Effect And Mechanism Of Neuronal IRAK1/4and Cerebral Ischemic Preconditioning-induced Activation Of Microglia1

BackgroudIschaemic stroke, resulting either from global or focal decreases in perfusion, areamong the most common and important causes of disability and death worldwide. Ischemicstroke induced brain injury is a multiphase process. Three main responses are involved inthis progress: excitotoxicity, inflammation, and programmed cell death (PCD). The shortduration of excitotoxicity after ischemic stroke does not provide an adequate time windowfor effective therapy in clinical practice. A more reasonable therapeutic window is likely tobe attained by placing emphasis on ameliorating the effects of the synergistic inflammationand PCD that are active for hours to days after ischemic stroke. Past studies have shownthat injury of neuron is the direct cause of the neurological deficits, and microglia playsignificant roles in the demise of brain tissue after cerebral ischemia. It is a rather recentfinding that neuron play a direct pivotal role in ischemic brain injury and functional deficits,at least at early time points. Furthermore, subsequent activation of microglia likelycontributes to the delayed inflammatory processes that occur within the infarct region.Based on these findings, two hypotheses were propose to protect against acute ischemicstroke-induced neuronal injury.Neuronal IRAK-1/4InhibitionToll like receptors (TLRs) are a major family of transmembrane pattern-recognitionreceptors (PRRs) that sense the invasion of pathogenic microorganisms and tissue injury, afunction thought to be limited to immune cells. However, it is increasingly clear that nearlyall cells within the body, including those within the CNS, express TLRs. The results ofrecent studies employing TLR-2and-4knockout mice suggest neuronal TLR-2and-4playa pivotal, negative role in hypoxia/ischemia(H/I)-induced brain injury and functionaldeficits. Meanwhile, the interleukin-1receptor associated kinases(IRAKs) are required for MyD88-dependent TLR activation. IRAK-1and IRAK-4have kinase activity, whereasIRAK-2and IRAK-M are catalytically inactive. Upon activation of upstream cognatereceptors, IRAK-4is thought to phosphorylate IRAK-1, resulting in the activation andautophosphorylation of IRAK-1and subsequent phosphorylation of downstream substrates.IRAKs have been studied in many immune cell types such as monocytes/macrophages,myeloid lineage cells,293T cells, and human umbilical vein endothelial cells(HUVEC).However, the neuronal function of IRAKs has not been well-studied. As IRAKs areinvolved in mediating pro-inflammatory responses in other cells, understanding the role ofIRAK-1and-4catalytic activities in neurons could be essential in the development oftherapeutics targeting this pathway. Based on the previous findings that endogenous andexogenous ligands may activate neuronal TLR-2/4to produce an inflammatory responseduring the first few hours after H/I-induced brain injury. We hypothesized that neuronalIRAK inhibition could have profound effects on adaptive immune responses and may be aneffective therapeutic target for H/I-induced brain injury.Pre-activation of MicrogliaPreconditioning is a procedure by which a noxious stimulus near to but below thethreshold of damage is applied to the tissue. Shortly after preconditioning or after a delay,the organ (and therefore the organism) develops resistance to, or tolerance of, the same,similar, or even different noxious stimuli given beyond the threshold of damage.Preconditioning thereby protects against subsequent injury. Cerebral ischemicpreconditioning (CIP) has been proved to be an innate neuroprotective mechanism.However, the function of microglia has not been well-studied in this area. Past studies haveshown that neuronal TLR-2and-4play a direct pivotal role in ischemic brain injury andfunctional deficits, at least at early time points. Furthermore, subsequent activation ofmicroglia likely contributes to the delayed inflammatory processes that occur within theinfarct region. However, our recent results, combined with growing evidence, suggest thatCIP could reprogram the activation of microglia in ischemic stroke. Another finding ofinterest is a significant relationship between the area of the activation of microglia and theneuroprotection in CIP. Based on these findings, we hypothesized that CIP could reprogramthe activation of microglia in ischemic cortex, leading to the induction of cytokines andother inflammatory mediators, which were involved in this neuroprotection. Part Ⅰ Effect and Mechanism of Neuronal IRAK-1/4Inhibition in Brain Injuryin Rats following Ischemic StrokeObjectiveTo investigate the expression levels of IRAK-1and-4， and the effects of IRAK-1/4inhibition on brain ischemic insult and neuronal hypoxia-induced injury.MethodsIn vivo and in vitro experiments were