| Stroke is a leading cause of adult morbidity,mortality and disability worldwide.Ischemic stroke is the major form of stroke,which is caused by cerebral thrombogenesis and interruption of blood flow into the brain.The ischemia and reperfusion further aggravated the process of neuroinflammation and neuronal injury.Therefore,thrombolytic and neuroprotective therapy is absolutely beneficial for stroke patients.Tissue plasminogen activator(tPA),as a powerful thrombolytic agent,is approved by the U.S.Food and Drug Administration(FDA).However,tPA may potentially increase the risk of intracerebral hemorrhage(ICH)complications and neurological injury.Compared with thrombolytic therapy,many factors have been reported to participate in neuronal damage repair after reperfusion.Evidences have proved that neuroinflammation has a crucial role in the pathogenesis of ischemic stroke.Neuroinflammation acts as a double-edged sword.On the one hand,it can obviously accelerate neuronal injury after ischemic stroke.On the other hand,neuroinflammation may potentially promote tissue remodeling and synaptic regeneration.Therefore,prevention of tPA negative effect and intervention of neuroinflammatory process are important strategies to against ischemic stroke.This study focused on two critical challenges of ischemic storke.First,we investigated the roles of glucose metabolic reprogramming in microglia on "classical Ml-like" polarization and pathogenesis of neuroinflammation.Second,we addressed the effect of retinoic acid(RA)on blood-brain barrier(BBB)integrality and tPA-induced cerebral hemorrhage.Part ? Glucose metabolic reprogramming drives microglial "classical M1-like" polarization and neuroinflammation in ischemic strokeMicroglia,considered as the sole resident macrophage in the brain,act essential roles in surveillance and elimination of synaptic,phagocytosis and modulation of inflammation in the CNS.Activated microglia is heterogeneous under multiple stimuli and environment,which can be initially categorized into two opposite types:classical M1 phenotype related to pro-inflammatory responses and alternative M2 phenotype corresponding with anti-inflammatory reactions and tissue remodeling.Although,as referred in recent recommendations,the M1/M2 definitions of microglia are oversimplified to describe the heterogenecity of activated microglia,such dichotomy is useful to accounting for microglial dual character in inflammation.Thus,in this study,we still used "classical M1-like" and "alternative M2-like" microglia to distinguish opposite effects of microglia in inflammation.Research has been indicated that neuroinflammatory response is regulated by activation of microglia and astrocytes and releases of chemoattractants and cytokines.Inflammatory is a key determinant of acute outcome and long-term prognosis in ischemic stroke.Microglia,considered as the resident macrophage in the CNS,directly participate in pathogenesis of inflammatory after activation.Therefore,understanding the mechanism of microglia polarization will benefit therapy for different inflammatory diseases including neuroinflammation as ischemic stroke.Increasing evidences support the critical role of metabolic reprogramming in macrophages activation.Rather than dependent on mitochondrial ATP production,"classical" like macrophages display high glycolysis rates,which can be induced by the expression of the glucose transporter 1(GLUT1)and pro-glycolytic 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3)enzyme.Conversely,obviously inhibition of glycolysis is observed in "alternative M2-like"macrophages.Furthermore,"alternative M2-like" macrophages have high rates of fatty acid oxidation(FAO)and oxidative phosphorylation.In the CNS,glucose is recognized as the main energy source.This suggested that reprogramming of glucose metabolism may participate in microglial polarization and pro-inflammatory effect.A strengthening of glycolysis is observed in LPS/IFN-y-stimulated microglia,which is dependent on the increase of hexokinase and lactate dehydrogenase activity,and high expression of GLUT 1.The function of monocarboxylate transporters(MCTs)is mainly to transport lactate outside of the cellular,which can be regarded as the