or CRF2(CRF receptor 2)in BV2 cells and adult male mice,which had constructed a distal middle cerebral artery occlusion(d MCAO)model.CRF not only accelerated microglial activity by promoting transcription and production of inflammatory factors,but also promoted the transformation of activated BV2 cells from a neuroprotective phenotype(M2)to a cytotoxic phenotype(M1),and these effects were mediated by the TLR4/My D88/NF-κB signaling pathway.These effects can be blocked by antalarmin but not by astressin-2B.CRF significantly aggravated the neurological deficit,increased infarction volume,and exacerbated neuronal injuries.Additionally,CRF significantly improved the levels of TNF-αand phospho-NF-κB in the ischemia penumbra.Finally,CRF significantly increased the proportion of CD16/Iba-1-positive cells and decreased the proportion of CD206/Iba-1-positive cells in the ischemia penumbra.These results provide evidence of the proinflammatory role of CRF in an ischemic stroke model and a possible underlying mechanism,which may facilitate the elucidation of potential treatment approaches for ischemic stroke.Part one Metabolic activation of microglia by CRFObjective:To evaluate the cytotoxic effects of CRF,antalarmin(CRF1antagonist)and astressin-2B(CRF2 antagonist)on BV2 microglia.To evaluate the effects of CRF,antalarmin and astressin-2B on the transcription and production of proinflammatory factors.To evaluate the effects of CRF,antalarmin and astressin-2B on phenotypic differentiation of BV2 microglia.Methods:BV2 microglia cells were divided into 6 groups:(1)Control group;(2)CRF(10 nmol/L)group;(3)antalarmin(100 nmol/L)group;(4)astressin-2B(100nmol/L)group;(5)CRF(10nmol/L)+antalarmin(100 nmol/L)group;(6)CRF(10 nmol/L)+astressin-2B(100 nmol/L)group.Cell counting kit(CCK-8)was used to measure the cell viability of the above groups after24 hours of intervention.ELISA was used to detect the production of IL-1βand TNF-αin the above groups after 24 hours of intervention.q RT-PCR was used to detect the expression changes of inflammatory cytokines(il1b,Tnf),M1 microglial marker genes(Nos2 and Ptgs2)and M2a microglial marker genes(Arg1 and Mrc1)in the above groups after 24 hours of intervention.The data were expressed as mean±standard deviation and analyzed by SPSS 26.0statistical software.P values<0.05 were considered statistically significant.Results:1 CCK8:Compared with the control group,there was no significant difference between the experimental group and the control group,indicating that the above concentrations of intervention had no toxic effect on BV2 cells for 24h;2 ELISA:Compared with the control group,the production of IL-1βand TNF-αwas significantly increased in the CRF and CRF+astressin-2B groups(P<0.01),while these effects were significantly inhibited in the CRF+antalarmin group;3 q RT-PCR:Compared with the control group,il1b and Tnf m RNA expressions were significantly increased in the CRF group and the CRF+astressin-2B group(**P<0.01,*P<0.05),and there was no significant difference between the CRF+antalarmin group and the control group;compared with the control group,the expression of Nos2m RNA in the CRF group was significantly increased(P<0.01);Ptgs2 m RNA expression was significantly increased in the CRF group and the CRF+astressin-2B group(P<0.01);Compared with the control group,the expression of Arg1 m RNA in the CRF group was significantly decreased(P<0.05),while the expression of Mrc1 m RNA in the CRF group was significantly lower than that in the control group(P<0.05),while there was no significant difference between the other groups and the control group.Summary:CRF can increase the transcription and production of inflammatory factors promoted by BV2 microglia and lead to the conversion of activated BV2 microglia from a neuroprotective(M2 type)to a cytotoxic(M1 type)phenotype.Part two Effect of CRF on inflammatory pathways in microgliaObjective:To investigate the effect of CRF on TLR4/My D88/NF-κB inflammatory pathway in BV2 microglia.Methods:BV2 microglia were divided into 6 groups:(1)control group;(2)CRF(10 nmol/L)group;(3)antalarmin(100nmol/L)group;(4)astressin-2B(100 nmol/L)group;(5)CRF(10 nmol/L)+antalarmin(100 nmol/L)group;(6)CRF(10nmol/L)+astressin-2B(100nmol/L)group.The BV2 microglia cells were collected after 24 hours of intervention,and the following indicators were detected by Western-blot:(1)TLR4;(2)My D88;(3)Phosphorylation of NF-κB in cytoplasm;(4)Phosphorylation of NF-κB in nuclear.Results:1 Compared with the control group,TLR4 protein levels in the CRF group and the CRF+astressin-2B group were significantly increased,respectively(P<0.01,P<0.05);2 Compared with the control group,My D88protein levels in the CRF group and the CRF+astressin-2B group were significantly increased(P<0.01,P<0.05);3 Compared with the control group,the level of phosphorylated NF-κB protein in the cytoplasm of the CRF group was significantly increased(P<0.01);4 Compared with the control group,the levels of phosphorylated NF-κB protein in the nucleus of the CRF group and the CRF+astressin-2B group were significantly increased(P<0.01,P<0.05).Summary:CRF acts on BV2 microglia CRF1 to activate the TLR4/My D88/NF-κB inflammatory signaling pathway.Part three CRF aggravates neuronal damage in ischemic stroke through inflammatory activation of microgliaObjective:Distal middle artery occlusion(d MCAO)model was established in male adult mice.The effects of