The Study Of The Role And Mechanism Of Calcitonin Gene-related Peptide In Neuronal Cell Death And MAPKs Pathway After Cerebral Ischemia And Reperfusion

The pathophysiology of occurrence and development of cerebral ischemiareperfusion injury is a complex process.Autophagy and apoptosiswhich hasplayed an important role especially cerebral ischemia. The central area ofischemia has a large number of neuronal necrosis, but few of cell necrosisoccurs in surrounding ischemic penumbra. The study found between cellnecrosis and apoptosis of this region there is a certain relationship. Earlyrecovery of blood supply reperfusion of ischemic tissue is an important issue tosave the penumbra of damaged neurons,. Neurons has relatively higher contentof CGRPwhich is important to the expansion of the cerebrovascular in cerebralischemia and reperfusion. The generation of CGRP plays an important role inthe protection of cells of neurons and reduces the apoptosis of neuronal cells incerebral ischemia and reperfusion, thereby reduce the infarct area of the cerebralischemia infusion, and can improve the function of neurons in the braintissue,and reduce the sequelae of cerebral ischemia and reperfusion, and enhencethe quality of life of patients with stroke, but the specific mechanism of action ofCGRP in the brain tissue is still not very clear.When the ischemia of brain tissue turned up, the neuronal cell occuresautophagy and apoptosis in ischemic and hypoxic conditions,. Autophagy is “adouble-edged sword "to cells. When mild autophagy occurs, it plays a positiverole, and maintain the normal function of the cell. When autophagy severely occurs, it affect cell survival, resulting death. Apoptosis plays an important rolein cerebral ischemia and reperfusion. Apoptosis mediates the death of theneurons of the central ischemic area. As cerebral ischemia-reperfusion injury,the occurrence of apoptosis involves complex molecular biological mechanisms,it affect the stability of the environment, and induces protease activation: Silkmitogen activated protein (MAPKs) signal transduction pathway and activationof calcium-regulating cycling-dependent kinase (CaMKs). Mitogen-activatedprotein (MAPKs) signal transduction pathway plays an important role inapoptosis. Currently the most studied of the MAPK signal transduction pathwayis extracellular regulated protein kinase (extra cellular regulated protein kinases,ERK) pathway, c-Jun amino-terminal kinase (c-Jun N-terminal kinase, JNK)pathway and P38pathway. The specific mechanism of action of three importantsignaling pathways of the MAPKs family in brain ischemia-reperfusion is stillnot very clear, and CGRP protective effect on the brain tissue. The relationshipbetween CGRP and three signaling pathways is also unclear.The rats as the study of the experimental cerebral ischemia and reperfusion,expression levels of the proteins of the brain tissue was detected and infarct sizeof TTC staining, and investigating the role of CGRP in cerebral ischemiareperfusion rat neuronal cells of brain tissue, as well as how to play a role? Therelationship among the JNK signaling pathway,,P38MAPK signaling pathwayand ERK signaling pathway?Method 1. Healthy adult male SD rats weighing250-300g. Improved cerebralArterial suture method to produce an animal model of focal cerebral ischemiareperfusion injury. Randomly divided into Sham group, MCAO group, MCAO+CGRP group, MCAO+CGRP+CGRP8-37group, MCAO+CGRP+ERKinhibitor group, MCAO+CGRP+PD98059group, MCAO+CGRP+P38inhibitor SB203580group.24animals in each group. After modeling success,given the intravenous administration of CGRP (3μg/kg), while intravenousCGRP inhibitor CGRP8-37(2.5mg/kg), intraperitoneal injection of ERKinhibitor PD98059(1mg/kg), intraperitoneal injection of P38inhibitor SB203580(5mg/kg, dissolved in5mg/ml in DMSO) for drug treatment.2. After successful modeling, observing the neurobehavioral score of brainfocal ischemia of the rat, and CGRP on rat brain focal ischemia neurobehavioralscore. Infarct size as well as the measurement of infarct size in the rat brain afterischemia and reperfusion by TTC staining3. According to western blot, observing protein expression of JNK, p-JNKand ERK, perk, P38, p-P38in rat brain tissue4. Immunohistochemical methods detect distribution of JNK and ERK, P38in rat brain tissueResult1. According to the neurobehavioral analysis of the rat brain focal ischemia,compared with the MCAO group, MCAO+CGRP group, the standard ofneurobehavioral rating is relatively low (P <0.01). Compared to MCAO+ CGRP group, neuroethology value of MCAO+CGRP8-37group, MCAO+CGRP+PD98059group and the MCAO+CGRP+SB203580group wassignificantly higher (P <0.01).2. According to the results of TTC staining, compared with the MCAOgroup, the infarct area of the brain tissues of MCAO+CGRP group decreasedsignificantly (P <0.001). Compared to MCAO+CGRP group, the infarct size ofbrain tissue of