special step of anaerobic glycolysis but not TCA cycle and oxidative phosphorylation.Enhanced expression of MCT1 and MCT2 has been detected in activated microglia in a rat ischemic stroke model.However,it is still unclear the effect of MCTs and glycolysis on microglial polarization and pro-inflammation.Therefore,the study mainly focused on whether MCT1 participates in microglial polarization through regulation of glycolysis and determines proinflammatory effect after ischemic stroke.?.Ischemic stroke increased expression of MCT1,MCT2 and MCT4 in "classical M1-like" microglia in miceMiddle cerebral artery occlusion(MCAO)is a classical ischemic stroke model to investigate neuronal injury and neuroinflammatory response after ischemia and reperfusion.Therefore,we first investigate microglial phenotype in ischemic stroke and expression of glycolytic related regulators,MCTs,in polarized microglia.The expression of "classical M1" markers,IObal and CD86,was detected using immunofluorescence in mice MCAO model.Compared with sham group,Iba1 and CD86 double-positive cells were significantly increased in the ischemic penumbra 1d and 3d after MCAO,which suggested that microglia rapidly polarized into "classical M1-lilke" phenotype after MCAO.Interestingly,compared with sham group,MCT1,MCT2 and MCT4 were largely increased in CD86-positive cells in the ipsilateral penumbra region 24 h after MCAO.?.MCT1,MCT4,and PFKFB3 were increased after LPS stimulation but not IL-4 in both BV2 microglia cell line and primary microglia1.LPS stimulation increased expression of MCT1,MCT4,and PFKFB3 in BV2 microglia cell line.To investigate the effect of MCTs and glycolysis on microglial polarization,BV2 microglial cell line was applied.The expression of glycolysis-related key determinants,MCTs and PFKFB3,were detected after BV2 cells stimulated with LPS and IL-4,respectively.RT-PCR results showed that LPS specially increased the expression of classical Ml microglia markers,iNOS,IL-1? IL-6 and STAT1,whileIL-4 largely increased the expression of alternative M2 phenotype markers,Argl and CD206.Importantly,stimulation with LPS,but not IL-4,specially increased the expression of MCT1,MCT4 and PFKFB3 in BV2 cells.2.LPS stimulation increased expression of MCT1,MCT4,and PFKFB3 in cultured primary microglia.Meanwhile,to further verify the above results,primary cultured microglia were applied in our experiments.Primary microglial cells were obtained from the brain of C57BL/6 mouse pups postnatal day 1-3 and stimulated with LPS and IL-4,respectively.The significant increases of MCT1,MCT4 and PFKFB3 were also found in LPS-stimulated microglia while no difference was found in IL-4-treated microglia.In conclusion,we indicated that there is a close link between high rate of glycolysis and microglial classical M1 polarization.?.MCT1 drives microglial M1 polarization via PFKFB3-enhanced glycolysis1.Knockdown of MCT1 inhibited the expression of LPS-induced classical M1 markers.To further investigate the role of MCTs in the classical M1 microglia polarization,the lentivirus mediated shRNA was applied in the followed experiments.BV2 cells were stimulated with different lentivirus and treated with LPS.Results showed that,compared with control group,knockdown of MCT1 significantly reduced LPS-increased expression of iNOS.However,knockdown of MCT2 or MCT4 has no effect on LPS-induced increase of iNOS.Meanwhile,MCT1 interference also decreases the expression of IL-1?,IL-6 and STAT1 after LPS stimulation.All above results suggested that MCT1 is necessary for LPS-induced microglial classical M1 polarization.2.Knockdown of MCT1 prevented LPS-induced high rate of glycolysis in microglia.To further verify whether MCT1 can direct regulate glycolysis rate in classical M1-polarized microglia,L-lactate assay kit was used in our experiments.Results showed that LPS stimulation increased a high release of lactate in BV2 cells.Knockdown of MCT1 significantly reduced LPS-induced the release of lactate.The level of lactate was detected no difference after interference of MCT2 or MCT4.Furthermore,knockdown of MCT1 also decreased the expression of PFKFB3 after LPS stimulation.The above results suggested that MCT1 directs classical Ml