| Background and objectivesAcute ischemic stroke is a major cause of adult disability and death at present. To cure cerebral infarction, thrombolysis and revascularization of the obstructed blood vessels to restore the cerebral blood flow are effective in clinic. However, these methods are effective and safe only in very earlier period of a few strokes. It has been demonstrated that cerebral ischemia-reperfusion injury (CIRI) is the main cause for the aggravation of cerebral injury and functional impairment.Inflammation and infection processes take a critical role in the pathology of stroke and CIRI. There are obvious inflammatory cells aggregations, up-regulation of cytokines expression. and increased expression of intercellular adhesion molecules during cerebral ischemia-reperfusion injury. Ischemia reperfusion induced tissue damage can be reduced in experimental models by a variety of anti-inflammatory agents.TLR4 is a number of the TLRs family. Recently, researches show that TLR4 plays an important role in cerebral ischemia-reperfusion injury. Among other factors, the renin-angiotensin system (RAS), especially angiotensinⅡ(AngⅡ). has been implicated in the pathogenesis and outcome of ischemic brain injury. Similar to the TLR4 signaling, AngⅡexerted proinflammatory effects in the brain by locally stimulating chemokines expression.The aim of the present study is to investigate the expression of TLR4 in the progress of CIR in vitro and in vivo. And to investigate the relationship between AngⅡand TLR4 signaling in the progress of CIR in CNS.Our study are divided into two parts:Experiments in vivoMethods:Typical rat thread model performed by middle cerebral artery occlusion (MCAO) was used to induce the reversible focal cerebral ischemia/reperfusion injury. Rats in focal CIR injury group were subject to one hour of ischemia followed by scheduled time of reperfusion. Rats in drug (captopril, losartan and simvastatin) treatment groups were treated with one of the studied drugs for 14 days before focal cerebral ischemia reperfusion injury. The behavioral tests were used to evaluate the damage to central nervous system.2,3,5-triphenyl tetrazolium chloride (TTC) staining method was used to assess the percentage of brain infarct area. RT-PCR analysis and immunohistochemisty were conducted to measure the expression of TLR4 mRNA and protein after I R and the influence of captopril, losartan and simvastatin in rat brain.Results:Compared with sham-operate group, rats subject with one hour of ischemia followed by 12 hours of reperfusion exhibited severe neural injury as shown in significant increase of neurological scores, significant increase of infarct area ratio. Pretreatment with captopril, losartan or simvastatin can reverse or attenuate those changes of CIRI to some extent. TLR4 expression in mRNA and protein levels was up-regulated after ischemia/reperfusion. Pretreatment of captopril, losartan or simvastatin could inhibit the upregulation of TLR4.Conclusions:(1) TLR4 expression was up-regulated after ischemia/reperfusion.(2) Captopril, losartan and simvastatin could alleviate the injury caused by CIR.(3) Captopril, losartan and simvastatin could inhibit TLR4 expression induced by CIR. The effects of captopril and losartan suggest the relationship between AngⅡand TLR4 signaling.(4) The mechanism of neuroprotective effects of captopril, losartan and simvastatin was connecting with TLR4 signaling.Experiments in vitroMethods:In a BV-2 microglial cell line, hypoxia/reoxygenation methods were used to mimic the cerebral ischemia reperfusion injury. MTT method and lactate dehydrogenase (LDH) leakage method were used for evaluations of cell viability and cell lysis respectively. RT-PCR analysis and Western blot analysis were conducted to measure the expression of TLR4 mRNA and protein after hypoxia reoxygenation injury or treatment with AngⅡ. The detrimental influence of AngⅡon the BV-2 cells and on the expression of TLR4 were researched. An AT1 blocker losartan and an AT2 blocker PD123319 were chosen in this study to reveal the mechanism of the effects of AngⅡ. The TNFαconcentrations in supernatants of BV-2 cells were measured by ILISA.Results:Losartan but not PD12339 could attenuate the injury on BV-2 cells caused by hypoxia/reoxygenation or AngⅡ. TLR4 expression in mRNA and protein levels was up-regulated after hypoxia/reoxygenation or AngⅡalone treatment. AngⅡcould sharpen the upregualiton of TLR4 expression after hypoxia reoxygenation. Losartan could inhibit the TLR4 expression induced by hypoxia/reoxygenation or AngⅡ, while PD123319 had no effects. TNFαlevel in supernatants of BV-2 cells were increased when the cells were treated with AngⅡ. And Losartan but not PD 12339 could decrease the TNFαlevel. Hypoxia/reoxygenation treatment significantly enhanced the LPS induced TNF-αexpression. Losartan but not PD123319 decreased the LPS induced TNF-αexpression in hypoxia reoxygenation injured BV-2 cells.Conclusion:1. Both hypoxia/reoxygenation and AngⅡcould upregulate the expression of TLR4 in BV-2 cells.2. The influence of AngⅡon the expression of TLR4 is independent and via AT1 receptor pathway.3. Losartan could attenuate the hypoxia/reoxygenation induced cell injury. The mechanism was connecting with TLR4 signaling.4. AngⅡcould induce inflammatory action in BV-2 cells. TLR4 singling may be a mediator of this effect.
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