MCAO+CGRP+CGRP8-37group, MCAO+CGRP+PD98059group, MCAO+CGRP+SB203580group was significantly increased (P<0.01).3. According to the results of the Western blot analysis, compared with theMCAO group, the p-JNK/JNK protein expression level of MCAO+CGRPgroup is significantly lower (P <0.05). Compared with MCAO+CGRP group,p-JNK/JNK protein expression of brain tissues of MCAO+CGRP+CGRP8-37group, MCAO+CGRP+PD98059group and MCAO+CGRP+SB203580group levels was significantly increased (P <0.05), especially in the MCAO+CGRP+PD98059group and MCAO+CGRP+SB203580group, p-JNK/JNKprotein expression level increases more apparently. Compared with the MCAOgroup, perk/ERK protein expression level of MCAO+CGRP group wassignificantly increased (P<0.05). Compared with the MCAO+CGRP group,perk/ERK protein expression of MCAO+CGRP+CGRP8-37group, MCAO+CGRP+PD98059group and MCAO+CGRP+SB203580group levels wassignificantly lower (P<0.05), especially in the MCAO+CGRP+PD98059 group reducing obviously. Compared with MCAO group, p-P38/P38proteinexpression of MCAO+CGRP group was significantly lower (P<0.05).Compared with MCAO+CGRP group, p-P38/P38protein expression of MCAO+CGRP+CGRP8-37group, MCAO+CGRP+PD98059group weresignificantly increased. And p-P38/P38protein expression level of MCAO+CGRP+SB203580group was significantly lower.4. Immunohistochemistry results of JNK and p38show that compared withthe sham group, the MCAO group found that the number of cells containingbrown particles increases significantly (P<0.05), the positive expressionincreased in brain tissue ischemia ischemic penumbra region’s position.Compared with MCAO group, the number of positive cells stained with JNKand p38antibody increased significantly, especially in the ischemic penumbraarea of the brain tissue. Compared with the MCAO+CGRP group, MCAO+CGRP+CGRP8-37, MCAO+CGRP+PD98059and MCAO+CGRP+SB203580group, the number of positive cells significantly reduced.Immunohistochemistry results of ERK show that compared with the sham group,the MCAO group found that the number of cells containing brown particlesincreases significantly (P<0.05), the positive expression increased in brain tissueischemia ischemic penumbra region’s position. Compared with MCAO group,the number of positive cells stained with ERK antibody increased significantly,especially in the ischemic penumbra area of the brain tissue. Compared with theMCAO+CGRP group, MCAO+CGRP+CGRP8-37, MCAO+CGRP+ PD98059and MCAO+CGRP+SB203580group, the number of positive cellssignificantly reduced.ConclusionThis study found that the rat brain of cerebral ischemia reperfusion modelproduces a certain amount of CGRP, but a small amount of CGRP can notprotect cerebral ischemia and reperfusion from damaging the neurons of thebrain tissue, when adding exogenous CGRP, the presence of a sufficient amountof CGRP neurons plays a protective role from cerebral ischemia-reperfusioninjury. In the brain tissue of cerebral ischemia and reperfusion, JNK signalingpathway, P38MAPKsignaling pathway and ERK signaling pathways areactivated simultaneously, the study reported that the activation of the JNKsignaling pathway mediated neuron apoptosis, P38MAPK signaling pathwaymay also cause inflammatory reactions and lead to the apoptosis of neuronalcells, the role in brain tissues after activation of the ERK signaling pathway isstill controversial, but reported in the literature, neuronal cells may play aprotective effect. We find by the present study, when increasing the exogenousCGRP, JNK and p38phosphorylation can be reduced, while CGRP at the sametime can promote the generation of ERK phosphorylation, also shows the sameresult in immunohistochemistry, when increasing the outer endogenous CGRP,in the brain tissue expression levels of p-JNK and p-P38are significantlyreduced, but perk expression levels are significantly increased. According toinfarct size calculated in the rat brain of ischemia-reperfusion injury, we find that the increase of exogenous CGRP played a significant role in the protectionof neurons in the brain tissue of the ischemic area, while in cerebral ischemiaand reperfusion, JNK signaling pathway, ERK signal path and P38MAPK signalpathway are activated, and appears. Large numbers of the brain CGRP caninhibit JNK signaling pathway and make activation of p38signaling pathway,and promote the activation of the ERK signaling pathway. CGRP of braintissue may make ischemic vasodilation of the area, so to restore the blood supplyof the ischemic area, and increase the supply of nutrients and oxygen to thebrain tissue, making JNK signaling pathway and P38MAPK signaling pathwaysof brain tissue apoptosis-related family of MAPKs are suppressed, so as toachieve protection of the role of neuronal cells. When Adding CGRP receptorantagonists, P38MAPK inhibitors and ERK blocker, even though the increaseof exogenous CGRP, nor CGRP on the neurons of the brain tissue play aprotective role.