polarization via up-regulation of glycolysis.3.Lactate inhibited LPS-induced microglial classical M1 polarization.To investigate whether MCT1 regulated microglial polarization through lactate transport,lactate was used to add to the BV2 cell medium.The result showed that additional lactate blocked LPS-induced the increase of iNOS and PFKFB3.This suggested that knockdown of MCT1 may increase the lactate concentration in the cytoplasm and further inhibit the glycolysis rate by a negative feedback manner.In short,above results revealed that MCT1 may participate in microglial classical Ml polarization via up-regulation of glycolysis.4.MCT1 regulated LPS-stimulated "classical M1-like" polarization via pro-glycolytic PFKFB3 enzymeTo further investigate the role of PFKFB3 in MCT1-promoted microlial classical polarization,lentirivus-mediated MCT1 shRNA and lenti virus-mediated overexpression of PFKFB3(Lenti-PFKFB3)were applied.Results showed that,under LPS stimulation,overexpression of PFKFB3 significantly rescued the decrease of iNOS mediated by Lenti-siMCT1.Furthermore,overexpression of PFKFB3 also prevented the effect of Lenti-siMCTl on the expression of IL-1? and IL-6.However,either overexpression of PFKFB3 or knockdown of MCT1 showed no effect on the expression of Argl and CD206.All above results suggested that MCT1 regulated LPS-stimulated "classical M1-like" polarization via pro-glycolytic PFKFB3 enzyme.?.Knockdown of MCT1 reduced the phosphorylation level of STAT1 after LPS stimulation in microgliaSTAT1,a potent transcription factor,has been reported to drive microglia and macrophage "classical" like polarization and inflammatory gene expression.Therefore,we addressed whether MCT1 can regulate STATI signaling.Corresponding to above results,compared with control group,MCT1 interference decreased LPS-induced expression of STAT1 protein 24 after LPS stimulation.Under LPS-stimulated condition,overexpression of PFKFB3 also rescued the decrease of STAT1 caused by MCT1 interference.As previous study,within 3 h after LPS stimulation although there was no significant change of total STAT1 protein,there was a rapid increase in the level of p-STAT1 in cultured microglia.Moreover,compared with vehicle group,knockdown of MCT1 inhibited LPS-induced p-STATl and overexpression of PFKFB3 blocked MCT1 interference-decreased p-STAT1 3 h after LPS stimulation.All these results further validated that MCT1-mediated glycolysis might direct microglial "classical" like polarization via up-regulation of phosphorylation of STAT1.?.MCT1-mediated microglial "classical M1-like" polarization aggravated oxygen-glucose deprivation-induced neuronal injuryOxygen-glucose deprivation(OGD)experiment has been regarded as an in vitro ischemic and reperfusion model.To investigate the effect of MCT1-mediated"classical M1-like" polarization on neuronal injury,cultured microglial BV2 cell line and neuronal SH-SY5Y cell line were subjected to OGD treatment.1.MCT1,MCT4 and PFKFB3 were increased in microglia after oxygen-glucose deprivation(OGD)condition.After treatment with OGD and reperfusion,the expression of iNOS,STAT1,IL-1?and IL-6 have a significant increase in BV2 cells,suggesting that OGD treatment transformed microglia into a "classical" like/pro-inflammatory phenotype.In addition,compared with control group,OGD treatment significantly increased the expression of MCT1,MCT4 and PFKFB3.2.Knockdown of MCT1 in microglia reduced neuronal injury after OGDThe 24-well transwell was used to coculture of SH-Y5Y and BV2 cells.After Lentivirus and OGD treatment,MTT assay was performed to analyze cell viability of SH-SY5Y cells.Compared with control group,OGD significantly decreased the cell viability of SH-SY5Y cells.Importantly,MCT1 knockdown in BV2 cells rescued the OGD-induced cell death of SH-SY5Y cells.However,knockdown of MCT2 showed no effect on neuronal injury after OGD condition.3.Knockdown of MCT1 in microglia attenuated the release of pro-inflammatory cytokines.To address whether knockdown of MCT1 can prevent the pro-inflammatory effect of microglia which initiates neuronal injury,the cytokine levels of IL-1?,IL-6 and TNF-? were detected using RT-PCR and Elisa.The results showed that,under OGD condition,knockdown