Effects Of Electroacupuncture On Cerebral C-jun N-terminal Kinase Expression After Cerebral Ischemia Reperfusion In Rats

Cerebral ischemia is one of the most common cause of human death and disability,as we all known,more than 50%vessels can spontaneously recanalization after cerebral infarction,and the mechanism has always been a research hot spot.Cerebral ischemia reperfusion injury is when the local brain blood supply obstacles caused by various reasons,when blood flow to restore recanalization time of cells in the body,corresponding a series of cascade reaction and pathophysiological process:excessive free radical formation,toxicity of excitatory amino acids,intracellular calcium overload,inflammatory cytokines and abnormal gene expression and so on,eventually lead to nerve cell apoptosis or necrosis.In the central area of ischemic brain injury,cells suffered severe swelling,necrosis because of the serious metabolic disorders and ion balance disorders,mainly characterized by cell necrosis.In the transitional place between normal tissue and ischemia central form "ischemic penumbra",half dark area in a few days after reperfusion,so called delayed neuron death(DND).DND mainly occurs in the hippocampus,striatum and cortical areas,where the development of irreversible injury is relatively slow,need a few days.characterized by cell apoptosis.If ischemia state can not get timely corrective and control,apoptosis of neurons in ischemic penumbra will being the part of Infarction.Therefore,study the cause of apoptosis related to signal transduction mechanism,intervene in the apoptosis process save ischemia penumbra with dying neurons,protect the ischemia penumbra from being infarction area,may provide a new prospect for CIR injury treatment.In recent years,the research shows that mitogen-activated protein kinase(MAPK)which was called contact hub cytoplasm and nuclei involved in cerebral ischemia reperfusion injury and play a key role in signal transduction of apoptosis,c-Jun N-terminal kinase(JNK)was called stress activated protein kinase(SAPK),As an important member of MAPK,belonging to the evolutionary conservative serine/threonine protein kinase,is one of the main signal transduction pathways within the central nervous system.JNK can be activated by many factors such as cytokine,growth factor and stress(ionizing radiation,osmotic pressure,heat shock and oxidative damage).A large number of experiments indicate that the activation of JNK signaling pathway relate to apoptosis with many systems.Such as through phosphorylation serine at the 63 and 73 of the c-jun N-terminal kinase and activation c-Jun and AP-1 resultly,promote the cell apoptosis or mediated apoptosis by phosphorylation Bcl-2 and the Bcl-xL,promote the mitochondrial cytochrome-C release,thus activating Caspase cascade to apoptosis.SP600125 is JNK inhibitors competitived ATP found in 2001.SP600125 can stop all subtypes of JNK catalytic area reversible to stop JNK activity,inhibit the c-Jun phosphorylation,stop the expression of TNF alpha,IFN-gamma,COX2 and IL-2.Many experiments have shown that the effect of acupuncture on ischemic cerebral apoplexy is good,but the mechanism of JNK signaling pathways about acupuncture on ischemic stroke in unclear yet.Therefore,this study try to study the apoptosis and c-jun amino terminal kinase signaling pathways with electro-acupuncture therapy on cerebral ischemia reperfusion injury in rats,explore the mechanism about protective effects against cerebral ischemia reperfusion injury by electro-acupuncture to provide scientific theoretical basis for clinical treatment.Objective:Observe Neurologic deficits after ischemia reperfusion in rats,pathological form of different times at Ischemic focal area,the neurons apoptosis,the expression of phosphorylation c-jun terminal kinase and the electro-acupuncture theray on them.further study the expression of JNK signaling pathways of treatment of electro-acupuncture theray after cerebral ischemia reperfusion,which has certain reference significance to the intervention mechanism of acupuncture on cerebral ischemia reperfusion.Methods:250 male SD rats were randomly divided into control group,model group,electric acupuncture(EA)group,JNK inhibitors group,inhibitors+ electric acupuncture(EA)group,50 in each group,each group is divided into five subgroups according to time,namely,2 h,6 h,1 d,3 d,7 d,10 about each subgroup,a total of 25 groups.Model group preparation of focal cerebral ischemia reperfusion model by Zea Longa methods,inhibitor group injection SP600125 10 ul(5 min after injection)before 30 min building in focal cerebral ischemia reperfusion model.Electric acupuncture group:cerebral ischemia reperfusion model were treated in electro-acupuncture after awake 90 min.EA group was applied at "Zusanli"(ST 36),"Sanyinjiao"(SP 6)and "Chize"(LU 5),"Hegu"(LI 4)20 min each time with dilatational wave,2/15 hz frequency,intensity is 2 ma.2 h,6 h,1 d of subgroups only one time.3 d and 1 week group were treat each day.Model group and inhibitors group wre grabbed to fixed,but not for electric acupuncture intervention at the same time every day.Main Outcome Measures:1.To evaluate the neurological behavioral changes of focal cerebral ischemia rats by the method of Longa's level 5 standard score method2.To observe the pathomorphism changes of focal cerebral ischemia rats