TLR4 Is Involved In Neuron Apoptosis Of The Cortex And Hippocampus In Cerebral Ischemia-reperfusion Mice

Cerebrovascular disease is a serious threaten to human health,especially ischemic disease,brain cell damage caused by cerebral ischemia-induced turn more serious after the restoration of blood perfusion,which called cerebral ischemia-reperfusion injury. Pathogenesis of cerebral ischemia-reperfusion injury involve energy metabolism, cellular acidosis,intracellular calcium homeostasis imbalance,oxidative stress, excitatory amino acid production,inflammation and apoptosis,and then a vicious circle is formed when all these segments overlap and interconnected each other.Apoptosis,also known as programmed cell death,which play an important role in ischemic brain injury and development,however its exact mechanism is unclear.New gene expression and certain "death protein" synthesis is necessary to activate the apoptotic process after cerebral ischemic injury.Expression of endogenous apoptosis-regulating genes may be play a key role in deciding the fate of ischemic neurons,caspase-3 is the caspase cascade "waterfall" and the most critical protein of regulator apoptosis in downstream in the large caspase family,and is the point of apoptotic process induced by all kinds of reason.Caspase-3 not only promote neuronal apoptosis during brain development,but also promote neuronal apoptosis caused by a variety of factors,which reveal that caspase-3 may be important effector molecules during ischemic neuronal apoptotic process.Toll like receptors(TLR4) is human Toll-like receptor proteins encoded by human Toll protein gene,which is homologous with the Drosophila Toll-like protein,and is one of transmembrane receptor of congenital immune system and pathogen pattern recognition receptors discovered newly,plays an important role in the regulation of cell phagocytosis in the acute inflammatory response,cell signal transduction and apoptosis.By mediated signal transduction of LPS infection by G-bacteria,TLR4 can induce NF-κB translocation and its corresponding gene activation,release pro-inflammatory factor and auxiliary costimulatory molecules,finally regulate inflammatory response.We found that TLR4 plays a very important role in cell apoptosis of brain ischemia-reperfusion injury.TLR4 is likely involved in a signal transduction pathway of cell apoptosis of brain ischemia-reperfusion injury,but which mechanism it participate is unknown.To study the relationship between TLR4 and apoptosis in the cerebral ischemia-reperfusion injury,the expression of Caspase-3 in cerebral cortex and hippocampus of mice was observed by immunohistochemistry,TUNEL and Western blot methods after TLR4 was blocked.We can further understand the signal transduction mechanism of cerebral ischemia-reperfusion injury and conclusion some theoretical basis for treatments by the resolution of these issues.Materials1.Experimental animals:90 Kunming mice were bought from China Medical University Laboratory Animal Center,weighing 20-30g.2.Experimental reagents:monoclonal antibody TLR4(Santa Cruz Inc.),rabbit anti-mouse polyclonal antibody Caspase-3(Beijing Zhong Shan Golden Bridge Biotechnology Co.,Ltd.),goat anti-rabbit secondary polyclonal antibody(Beijing Zhong Shan Golden Bridge Biotechnology Co.,Ltd.),TUNEL assay Kit(Roche company),immunohistochemical SP kit and DAB kit(mai new companies).β-actin, goat anti-rabbit secondary antibody labeled by horseradish peroxidase,goat anti-rabbit secondary antibodies labeled by FITC were purchased from Beijing Zhongshan Company.3.Experimental instruments:low temperature refrigeration centrifuge,spectro photometer,automatic running gel imaging system,GP electrophoresis apparatus, upright fluorescense microscope,-80℃deep freeze refrigerator,homeothermia freezing microtome,Motic Images Advanced 3.2 image analysis system,Homogenate machine;centrifuge;plus injector.Methods1.Cerebral ischemia-reperfusion injury animal model was prepared by bilateral carotid artery occlusion.2.Animal treatment and packet animals were randomly divided into 3 groups (30/group):sham operation group(Sham);ischemia-reperfusion group(I/R);TLR4 blocking group(TLR4).The transient forebrain ischemia were caused by bilateral common carotid artery occlusion for 12 minutes in I/R group and TLR4 group, bilateral common carotid arteries of Sham group were exposed for 12 min,and then established 6h,12h,24h,48h,72h five time points after reperfusion.TLR4 blocking group was inject the TLR4 antibody(10mg/ml) into right common carotid artery by a micro-syringe after ischemic.3.To observe the pathological variation of hippocampus and cortex tissue by HE dyeing4.the expression of Caspase-3 was detected by immunohistochemistry in cerebral cortex and hippocampus of mice in various groups.5.the expression of Caspase-3 was detected by western blot in cerebral cortex and hippocampus of mice in various groups.6.Apoptosis was detected by TUNEL in cerebral cortex and hippocampus of mice in various groups.7.image analysisThe results of western blot was scan by Chemi Imager 5500V2.03 software,IDV value was quantitative analysis by Fluor Chen2.0 Fluor Chen2.0.Photograph collection and quantitative analysis of immunohistochemistry by Motic Images Advanced 3.2 image analysis system.TUNEL in situ detection of apoptosis results in the determination of high-power microscope field of vision hippocampus 5 the average number of apoptotic cells.The mean number of apoptotic cells detected in hippocampus 5 by TUNEL was observed by high-power microscope.8.statistical treatmentAll values are mean±standard deviation indicated that the experimental data using