performed in this study.1. Models of focal cerebral infarct were established by occlusion of middle cerebralartery (MCAO) in SD rats.2. Rats were randomly divided into control group、sham surgery group、vehicle groupand treatment group.3. The mRNA and protein expressions of IRAK-1and-4in cerebral cortex weredetected by Reverse transcriptase reaction-polymerse chain reaction(RT-PCR) method、Western blot(Wb) analysis and laser scanning confocal microscope (LSCM).4. The neurological deficits were evaluated in0-4scales. The infarct size wasmeasured by2%2,3,5-triphenyltetrazolium chloride (TTC) staining. The mortality of eachgroup was also calculated.5. B35cell anoxia model via cobalt chloride (CoCl2) was established.6. The mRNA and protein expressions of IRAK-1and-4in B35cell were detected byRT-PCR method、Wb analysis and LSCM.7. Cell viability was assessed by Cell Counting Kit-8(CCK8) method and anti-Microtubule-associated protein (MAP-2) antibody staining; cell PCD was evaluated by flowcytometric analysis.8. NF-κB activation was assessed by LSCM; TNF-α and IL-6releasement wereexamined by ELISA; and the expression of T-JNK、p-JNK及c-Casepase3were determinedby western blot. ResultsIn vivo1. Cerebral cortex expresses IRAK-1and-4and responds to MCAO injury.2. IRAK-1/4inhibition decreases mortality rate, functional deficits, and ischemicinfarct volume in MCAO-treated animals.In vitro1. B35cells express IRAK-1and-4and respond to CoCl2-induced hypoxic injury.2. IRAK-1/4inhibition attenuates CoCl2-induced cytotoxicity in B35cells.3. IRAK-1/4inhibition prevents CoCl2-induced apoptosis in B35cells.4. IRAK-1/4inhibition in B35cells reduces CoCl2-induced NF-κB activation andproduction of TNF-α and IL-6.5. IRAK-1/4inhibition mediates JNK and capsase-3activation in B35cells.ConclusionBrain ischemic injury increased the expression of IRAK-1and-4in cerebral cortex，and IRAK-1/4inhibition at the early stage could effectively protect the neuron against death.The mechanism may be related with down-regulation of the inflammation response resultedfrom stroke. Therefore, application of an IRAK-1/4inhibitor may be effective in reducingH/I-induced brain injury and could be an important basis for future attempts to improveH/I-induced brain injury therapy. Part Ⅱ Effect and Mechanism of Cerebral Ischemic Preconditioning-inducedActivation of Microglia in NeuroprotectionObjectiveTo investigate the role of microglia in the neuroprotection of CIP, the feature ofmicroglia pre-activation, the differentiation of distinct microglia, i.e.,“classically activated”proinflammatory (M1) or “alternatively activated” anti-inflammatory (M2) cells, and themechanism of neuroprotection.MethodsIn vivo and in vitro experiments were performed in this study.1. Model of focal cerebral infarct was established by MCAO, and model of CIP wasestablished by transient MCAO(tMCAO).2. Rats were randomly divided into control group、sham surgery group、vehicle group、CIP group、treatment group.3. The neurological deficits were evaluated in0-4scales. The infarct size wasmeasured by TTC staining. The mortality of each group was also calculated.4. The feature of microglia pre-activation was observed by LSCM.5. The mRNA expressions of M1(CD16、CD32、iNOS)/M2(TGF-beta、Ym1/2) incerebral cortex were detected by RT-PCR method.6. The mRNA expressions of IκB-α, TNF-α in cerebral cortex were detected by in situhybridization method.7. PC12+BV2cell Co-culture ischemic/CIP model via Oxygen-Glucose Deprivation(OGD)/Transient Oxygen-Glucose Deprivation (tOGD) was established.8. Cell viability was assessed by CCK8method and LDH analysis.9. The protein expressions of CD11b in cerebral cortex in vivo and microglia in vitrowere measured by LSCM.10. The mRNA expressions of M1(CD32、iNOS、CD86)/M2(CD206、IL-10、CCL-22)in cerebral cortex were detected by Realtime-PCR method. ResultsIn vivo1. tMCAO decreases mortality rate, functional deficits, and ischemic infarct volume inMCAO-treated animals. Otherwise, minocynine could inhibit this neuroprotection.2. tMCAO could reprogram the activation of microglia in ischemic cortex.3. tMCAO could reprogram the mRNA expression of M1(CD16、CD32、iNOS)/M2(TGF-beta、Ym1/2)in ischemic cortex.4. tMCAO could increase the mRNA expression of IκB-α, and keep TNF-α at thelower level in ischemic cortex.In vitro1. tOGD attenuates OGD-induced cytotoxicity in PC12cells.2. tOGD could decrease the protein expressions of CD11b in OGD-treated BV2.3. tOGD could reprogram the mRNA expression of M1(CD32、iNOS、CD86)/M2(CD206、IL-10、CCL-22)of BV2in OGD culture.ConclusionCIP could reprogram the activation and M1/M2gene expression ofmicroglia/macrophage in ischemic stroke, and leading to the inhibition of TLR2/NF-κbactivity and inflammatory response following rat ischemic brain injury, which may beinvolved in this neuroprotection. Our work may confer a new therapeutic target of ischemicstroke.