with HE and TTC staining by electron microscope.3.To observe the cell apoptosis at different times of focal cerebral ischemia rats by TUNEL method4.To observe the expression of phosphorylation c-jun terminal kinase at different times of focal cerebral ischemia rats by immune histochemical method.Data processing:all the data statistics are conducted on SPSS 13.0.Data are used by the form of(x±s).One-way ANOVA and LSD were used to analyze all experimental data.P<0.05 means difference was statistically significant.Results:1.Rats appear different degree of nerve function defect after cerebral ischemia reperfusion.At the 2th hour after cerebral ischemia reperfusion,the rats appear the most serious neurologic deficits,with time gone,the nerve function defect symptoms gradually reduce,at the 3th and 7th day,the nerve function defect symptoms reduce obviously.Neural function defect score comparison:At the 2th hour and 6th hour after cerebral ischemia reperfusion,EA group,JNK inhibitor group,JNK inhibitor +EA group were no significant difference comparison to the model group;At the 1th day after cerebral ischemia reperfusion,EA group and JNK inhibitor group were higher than that in I/R group(P<0.05),JNK inhibitor+EAgroup was significantly higher than I/R group(P<0.01);At the 3th day and 7th after cerebral ischemia reperfusion,EA group,JNK inhibitor group and JNK inhibitor+EAgroup were significantly higher than I/R group(P<0.01).2.Morphological comparison:brain structure of normal rats are clear and complete,nerve cells arranging rules,large and round nucleus at high magnification.Brain structure of I/R rats are fuzzy,signs of nerve cell necrosis,disappear of neurons.The structure of the visible screen mesh,part of the nerve cells arranged disorder,deep into the nucleus pycnosis,nucleolus disappeared,neuronal cytoplasm condensed,paint,cytoplasm vacuoles degeneration.The nucleus pulp is not easy to distinguish,cell weeks gap to expand.Cerebral infarction comparison:At the 2th hour,6th hour and 1th day after cerebral ischemia reperfusion,EA group,JNK inhibitor group,JNK inhibitor + EA group were no significant difference comparison with I/R group.At the 3th day and 7th day after cerebral ischemia reperfusion,EA group,JNK inhibitor group were lighter degree infarction compared with I/R group(P<0.05),JNK inhibitor + EA group were significant lighter degree infarction compared with I/R group(P<0.01).3.Apoptosis:After cerebral ischemia reperfusion,rats appear cell shrinkage,smaller volume,normal contact between cells decreased,core chromatin extensive enrichment.At the 2th hour after cerebral ischemia reperfusion,apoptotic cells increased and peaked at the 1th day,and then apoptotic cells retreated.Apoptotic cells decreased at the 3th day after reperfusion and relatively low at the 7th day after reperfusion.Compared with sham group at the same time,the difference was statistically significant(P<0.01);At the 2th hour and 6th hour after cerebral ischemia reperfusion,EA group,JNK inhibitor group and JNK inhibitor + EA group of cells apoptosis rate were lower compared with I/R group(P<0.05).At the 1th day after cerebral ischemia reperfusion,EA group,JNK inhibitor group and JNK inhibitor+ EA group of cells apoptosis rate were lower significantly compared with I/R group(P<0.01).At the 3th day after cerebral ischemia reperfusion,EA group,JNK inhibitor group of cells apoptosis rate were lower compared with I/R group(P<0.05),and JNK inhibitor + EA group of cells apoptosis rate were lower significantly compared with I/R group(P<0.01).At the 7th day after cerebral ischemia reperfusion,JNK inhibitor + EA group of cells apoptosis rate were lower compared with I/R group(P<0.05)4.Comparison of p-JNK levels:At the 2th hour after ischemia reperfusion in rats,the expression levels of p-JNK began to rise and higher at the 6th hour and reach peak at the 1th day and then the expression levels of p-JNK gradually decline.At the 3th day and 7th day,the expression levels of p-JNK decreased to the lower level.I/R group were significantly lower than sham group at the same time(P<0.01).At the 2th hour and 3th day after reperfusion,the expression levels of p-JNK decreased in EA group,JNK inhibitor group and inhibitor + EA group are lower compared with I/R group(P<0.05).At the 6th hour and 1th day after reperfusion,the expression levels of p-JNK in EA group,JNK inhibitor group and inhibitor + EA group were significantly lower compared with I/R group(P<0.01).At the 7th day after reperfusion,the expression levels of p-JNK in inhibitor + EA group were lower compared with I/R group(P<0.05).Conclusion:1.Electro acupuncture with "chize" and "Hegu","Zusanli"and "Sanyinjiao"can obviously improve neural function defect scale and brain tissue morphology and reduce the apoptosis index of cerebral ischemia reperfusion rats.2.Electro acupuncture with "chize" and "Hegu","Zusanli"and "Sanyinjiao"can inhibits the expression of p-JNK.3.The expression of cell apoptosis rate and p-JNK peaked at 1d after ischemia reperfusion.After electro acupuncture treatment,the expression of cell apoptosis rate and p-JNK decline obviously at the 1th day.4.The experiment results suggest that "acupuncture stimulation—inhibition of JNK signal regulation—reduce apoptosis",might be one of key mechanisms about acupuncture treatment for cerebral ischemia reperfusion injury.