statistical software SPSS13.0 analysis,and analysis of variance is used to carry out the inter-grou p comparison.Results1.To observe the result by histopathology and The results of nerve pathological injury evaluation.To observe by HE dyeing:S group:in the fight side's hippocampus 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.The nerve pathological injury evaluation was low in S group in the right side's hippocampus 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).2.The exprssion of Caspase-3 and TUNEL result in cortexImmunohistochemistry showed:the mean optical density(MOD) of Caspase-3 in the cerebral cortex is higher in ischemia-reperfusion group than in sham group(P＜0.05);compared with the ischemia-reperfusion group,the mean optical density(MOD) of Caspase-3 decreased significantly in the cerebral cortex of mice in TLR4 blocking group.Western blot showed:compared with the sham-operated group,the ratios between integrated density value(IDV) for Caspase-3 protein band and IDV forβ-actin protein band increased markedly in cerebral cortex of the mice of ischemia-reperfusion group; however,the ratios between IDV for Caspase-3 protein band and IDV forβ- actin protein band is lower markedly in TLR4 blocking group than in ischemia-reperfusion group(P＜0.05).TUNEL showed:the number of apoptotic cells is higher in the cerebral cortex of the mice in ischemia-reperfusion group than in sham-operated group,however the number of apoptotic cells is lower in the cerebral cortex of the mice in TLR4 blocking group than in ischemia-reperfusion group.3.The exprssion of Caspase-3 and TUNEL result in HippocampusImmunohistochemistry showed:the mean optical density(MOD) of Caspase-3 in the cerebral cortex is higher in ischemia-reperfusion group than in sham group(P＜0.05);compared with the ischemia-reperfusion group,the mean optical density(MOD) of Caspase-3 decreased significantly in the cerebral cortex of mice in TLR4 blocking group. Western blot showed:compared with the sham-operated group,the ratios between integrated density value(IDV) for Caspase-3 protein band and IDV forβ-actin protein band increased markedly in cerebral cortex of the mice of ischemia-reperfusion group; however,the ratios between IDV for Caspase-3 protein band and IDV forβ- actin protein band is lower markedly in TLR4 blocking group than in ischemia-reperfusion group(P＜0.05).TUNEL showed:the number of apoptotic cells is higher in the cerebral cortex of the mice in ischemia-reperfusion group than in sham-operated group,however the number of apoptotic cells is lower in the cerebral cortex of the mice in TLR4 blocking group than in ischemia-reperfusion group.DiscussionTLRs is a classic recognition receptors discovered recently,and widely distributed in the surface of immune cells.Janeway,Medazhitov and Rock firstly found that Toll like receptors(TLR4) is human Toll-like receptor proteins encoded by human Toll protein gene,which is homologous with the Drosophila Toll-like protein.Now,TLR4 has been a hot research spots in all kind of pathogenesis.Lehnardt S has confirmed that TLR4 signal transduction pathway is relate to neuronal degeneration in the central nervous system.Gao Y has found that MyD88 signaling pathway mediated by TLR4 has been involved in the inflammatory response and apoptosis in cerebral ischemia-reperfusion injury of mice.The regulation of apoptosis is not yet clear in cerebral ischemia-reperfusion injury of mice,however it is certainly that hydrolase family protein,Fas,bcl-2 family must play an important role,and there is a common characteristic which is the activation of proteolytic enzymes in cerebral ischemia-reperfusion injury of mice,and the core is the activation of Caspase-3 in whole the process.The level and time of apoptosis can be to know by the detection of Caspase-3.Immunohistochemistry show that the expression of Caspase-3 is significantly higher in ischemia-reperfusion group than in sham-operated group and increased with the time elongation,reach its peak at 48h.,while the expression of Caspase-3 is significantlylower in the TLR4 blocking group than in ischemia-reperfusion group at the same time point,and the result of Western Blot is the same as Immunohistochemistry,which imply that TLR4 might be an important role to the expression of Caspase-3 in the cerebral cortex and hippocampus of mice in ischemia-reperfusion injury.Apoptosis cells is significantly higher in ischemia-reperfusion group than in sham-operated group,while the number of Apoptosis cells is significantly lower in the TLR4 blocking group than in ischemia-reperfusion group,which reveal that TLR4 might invole Apoptosis in the brain cortex and hippocampus of mice in ischemia- reperfusion injury.Liu TH found Caspase-3 play an important role in apoptosis in cerebral ischemia-reperfusion,as a result,apoptosis of nerve cells in hippocampus of mice can reduced after TLR4 was blocked in cerebral ischemia-reperfusion injury,but how TLR4 partcipates apoptosis is need to be further studied.Cerebral ischemia is clinically common diseases,anastate reperfusion is necessary treatment measures in ischemic district.Our research provides a reference to how to alleviate the damage and nerve cells apoptosis in ischemia-reperfusion,and provide a new therapeutic approach for ischemic diseasesConclusionsThe expression of Caspase-3 and cells apoptosis significantly increased in cortex and hippocampus in ischemia-reperfusion injury of mice compared with sham,but decreased in TLR4 blocked group than in ischemia-reperfusion injury of mice,which indicates that TLR4 might be involved in cell apoptosis in cortex and hippocampus of ischemia-reperfusion mice.