Rofe Of TLR4-mediated MyD88-dependent Signaling Pathway During Cerebral Ischemia-Reperfusion Injury In Mice

ObjectiveIschemia reperfusion injury is referred to as idem effect and hypoxia of partial tissue organs caused by all kings of reasons,leading to ischemia injury in the cell tissue, and when blood reperfusion is recovered under certain conditions,the function dysbolism and disorganization of cells are not improved,instead,they are worsened. Complicated pathological and physiological changes happen during ischemia reperfusion.Ischemia reperfusion can save dying cells,while it can also worsen the injury and make the cells dead.Ischemic cerebrovascular disease is one of the principal diseases in the world which may cause patients die and become disabled.So how to decrease the cerebral ischemia reperfusion injury has become a key problem needed to be resolved in the process of curing ischemic cerebrovascular disease.The fact that cerebral ischemia reperfusion causes cerebral cells injured is discovered by studies recently a quick cascade reaction in which there are many links and inflammatory reaction is one of the important mechanisms of cerebral ischemia reperfusion injury.More and more reports showed that even though there is no infection,the signal given by damage and stringent state cells can cause immune reaction.TLR4(Toll-like receptor 4,TLR4) is a pattern recognition receptor(PRRs). It can recognize pathogen and switch on inherent immunity when pathogen begins to intrude the mechanism.TLR4-mediated MyD88(myeloid differentiation protein 88 ) signaling pathway plays an important role in anti-infection.Nowadays,the study on TLR4-mediated MyD88 signaling pathway is mainly focused on anti-infection immune reaction.Inflammatory reaction plays an important role in cerebral ischemia reperfusion injury,while,there is no report about whether TLR4-mediated MyD88 signaling pathway is activated and work in cerebral ischemia reperfusion injury.In the process of switching on immune response to resist the invasion of pathogen,TLR4-mediated MyD88 signaling pathway can regulate the expression of many inflammation related genes.TLR4 being activated causes the combination of autospecific dimerization and MyD88.MyD88 combines with interleukin-1receptor-associated kinase(IRAK),which leads to the self phosphorylation of IRAK,thus activate tumor necrosis factor receptor-associated factor 6,(TRAF6),making the mitogen-activated protein kinase (MAPK) family activated,in which nuclear factor-κB(NF-κB) induces kinase activate kinaseα,βof I-κB family,making I-κB degrade because of phosphorylation and NF-κB shift to cell nucleus,then switch on the transcription of some inflammatory cell factors,such as TNF-α,IL-1 and IL-6.This study is intended to observe whether TLR4-mediated MyD88 signaling pathway is activated in the process of cerebral ischemia reperfusion in the mouse hippocampus CA1 region and cortex of parietal lobe;the variation of correlated signaling molecule,brain tissue inflammatory reaction and neurone injury after TLR4 antibody is adopted to close and block TLR4 receptor in TLR4-mediated MyD88 signaling pathway;to discuss the molecule mechanism in the process of cerebral ischemia reperfusion to provide new study and treatment method in order to decrease cerebral ischemia reperfusion clinically.Methods1.Grouping of experimental animals144 healthy Kunming mice,male and female,weighing 18-20 grams,supplied by Experimental Animal Center of China Medical University,were divided into 3 groups at random:(1)sham operated group(S group n=48);(2)ischemia reperfusion model group(I group n=48);(3) TLR4 blocking group(T group n=48).The above 3 groups were subdivided into post sham operation or reperfusion 12h,24h,48h and 72h groups.2.The preparation of mice cerebral ischemia reperfusionThe following method is adopted:first,block the blood current of mice' both sides' common carotid arteries,then,reperfusion is conducted.After the blood current was block,the mice with such phenomena were chosen as samples:heart beating quickly,breath extent becoming deeply and breath frequency firstly becoming quickly, then,slowing down.T group:TLR4 antibody(10μg/ml) were infected into the right common carotid arteries;I group:the same amount physiological saline was infected;S group:only both sides' common carotid arteries were separated.3.The preparation of tissue sectionThe mice of above groups were fixed by paraform,then,the brain tissue was taken out,imbedded by paraffin according to routine methods and successive brain coronal slice(7μm) was made by section cutter.4.To observe the pathological variation of hippocampus and cortex tissue by HE dyeing;to analyze the image and deal with it by statistics.5.The nerve pathological injury evaluation and statistical disposal of hippocampus CA1 region and cortex of parietal lobe neuron.6.To test the expression of TLR4,MyD88,TNF-α,IL-1βby Western blot;to analyze the image and process the data.7.To test the expression of slice TLR4mRNA and MyD88mRNA by TLR4,MyD88 in-situ hybridization detection kit;to analyze the image and deal with it by statistics.8.To test the NF-κB combination activity by Electrophoretic Mobility Shift Assay(EMSA);to deal with it by statistics.9.To test the expression of TNF-αand IL-1βby immunohistochemistry stain;to analyze the image and deal with it by statistics.Results1.To observe the result by histopathologyTo observe by HE dyeing:S group:in the right side's hippocampus CA1 region and cortex of parietal lobe,the cells form was normal,the cells were in good