CRF,antalarmin or astressin-2B on neurological function,cerebral infarct volume and neuronal damage were detected by lateral ventricle injection.In addition,the levels of TNF-αand phosphorylated NF-κB,the proportion of CD16/Iba-1 positive cells and the proportion of CD206/Iba-1 positive cells in the cerebral ischemic penumbra were detected by immunofluorescence technique.Methods:Focal cerebral cortical ischemia was induced by permanent occlusion of the middle cerebral artery(MCA)and common carotid artery(CCA).Male adult mice were subjected to permanent ligation of the right CCA and electrocoagulation of the right distal MCA.Sham-operated mice were exposed to the CCA but not to occlusion and subsequently to the distal MCA but not to electrocoagulation.The mice were randomly divided into 5groups,10 mice in each group.After successful modeling,the mice in each group were given the following interventions:(1)Sham operation group(Sham);(2)Middle cerebral artery occlusion group(d MCAO-Con);(3)Middle cerebral artery occlusion+CRF group(d MCAO-CRF);(4)Middle cerebral artery occlusion+antalarmin group(d MCAO-antalarmin);(5)Middle cerebral artery occlusion+astressin-2B group(d MCAO-astressin-2B).According to the experimental design,the mice were assessed for neurobehavioral deficits using the Bederson test on day 2 after cerebral ischemia and then killed by rapid dislocation under deep anesthesia.The infarct volume was measured by 2,3,5-triphenyltetrazolium chloride(TTC)staining.H&E staining and Nissl staining were used to observe the morphological changes of neurons in the ischemic penumbra.The proportion of Iba-1,TNF-α,P-NF-κB,CD16/Iba-1 and CD206/Iba-1 positive cells in ischemic penumbra were detected by immunofluorescence technique.Results:1 Compared with the Sham group,the neurological deficit score of the d MCAO-Con group was significantly increased(P<0.01).Compared with the d MCAO-Con group,the neurological deficit score of the d MCAO-CRF group was significantly increased(P<0.05),indicating that the neurological function was worse.There was no significant difference in neurological deficit scores among d MACO-Con,d MACO-antalarmin and d MACO-astressin-2B.2 Compared with the d MCAO-Con group,the infarct volume was significantly increased in the d MCAO-CRF group(P<0.01);However,d MACO-antalarmin significantly reduced infarct volume(P<0.05).3 In the ischemic penumbra,H&E staining positive cells(H&E~+)had clear outline,compact structure,and intact nucleoli.The neurons with positive Nissl staining(Nissl~+)had abundant cytoplasm,intact cell body and dense nucleus.The proportion of H&E~+cells in Sham group and d MCAO-Con group were86%±4.2%and 65%±5.8%,respectively,and the difference between the two groups was statistically significant(P<0.01).Compared with the d MCAO-Con group,the proportion of H&E~+cells in the d MCAO-CRF group was significantly low to 51.7%±6.8%(P<0.05);The percentage of Nissl~+cells in Sham group and d MCAO-Con group were 84.3%±4.3%and 63.3%±4.3%,respectively,and the difference between the two groups was statistically significant(P<0.01).Compared with the d MCAO-Con group,the proportion of Nissl~+cells in the d MCAO-CRF group was significantly decreased to50%±5.1%(P<0.05).4 Compared with the d MACO-Con group,the expression of Iba1 and TNF-αwas significantly increased in the d MACO-CRF group,and significantly decreased in the d MCAO-antalarmin group.Compared with the d MACO-Con group,the expressions of Iba-1 and P-NF-κB were significantly increased in the d MCAO-CRF group and decreased in the d MCAO-antalarmin group.5 Compared with the Sham group,the d MCAO-Con group had a significant increase in the proportion of CD16/Iba-1 positive cells(P<0.01).The proportion of CD16/Iba-1 positive cells in the d MCAO-CRF group was significantly higher than that in the d MCAO-Con group(P<0.01),and the proportion of CD16/Iba-1 positive cells in the d MCAO-antalarmin group was significantly lower than that in the d MCAO-Con group(P<0.01).Compared with the Sham group,the proportion of CD206/Iba-1 positive cells in the d MCAO-Con group was significantly increased(P<0.01),however,the proportion of CD206/Iba-1 positive cells in the d MACO-CRF group was significantly lower than that in the d MCAO-Con group(P<0.01).The proportion of CD206/Iba-1 positive cells in the d MCAO-antalarmin group was significantly higher than that in the d MACO-Con group(P<0.01).Summary:CRF significantly aggravate the damage of neurological function after ischemic stroke,increase the volume of cerebral infarction and lead to the damage of neurons.CRF1 receptor antagonist antalarmin can reverse some of the above effects,and has a certain protective effect in brain injury after cerebral ischemia.Conclusions:CRF can activate BV2 microglia by promoting transcription and production of inflammatory factors,which lead to the transformation of activated BV2 cells from a neuroprotective phenotype(M2)to a cytotoxic phenotype(M1).CRF significantly aggravates ischemic stroke injury via increasing inflammatory microglia activation,which is correlated to the promotion of TLR4/My D88/NF-κB pathway and the augment of M1 type neurotoxic microglia activation.Therefore,our results seem to support that the release of CRF and the activation of microglia are mutually reinforcing,and either local release of CRF or peripheral release of CRF appears to be primarily contributing to the effects on ischemic stroke.