of MCT1 in microglia significantly decreased the expression and release of IL-1?,IL-6 and TNF-?.Part ? Retinoic acid ameliorates blood-brain barrier disruption and tPA-induced cerebral hemorrhage in ischemic strokeThe intact blood-brain barrier(BBB)is essential for maintenance of physiological environment of synaptic and neuronal functions.The structure of BBB is composed of brain endothelial cells(BEC)and the intricate junctional complex,astrocyte end-feet and pericytes.BBB disruptions have been reported after ischemic stroke in both stroke patients and animal models.The decreases of BEC junctional complex-related proteins,including ZO-1,VE-cadherin,Occludin and PECAM-1,were observed in ischemic stroke,which directly led to BBB injury.In ischemic stroke,tPA is an effective thrombolytic agent and has been approved by the FDA.However,TPA has been proved to be sufficient and necessary for BBB disruption in ischemic stroke.TPA activated platelet-derived growth factor ?-receptors(PDGFR-a)via PDGF-CC and lead to BBB injury.Due to the negative effect on BBB,tPA is only allowed to use only within 3 hours after onset of ischemic stroke in a few stroke patients.Otherwise,late tPA treatment will increase the risk of intracerebral hemorrhage(ICH)complications.Therefore,it is a strategy of great importance to prevent tPA-aggravated BBB injury during thrombolysis in ischemic stroke.Retinoic acid(RA),a bioactive derivative of vitamin A,plays pleiotropic roles in early organogenesis in vertebrate embryo.Evidences proved that radial glial cell-derived RA is necessary for the BBB formation through regulating the expression of VE-cadherin,ZO-1 and Occludin in embryo stage.However,it is still unclear whether the effect of RA in BBB protection after ischemic stroke.It is unknown whether RA can prevent the negative effect of tPA on ICH.Therefore,this study aimed to address the effect of RA on BBB permeability and tPA-induced ICH under ischemic stroke.I.RA pretreatment attenuated BBB permeability and infarction following MCAO1.RA specially increases the expression of BBB-targeted proteins,ZO-1 and VE-cadherin in adult rats.It has been proved the effect of RA during BBB formation in mouse embryos.To investigate the effect of RA on the expression of BBB-targeted proteins in vivo,the adult rats were received intraperitoneal injection of RA for 4 consecutive days.Dorsolateral cortex region of rat was obtained to quantify mRNA levels of BBB-targeted proteins using real-time quantitative PCR.Our results showed that there is a significant increase of ZO-1 and VE-cadherin after treatment with either 5 or 25 mg/kg RA,compared with control group.However,RA has no effect on the expression of Claudin-5,Occludin and PECAM-1.2.RA reduced MCAO-induced BBB disruption using intravenous injection of Evans blue(EB)dye.To investigate the effect of RA pretreatment on BBB injury,MCAO was chosen as the ischemic stroke model in our experiments.Our results showed that the extravasation of EB dye was increased after MCAO.RA pretreatment significantly attenuated MCAO-induced the extravasation of EB dye,which suggested that RA could prevent MCAO-induced BBB injury.3.Pretreatment with RA ameliorated infarction area after MCAO using TTC staining.It has been proved that BBB protection can ameliorate infarction area and neurological deficits in ischemic stroke.To investigate whether the effect of RA on BBB protection could subsequently have a neuroprotective effect,Infarction area was measured by TTC staining 5 h after MCAO.The results displayed that RA reduced cerebral infarction in MCAO rats compared with sham group,which suggested that RA also has a neuroprotective effect during ischemic stroke.4.RA pretreatment reduced tPA-induced aggravation of BBB permeability following MCAO.To further verify whether RA can rescue tPA-induced aggravation of BBB disruption in ischemic stroke,rats were subjected to tPA administration after MCAO.After EB injection and diffusion,we found that tPA significant increase MCAO-induced the extravasation of EB dye outside of the vascular.RA pretreatment significantly decreased tPA-aggravated the EB extravasation in MCAO rats,which suggested that RA can protect tPA-induced aggravation of