Dexmedetomidine Regulates Cerebral Ischemia Reperfusion Injury In Rats Via PPAR-?-TLR4-NF-?B

Part one:Establishment and morphology of cerebral ischemia-reperfusion model in ratsObjective:Based on the model of cerebral middle artery occlusion and cerebral ischemia-reperfusion injury in rats,we try to explore the specific manifestations of brain tissue and cell damage,and provide the evidence for subsequent inflammatory and apoptosis effects from the point of view of cytomorphology analysis.Materials and Methods:40 healthy SD male rats(the average weight:280 ±18 g)were selected from the animal management association of our institute and were divided into two groups randomly,the Sham group and I/R group.No surgical measures were carried out on the Sham group and middle cerebral artery occlusion model in rats of I/R group was made by using suture embolization method.After 90 minutes of ischemia,the thread was removed to make blood flow reperfusion.Cerebral blood flow meter was used to detect blood flow in the distribution area of middle cerebral artery.The model was successfully established when more than 70%rats reperfusd blood flow after ischemia for 90 minutes.HE and Nissl cell staining were used to detect the inflammatory infiltration of brain cells.SEM was used to detect the surface damage of brain tissue.Transmission electron microscopy was used to detect changes of intracellular organelles.Mitochondrial membrane potential was detected to analyze the energy barrier of the mitochondrial transmembrane.Results:The rats in sham group developed well and no death happened.The actual survival rate of rats in I/R group was 85%,3 rats died,4 rats did not bleed in the cerebral blood flow image after dissection,and the success rate of the model group was 65%.HE staining analysis:the cells were arranged uniformly,with a large number and smooth structure in the Sham group.In the I/R group,the number of vacuoles increased,the morphological regularity was poor,and the tissue fluid was more.Nissl cell staining analysis:the cells were evenly distributed,with a large number and smooth structure in the Sham group.In the I/R group,the cell density of cortical tissue and hippocampal tissue was sparse,and the cells showed pyknosis,and the volume decreased gradually.SEM scaning analysis:the cell growth pattern was normal,and the overall arrangement was dense with a large density,regular shape and clear outline in the Sham group.In SEM images of the hippocampus,the connections between cells were better.In the I/R group,the cell morphology changed,the connectivity and the number of microvilli gradually decreased,and the cell bulges were more serious.TEM scanning analysis:the cell contents in Sham group were not changed,and their texture was even and growing well.In the I/R group,there was a certain degree of lipid deposition in the cells,and the cells showed obvious vacuole appearance.In the hippocampal tissue,a number of lysosomes and phagocytes appeared in the cell body,and the mitochondria and golgi bodies in the hippocampal tissue were enlarged and the lesion was more severe.Mitochondrial transmembrane potential analysis:the mitochondrial membrane potential of the cortex and hippocampal tissue in the Sham group was all red fluorescence.The mitochondrial membrane potential of the cortex and hippocampal tissue in the I/R group was abnormal,the fluorescence was yellow-green,and the internal potential depolarization was severe.Conclusion:The establishment of cerebral ischemia-reperfusion model in rats caused substantial damage to neurons,increased cell space,nuclear and cytoplasmic shrinkage,and different degrees of damage to organelles;the overall number of cells decreased.There was a certain degree of apoptosis and necrosis;the abnormal mitochondrial transmembrane potential suggested that an unscheduled apoptosis process was occurred in cells,which provided morphological basis for the following two parts of the study.Part two:DEX regulates the expression of brain immune factors and apoptosis in I/R ratsObjective:On the basis of the first part of the morphological research,the inflammatory and apoptotic effects of cerebral ischemia-reperfusion injury in rats and the inhibitory effect of dexmedetomidine were discussed,so as to provide help and specific strategies for clinical research and treatment.Materials and Methods:40 healthy SD male rats(the average weight:276 ±16 g)were selected from the animal management association of our institute and were divided into four groups randomly,the Sham group,I/R group,Dex1 and Dex2 group.The rats in the last three groups were made cerebral ischemia and reperfusion model