| BachgroudCerebrovascular disease has become one of the top three fatal diseases and the first leading disease worldwide, with more than70%people over65years old who have experienced a stroke onset,.With acceleration of the ageing of society, the number will continue to increase.among all the patients,85%of which are ischemic stroke.Although pathophysiological mechanisms of ischemic stroke is complex,now studies suggest that inflammation plays a key role in ischemic brain injury.Abruptly interrupted blood supply initiates the ischemic cascade within a few seconds in brain tissue, resulting in cell membrane damage and neuron death culminated in the core area of infarction,,While the large area adjacent to core called the Penumbra,if blood flow restored quickly,will successfully survived normally. Severe hypoperfusion of cerebral blood flow, excitotoxic and oxidative damage as a result, in turn causing microvascular damage, blood-brain barrier dysfunction and then initiate inflammation after focal ischemia, increase the cerebral ischemic injury and permanent brain damage.TLRs are one of the most important pattern recognition receptors families.TLRs have extracellular Leucine-rich repeat sequences for identifying pattern related molecullar pathogens, also have intracellular structure contains of TIR domains used to activate intracellular signaling. Recent studies confirm that especially TLR4play a key role in the inflammatory response and inducible inflammation mediator after focal cerebral ischemia. In TLR4gene deficiency mice the cerebral infarction of brain injury and neurological damage were significantly reduced compared to the wild type mice. TLR4activating caused NF-κb nuclear translocation, that is,TLR4/NF-κb signaling induces expression of inflammatory factors such as TNF-alpha, IL6,resulting in inflammatory reaction.In addition, studies have confirmed that lipopolysaccharide can induced BIT, with suppression TLR4/NF-K b signaling system through negative feedback loop, hence enhanced TLR4-TIRF signal axis,increased expression of anti-inflammation IFN-β, makes inflammatory response weak. So we administrated IPC prior to permanent middle cerebral atery occlusion rat model(pMCAO), detected TLR4signaling axis-related expression of inflammatory factor and the influence of IPC on the TLR4signaling axis, to further explore the endogenic neuroprotection mechanisms of BIT, to search new target strategies for acute cerebral infarction treatment, are of great significanceMethods1. SD rats were randomly divided into experimental group (IPC+pMCAO), the control group (SS+pMCAO), the sham surgical group (SS+SS).2. IPC(lOmin) were performed48h before pMCAO established.3. The neurological functional scoring and the severity of brain damage24h after pMCAO were evaluated.4. Cerebral infarction volume and brain water content were evalued with dry-wet method.5. RT-PCR was used to detecte the TLR4mRNA,NF-K bp65mRNA,.6. Immunohistochemistry was used to study the expression of protein TLR4, NF-κB,TRIF at the designated timepoint6h,12h,24h,48h and72h after pMCAO..7. ELISA technology was performed to detect the TNF-a content at the designated timepoint6h,12h,24h,48h and72h after pMCAO.Results1. Results show that neurological function score in the experimental group were reduced significantly compared with the control group (p<0.01).2. Histological study of cerebral ischemic damage in the experimental group were also mitigated compared with the control group,with less inflammatory cells infiltration.3. Brain water content were less in the experimental group than in the control group (p<0.01), indicate that BIT was induced by IPC.4. Determination of RT-PCR found that TLR4mRNA,NF-p65κb mRNA at6h were visible in the experimental group and the control group, peaked at24h, thereafter gradually declining,the expression were significantly reduced in the experimental group compared with the control group.5. Immunohistochemicaly examined that TLR4,NF-κB,TRIF expression was higher in experimental group than that in the control group.6. ELISA analysis shown in experimental group TNF-alpha levels was significantly lower than the control group there was statistically significant difference (p<0.01)ConclusionLiteratures have shown that IPC can induce BIT, but impacts on the expression of TLR4signaling axis after pMCAOon are still not clear. The study confirmed that:1. IPC can induce BIT,significantly reduced inflammatory responses in brain tissue after cerebral infarction, narrowing the area of cerebral infarction, induces the production of BIT.2. While IPC inhibitated TLR4/NF-K b signaling after pMCAO, accordingly reduced the production of Inflammatory Cytokines.3. In addition, through the activity of negative feedback mechanism, IPC significantly increased the TRIF, an increased the anti-inflammatory