The Research Of Regulatory Mechanism Of MyD88 Signaling Pathways Mediated By Toll-like Receptor-2 In Brain Ischemic Injury After Cerebral Ischemia And Reperfusion

In many countries,stroke has become a major cause of death.In the counties of China,cerebrovascular disease is the second cause of death;in the cities of China,cerebrovascular disease is the third cause of death.Disability and dysfunction after stroke bring patients and families a great burden.Most of the stroke patients were ischemic stroke,and a small proportion was hemorrhagic stroke.The transmembrane TLRs distributed in immune cells,vascular endothelial cells,and brain tissue-specific cells may mediate inflammatory responses after cerebral ischemia.Studies have shown that TLR2 is up-regulated several hours after cerebral ischemia and may be involved in neuronal damage after cerebral ischemia.Ziegler and other studies have shown that MCAO after injection of anti-TLR2 closed monoclonal antibody T2.5,Immunohistochemistry found that the number of CD11b positive cells decreased,it also inhibit leukocyte aggregation and microglia migration;At the same time leading to NeuN positive cells increased after cerebral ischemia,it suggest that inhibition of TLR2 has neuroprotective effects.The medullary differentiation factor(MyD88)is an important transduction protein in the TLR signaling pathway,and its dependent signaling pathways and regulatory gene products play a key role in innate immunity and adaptive immunity.Matrix metalloproteinase 9(MMP-9)can degrade the basal membrane components of the cerebrovascular,leading to increased BBB permeability,leukocyte invasion,brain edema and hemorrhagic transformation.Objective:Explore the alternative relationship among TLR2(toll-like receptor 2,TLR2)?MyD88(Myeloid differentiation factor 88,MyD88)and MMP-9(matrix metalloproteinase-9.MMP-9)after brain cerebral ischemia(2h)and reperfusion.Then,exploring the possible role of TLR2?MyD88 and MMP-9 in brain injury formation after brain cerebral ischemia and reperfusion.Methods:Underwent the model of right middle cerebral artery occlusion(MCAO)2 hours and reperfusion in Sprague-Dawley rats(250g-280g).Rats in this research were divided into three groups randomly:the MCAO group?the sham group?the T2.5-treated group(T2.5 antagonist,0.1212ug/g,was injected through jugular vein when rats began to reperfusion after 2h MCAO).We chose the ischemic brain tissue of three groups was selected to carry out the following experiment in this research at the different reperfusion time points after MCAO.Firstly,we use Western Blot to observe the expression level of TLR2?MyD88 and MMP-9 at 1h?2h?3h?6h?12h?24h after brain ischemia and reperfusion in infracted cortex of brain tissue in sham group and MCAO group(There are five different rats at each time point,n=5).Secondly,we use TLR2 antagonist T2.5 via jugular vein injection at the beginning of reperfusion after MCAO,then use Western Blot to observe the expression changes of TLR2?MyD88 and MMP-9 at 24h after brain ischemia and reperfusion in infracted cortex of brain tissue in MCAO group and T2.5-treated group(There are five different rats at 24h,n=5).Lastly,we test the infarct area(TTC staining)?brain cerebral edema(wet-dry weighting method)?BBB permeability(Evan's blue)and neurological function score test at 24h after brain ischemia and reperfusion in three groups(sham group?MCAO group and T2.5-treated group)(every measure has five rats,n=5).Results:1?Western Blot results showed,the expression level of TLR2 in MCAO group began to increase at 6h after brain ischemia and reperfusion and the differences were statistically significant compared to the sham group(p<0.05).This trend lasted for 24 hours after brain ischemia and reperfusion(p<0.05).2?Western Blot results showed,the expression level of MyD88 in the MCAO group began to increase at 6h after brain ischemia and reperfusion and the differences were statistically significant compared to the sham group(p<0.01).This trend lasted for 24 hours after brain ischemia and reperfusion(p<0.05).3?Western Blot results showed,the expression level of MMP-9 began to increase at the 24h after brain ischemia and reperfusion,the decreased extent compared to the sham group were statistically significant(p<0.05).4?At 24h after brain ischemia and reperfusion,we tested BBB permeability?brain cerebral edema?infracted area and neurological score.All of these in the MCAO group were higher when compared to the sham group and the differences were statistically significant(p<0.01?p<0.01?p<0.001?p<0.001).5?Western Blot results showed,compared to MCAO group,the expression level of TLR2?MyD88 and MMP-9 were lower in T2.5-treated group at 24h after brain ischemia and reperfusion and the differences were statistically significant(p<0.05).At 24h after brain ischemia and reperfusion in T2.5-treated group,the BBB permeability?the infracted area?the brain cerebral edema and neurological score were lower than MCAO group and the differences were statistically significant(p<0.01?p<0.05?p<0.01?p<0.01).Conclusion:1?At 6 h after cerebral ischemia reperfusion,the expression level of TLR2 and MyD88 increase immediately;24h after cerebral ischemia reperfusion,the expression level of MMP-9 begin to increase,the above factors may play a role in the process of cerebral ischemia-reperfusion injury,such as the regulation of BBB permeability,the brain cerebral edema,cerebral infarction volume and neurological function score.2?The TLR2 antagonist T2.5 finally reduce BBB permeability.and alleviate cerebral infarction,cerebral edema and neurological deficits to improve brain ischemia and reperfusion injury by decreasing the expression level of MMP-9 which may be inhibited through MyD88 signaling pahway.