for 24 h,and different treatments were performed later.The rats in the I/R group were injected the same amount of saline;the rats in the Dexl and Dex2 groups were intraperitoneal injected with dexmedetomidine solution(10?g/kg,20?g/kg,i.p.,respectively).Immunohistochemical method was used to analyze the expression of inflammatory factors IL-6,IL-8,TNF-? and GFAP;ELISA was used to detect IL-6,IL-8,TNF-? and GFAP.Cell apoptosis was measured by flow cytometry.DNA ploidy analysis was employed to analyze cell cycle;apoptosis expression and ratio were detected by flow cytometry.Western Blot was used to detect Atg5,caspase-3,Bax and Bcl-2.RT-PCR was used to analyze the mRNA levels of apoptosis-related protein Bax and Bcl-2.Results:Cell inflammatory effect analysis:(1)The levels of IL-6,IL-8,GFAP,TNF-? in the Sham group,Dex2 group,Dexl group,I/R group increased successively,the difference was statistically significant(P<0.05).(2)Immunohistochemical analysis:the expression of immune factors of the brain tissues in the Sham group,Dex2 group,Dexl group,I/R group increased successively:Sham group<Dex2<Dex1 group<I/R group(P<0.05).Apoptosis effect analysis:(1)the apoptosis rates of neurons in the Sham group,Dex2 group,Dexl group,I/R group were 4.87%,6.45%,12.33%,17.80%,respectively,the difference was statistically significant(P<0.05).(2)Cell cycle analysis:The cell cycle of neurons changed substantively,and the blocking effect strengthened obviously and mainly stagerated at G2 phase after ischemic reperfusion.The blocking effect of the neuronal cell cycle weakened significantly after Dex treatment,and the blocking effect of G2 phase decreased gradually,the proportion decreased to(26.55±3.89)%and(54.05±5.66)%.(3)Bax and bcl-2 mRNA expression analysis:Bax mRNA expression in the Sham group,Dex2 group,Dexl group,I/R group decreased successively,while bcl-2 mRNA expression increased successively(P<0.05).(4)Western-blot analysis:Caspase-3,Bax and Atg5 protein expression in the Sham group,Dex2 group,Dex1 group,I/R group decreased successively,the difference was statistically significant(P<0.05).Conclusions:Injury,apoptosis and the inflammatory effect of neuronal cells occurred in the cerebral ischemia-reperfusion model.Dexmetomidine treatment impaired injury,apoptosis,and palyed anti-inflammatory and cell repair roles,which was worth further clinical research.Part three:DEX regulates PPAR-?-TLR4-NF-?B signal transduction in rats with cerebral ischemia reperfusion injuryObjective:On the basis of the process of apoptosis and inflammatory reaction of the cerebral ischemia-reperfusion model,PPAR-?-TLR4-NF-?B signaling pathway regulated specific mechanism in the process of cerebral hemorrhage.We explored the mechanism to find potential targets to treat cerebral hemorrhage.Materials and Merhods:The same groups and treatment were carried out as the second part.Immunohistochemical method and RT-PCR were used to detect TLR4,TRIF,PPAR-y expressions.Western blot was used to detect TLR4,TRIF,NF-?B,PPAR-y,p65,1?B?p-p65?p-1?B expression.Results:Immunohistochemical results:the expression of TLR4 increased,but decreased after dexmetomidine treatment.The expression of PPAR-y and TRIF was negatively correlated with TLR4 and PPAR-y.RT-PCR results showed that TRIF increased,but decreased after dexmetomidine treatment in a dose-dependent manner.Dexmetomidine increased PPAR-y expression in the brain tissue,which could effectively inhibit the expression of TLR4/NF-?B signaling pathway.TLR4 increased when cerebral ischemia reperfusion developed,and dexmedetomidine decreased TLR4 expression in a dose-dependent manner.Transcription factor TLR4,TRIF,TLR4/NF-?B in the Sham group,Dex2 group,Dexl group,I/R group increased successively,the difference was statistically significant(P<0.05).The expression of immunosuppressive factors LKB and p65 decreased after cerebral ischemia reperfusion developed,and the inflammatory response and apoptosis enhanced.However,the immunosuppressive factors in the signal circuit increasedvat different degrees after dexmetomidine treatment in a dose-dependent manner.PPAR-? analysis indicated that the expression in the I/R groupm Sham group,Dex1 group,Dex2 group increased successively,the difference was statistically significant(P<0.05).Concluisons:TLR4/NF-?B signaling pathway plays a regulatory role in cerebral ischemia reperfusion model in rats,which can effectively increase its inflammatory effect and accelerate the apoptotic process.Dexmedetomidine can effectively protect nerve function via inhibting TLR4/NF-?B signal pathway,inflammatory reaction and cell apoptotic process.It has better clinical development.