arrangement and the form was complete.I group:the cells arrangement was scattered and cavitation existed in some endochylema.Some neuron swelled and distributed unevenly;cell nucleus contracted and was stained deeply;nucleoli disappeared.T group:the pericaryon swelling was reduced distinctly and the cell form was improved significantly.2.The results of nerve pathological injury evaluationThe nerve pathological injury evaluation was low in S group in the right side's hippocampus CA1 region and cortex of parietal lobe neuron at different time points. The evaluation of I group and T group was increased at different extent.Compared with S group,the difference was significant(P＜0.05).The evaluation of T group was lower than I group at different time points(P＜0.05).3.To test the expression of TLR4 and MyD88 by Western blot and the results of quantitative analysisIn the right side's hippocampus CA1 region and cortex of parietal lobe,TLR4 protein expression of I group was distinctly higher than that of S and T group.IDV (integrated density value) of TLR4 expression strap in I group was higher than that of T group(P＜0.05).MyD88 protein expression in I group was distinctly higher than that of T group and S group.IDV of MyD88 expression strap in I group was higher than that of T group(P＜0.05).4.In situ hybridization of TLR4mRNA and MyD88mRNA and the results of quantitative analysisThere was clear TLR4mRNA expression in the right side's hippocampus CA1 region and cortex of parietal lobe in I group and T group.TLR4mRNA positive reaction product average optical density value in T group was lower than that of I group (P＜0.05).TLR4mRNA positive reaction product average optical density value in I group was higher than that of S group(P＜0.05).There was clear MyD88mRNA expression in the right side's hippocampus CA1 region and cortex of parietal lobe in I group and T group.MyD88mRNA positive reaction product average optical density value in T group was lower than that of I group(P＜0.05).MyD88mRNA positive reaction product average optical density value in I group was higher than that of S group(P＜0.05).5.NF-κB combination activity and the results of quantitative analysisNF-κB combination activity in the right side's hippocampus CA1 region and cortex of parietal lobe in I group was distinctly higher than that of T group and S group(P＜0.05).IDV of NF-κB strap in I group was higher than T group.6.To test the expression of TNF-αand IL-1βby Western blot and the results of quantitative analysisWestern blot was adopted to detect the IL-1βexpression in the right side's hippocampus CA1 region and TNF-αexpression in cortex of parietal lobe of mice of every group.Western blot analysis showed that IL-1βprotein expression in I group was distinctly higher than that of T group and S group.IDV of IL-1βexpression strap in I group was higher than that of T group(P＜0.05).The analysis of TNF-αprotein expression in cortex of parietal lobe of mice of every group showed that TNF-αprotein expression in I group was distinctly higher than that of T group and S group.IDV of TNF-αexpression strap in I group was higher than that of T group(P＜0.05).7.The immunohistochemistry stain of TNF-αand IL-1βand the results of quantitative analysisThe immunohistochemistry method was adopted to detect IL-1βexpression in the right side's hippocampus CA1 region of mice of every group at different time points. IL-1βexpression was seldom in S group,IL-1βimmune dyeing was darkened in T group and IL-1βexpression in I group was more than that of T group.IL-1βpositive reaction product average optical density value in I group was higher than that of T group and S group.IL-1βpositive reaction product average optical density value in I group was higher than the average value of T group(P＜0.05).The results of TNF-αexpression in cortex of parietal lobe of rats of every group at different time points were following:TNF-αexpression in cortex of parietal lobe was seldom in S group,TNF-αimmune dyeing was darkened in T group and TNF-αexpression in I group was more than that of T group.TNF-αpositive reaction product average optical density value in I group was higher than that of T group and S group.TNF-αpositive reaction product average optical density value in I group was higher than the average value of T group (P＜0.05).Conclusion1.TLR4 is activated by cerebral ischemia reperfusion(CIR) in hippocampus CA1 region and cortex of parietal lobe in mice,which indicates that TLR4 may be involved in the pathogenesis of.CIR damage.2.Neuronal damage caused by CIR was palliated by blocking TLR4 in hippocampus CA1 region and cortex of parietal lobe,.which indicates that TLR4 is involved in the pathogenesis of.CIR damage3.The expression of MyD88 was increased after TLR4 was activated by CIR, while the expression of MyD88 was decreased after TLR4 was blocked by TLR4 antibody in hippocampus CA1 region and cortex of parietal lobe,which means that TLR4 participates in the pathogenesis of.CIR damage through upregulating the expression of NF-κB,IL-1βand TNF-α.4.NF-κB was activated and inflammatory factors IL-1βand TNF-αwas overexpressed in CIR,NF-κB activity and the expression of IL-1βand TNF-αwas decreased significantly while TLR4 was blocked by TLR4 antibody;which indicates that TLR4 is involved in the pathogenesis of.CIR damage through upregulating the expression of MyD88.5.TLR4-mediated MyD88 signaling pathway was activated in CIR,the inflammatory reaction was palliated while TLR4 was blocked,which means that TLR4 -MyD88 signaling pathway participates in the pathogenesis of.CIR damage...