BBB disruption during ischemic stroke.?.RA pretreatment prevented the decrease of VE-cadherin and ZO-1 proteins following ischemic strokeZO-1 and VE-cadherin are two crucial proteins of the junctional complex between the BEC.Both ZO-1 and VE-cadherin play essential roles to maintain the integrity of BBB.To observe the effect of RA on ZO-1 and VE-cadherin in ischemic stroke,we used immunofluorescence and western bolt methods to analyze the expression of the two proteins.Our results showed that MCAO significantly decreased the levels of ZO-1 and VE-cadherin compared with sham group.RA pretreatment rescued MCAO-induced decrease of these two proteins in ischemic stroke.This suggested that RA might protect BBB through regulating the expression of ZO-1 and VE-cadherin during ischemic stroke.?.RA protective effect on BBB is dependent on increase of ZO-1 and VE-cadherin expression via RAR? in cultured RBE4 cells1.RARa antagonist blocked RA protective effect on BBB and RA-induced increase of ZO-1 and VE-cadherin under OGD condition.To investigate the mechanism of the BBB protection effect of RA,the cultured rat brain microvascular endothelial cell line RBE4 was used in our experiments.The RBE4 cells were treated with RA and RA receptor(RAR)antagonists before oxygen-glucose deprivation(OGD)treatment.The diffusion of FITC-dextran can reflect the BBB permeability.RA treatment reduced the diffusion of FITC-dextran in OGD condition,suggesting that RA plays a protective effect on BBB.The RAR?antagonist,but not RAR? inhibitor,prevented RA-decreased diffusion of FITC-dextran,which suggested that RA protect BBB permeability via RAR? under OGD condition.Furthermore,we found that RA increased the expression of ZO-1 and VE-cadherin and had no effect on claudin-5.RARa antagonist blocked RA-induced increase of ZO-1 and VE-cadherin.2.RA attenuates BBB permeability through up-regulation of ZO-1 and VE-cadherin.To verify whether RA protects BBB through regulating ZO-1 and VE-cadherin,we used the siRNA oligonucleotides of ZO-1 and VE-cadherin.The two ANOVA analyses showed that there are significant RA x ZO-1 and RA x VE-cadherin interactions.Both ZO-1 siRNA and VE-cadherin can partially decrease RA protective effect on BBB permeability.Thus,we demonstrated that RA induced the expression of ZO-1 and VE-cadherin via RARa,further prevented BBB disruption in ischemic stroke.?.RA administration immediately after the onset of MCAO ameliorated tPA-induced intracerebral hemorrhage and neurological dysfunction1.RA reduced tPA-induced intracerebral hemorrhage in ischemic stroke To investigate whether RA can reduce tPA-induced ICH during ischemic stroke,an animal model using d-glucose injection was used to increase tPA-induced hemorrhage according to the references.Compared with vehicle group,injection of tPA significantly increased hemorrhage in the ipsilateral area.Administration of RA after onset of MCAO significantly decreased tPA-induced hemorrhage.suggesting that RA can be used together with tPA to cue ischemic stroke and reduce the risk of ICH.2.RA treatment immediately after the onset of MCAO prevented the decrease in VE-cadherin and ZO-1 protein levels 24 h after ischemic strokeEvidences have been showed that tPA disrupts BBB integrity through decrease of junctional complex,such as ZO-1,Claudin-5 and VE-cadherin in ischemic stroke.We detected whether RA can prevent tPA-induced decrease of ZO-1 and VE-cadherin.We observed that RA significantly rescued tPA-induced disruption of ZO-1 and VE-cadherin.This suggested that RA prevent tPA-aggravated BBB injury and ICH through an indirect pathway.3.RA treatment improved tPA-induced neurological dysfunction after MCAO ischemic stroke.To investigate the effect of RA treatment on tPA-aggravated neurological injury after ischemic stroke,neurological scores(0-4 point)and the tape removal tests were used in the followed experiments.Our results showed that tPA injection significantly increase neurological scores and tape removal times.RA obviously ameliorated tPA-aggravated neurological scores and tape removal times.This suggested that RA also can reduce tPA-aggravated neurological injury in ischemic stroke. |
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