factors,IFN-(3,thereby reducing the degree of ischemia-induced brain tissue injury.4. These results, including inhibiting inflammatory TLR4/NF-K b signaling pathway, TRIF pathway activation reducing pro-inflammatory factor, increasing in anti-inflammatory factor indicated that TLRs signaling axis were involved in the mechanisms of endogenic neuroprotection of BIT induced by IPC.5. TLR4inflammatory signaling pathway activation played a key role on cerebral ischemia injury, and involved in the BIT-induced processes.6. TLR4signaling pathway is the probably target for a new therapeutic strategy of acute ischemic stroke.Although the exact molecular mechanism of BIT is not clear, it has been initially confirmed that requirement of new protein synthesis, promoting inflammatory activation of the transcription factor NF-κb and productive inflammatory cytokine such as TNF-a, IL-1β and IL-6. Pretreatment with small dose of LPS can prevent brain tissues injury after MCAO and remit inflammatory response, inhibitate peripheral neutrophils and macrophages/microglia infiltration,reduce TNF-aneural toxicity after cerebral ischemia, so LPS pretreatment is potential effective agent for cerebral infarction treatment. So the important role of TLR4inflammatory injury after cerebral ischemia helps to elucidate the neuroprotective mechanism of pretreatment with LPS. LPS signal transduction after pretreatment weakened TLR4/NF-κb signaling axis through a negative feedback loop, preventing TLR4-MyD88interaction, and sequentially inhibitated signaling, reduced the generation of Inflammatory Cytokines,while when again exposed to LPS,stroke-induced TLR4signaling would transfer from NF-κb to IRF3axes induction, promoting releasing of anti-inflammatory factors of IFN-β.And similarly, CpG pretreatment caused the same kind of anti-inflammatory response via signals axis, induced cerebral ischemic tolerance and cross tolerance between TLR4and TLR9, it was speculated that role of pretreatment with LPS and CpG share a common signaling pathway. So pre-stimulus before ischemic stroke may reprogramming TLR activation. LPS and CpG pretreatment can respectively caused TLR4and TLR9activation, caused minor inflammatory reaction, with a small TNF-alpha increase, at this time restrained TLR/NF-κb signal axis expression through a negative feedback suppression factor to regulate inflammation responds,only recognize endogenic ligends generated by minor ischemic stroke. in this new cell environment, TLR4activates was blocked, failed to activate the NF-κb signaling pathways to produce inflammatory cytokine such as TNF-a,leading to reduced cerebral ischemic injury, and stroke-induced TLR4would shifted form NF-κb activation to IRF3induction, produce downstream anti-inflammatory factors products, IFN-p.Therefore, activation TLRs and its signaling pathway before the cerebral ischemia may provide potential strategies for treatment of cerebral infarction. In addition to LPS and CpG,TLRs of exogenous ligand HMGB1, as proved in a mouse model of cerebral ischemic stroke, intraperitoneal injection of HMGB1antibodies against can significantly reduce the cerebral ischemic injury.Ischemic stroke involves a extremely complicated pathophysiological mechanism and clinical treatment are also very limited, currently only recombinant tissue-plasminogen activator(r-tPA)for thombolysis is approved and firmed effective. but its application time window is very short (3hours), has risk of brain bleeding (6%) and then the reperfusion reperfusion injury,Even in advanced country such as USA,the large majority of acute stroke patients canot go to hospital within3hours of onset,consequently have no chance to use t-PA. BIT is the focus of research in recent years, and it has been observed from clinical phenomena that BIT existent in human brain. Inflammation reaction after focal cerebral ischemia is a significant cause of brain damage, being a key component of the innate immune system of central nervous system, TLRs have played a key role in inflammatory injury after cerebral ischemia.This research found that IPC48h prior to pMCAO induced BIT, changed TLR4signaling and related downstream inflammatory cytoking, IPC was involved in the neuroprotection mechanism by decreased the TLR4/NF-κb signaling related inflammatory products and reduced the ischemic brain damage,on the other hand,IPC prestimuli shift TLR4from NF-κb induction to TRIF induction,therefore increased generation of anti-inflammatory cytokine, IFN-β.So,TLRs reprogramming is the way to provide neuroprotection.lt has been well documented that TLR2,TLR4,TLR9and their ligand HMGB1contribute to ischemic brain injury.LPS,CpG, HMGB1antibody appears to be the promising precondition agent to induce protection to acute ischemic brain damage. |
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