Based On JNK Signaling Pathway, The Regulation Of Electroacupuncture On Neuronal Apoptosis In Mice With Cerebral Ischemia-reperfusion Was Investigated

ObjectivesWith the cerebral ischemia reperfusion injury(CIRI)on pathological mechanism deeply,c-Jun N-terminal kinase(JNK)an important role in the signaling pathway in the apoptosis process has attracted more and more attention.It has gradually become one of the hotspots of research.In this study,the model of cerebral ischemia reperfusion in mice was made by bilateral common carotid artery ligation(BCCAo/2-VO).In this study,we explored the mechanism of electroacupuncture at Baihui Point(GV20),Geshu Point(BL17)and Shenshu Point(BL23)to regulate the JNK signal transduction pathway of caspase independent mitochondrial pathway in CIRI damage brain neuron apoptosis from the different links of JNK signal transduction mitochondrial pathway.MethodsThe adult male C57BL/6 mice of grade SPF were divided into 3 groups(each group)by random number table method:sham operation group,model group and electroacupuncture group.The sham operation group only separated the bilateral common carotid arteries,did not block blood flow,and did not bleed at the tip of the tail.In the model group and the electroacupuncture group,the cerebral ischemia reperfusion injury model was made by bilateral common carotid artery ligation.The mice in the electroacupuncture group were given the electroacupuncture intervention at Baihui Point(GV20),Geshu Point(BL17)and Shenshu Point(BL23).We choose a disposable sterile stainless steel needle.The needle handle electric acupuncture apparatus using continuous wave,frequency of 3 times/sec.The duration of electroacupuncture was 10min,and was performed after the model was successfully made for 2h.The sham operation group and the model group did not intervene,only receiving the same fetching stimulation as the electronic group.The neurological function of the mice was evaluated by 5 score score and stroke index score at 2h and 24h after the success of the model.MRI,MRA and HE staining in each group of mice were detected by animal nuclear magnetic resonance.The morphological changes of brain tissue in each group were detected,and the apoptosis of neurons in each group was determined by TUNEL.Flow cytometry(FCM)was used to detect the apoptosis of nerve cells in each group,and to detect the activation of JNK in the brain tissues of each group.Immunohistochemical technique was used to detect the expression of NSE and AIF in the brain tissues of all the mice.And the expression of Bax,Bcl-2,Caspase-3 and Caspase-9 protein in the cortex and hippocampus of mice was detected by Western Blot.Results1.The effect of Electroacupuncture on neural function score in mice with cerebral ischemia reperfusion2 hours after modeling by "5 points method" and "stroke index score" evaluation method,the model group was significantly higher than that of sham operation group(P<0.05),EA group and model group were not significantly different(P>0.05);model 24 hours after the "5 points method" and "stroke index score method,model group was significantly higher than that of sham operation group(P<0.05),EA group were significantly lower than model group(P<0.05).It can be seen that after 24 hours electroacupuncture intervention can obviously improve the neurological function score of cerebral ischemia reperfusion mice.2.The effect of Electroacupuncture on brain magnetic imaging and brain tissue morphology in cerebral ischemia reperfusion miceIn the sham operation group,MRI did not show obvious infarction and edema.The MRA vessels in the sham operation group were clear,and there was no obvious abnormality.The MRI edema phenomenon in the model group was obvious(white bright area),the edema volume was 52.8mm3,and the MRA vessels in the model group were fuzzy,showing obvious stenosis and interruption.The pathological changes in hippocampal CA1 area of each group were observed by HE staining.The hippocampal pyramidal cells in sham operation group were arranged orderly,the cell morphology was normal,the light staining was clear,the nucleolus was clear,and had a complete frame structure.However,the cells in the CA1 region of the hippocampus of the model group were arranged in disorder and sparsely,and the loss and deformation of the nerve cells,nuclear condensation,and deep nucleolus staining were observed.The morphological changes in the electroacupuncture group were similar to those in the model group,but the cells in the model group were more regular and the nucleolus were clearer.In addition,edema,nerve cell loss and deformability were reduced.3.The effect of Electroacupuncture on neuronal apoptosis,JNK activation in miceThe apoptotic values of hippocampus and cortical cells in model group were significantly higher than those in sham operation group(P<0.05),and the apoptotic values in hippocampus and cortex of electroacupuncture group were significantly lower than those in model group(P<0.05).The fluorescence intensity of the activation state of JNK in hippocampus and cortex of mice showed that there was no significant difference in the fluorescence intensity of JNK activation state of the mice in each group(P>0.05).However,the fluorescence intensity of JNK activation in the hippocampus of the electroacupuncture group was lower than that in the model group(P<0.05).4.The effect of Electroacupuncture on the expression of NSE and AIF in the hippocampus and cortex of miceThere was no NSE positive cells in hippocampus and cortex of sham operation group.NSE expression in hippocampus and cortical tissue of model group was positive,and there were NSE positive cells in hippocampus and cortex.Compared with model group.NSE positive cells in hippocampus and cortex decreased significantly in EA group(P<0.05).There was no AIF positive cells in hippocampus and cortex of sham operation group.AIF expression in hippocampus and cortical tissue of model group was positive,and there were AIF positive cells in hippocampus and cortex.Compared with model group,AIF positive cells in hippocampus and cortex decreased significantly in EA group(P<0.05).5.The effect of electroacupuncture on the expression of related proteins Bax,Bcl-2,Caspase-3 and Caspase-9 in JNK signaling pathwayThe gray value of Bax and Caspase-3 in hippocampus and cortex of electroacupuncture group was significantly lower than that of model group(P<0.05).The gray value of Bcl-2 in hippocampus and cortex of electroacupuncture group was significantly higher than that in model group(P<0.05);however,the gray value of Caspase-9 in electroacupuncture group was not significantly different from that in model group(P>0.05).Conslusion1.Electroacupuncture intervention can effectively improve the score of 5 score and stroke index score in mice with cerebral ischemia reperfusion.2.Electroacupuncture intervention can reduce the loss of neurons in the cortex and hippocampus of mice,the contraction and abnormal deformation,and reduce the apoptosis of nerve cells.3.Electroacupuncture intervention can inhibit the activation of JNK in hippocampal cells of CIRI mice,up regulate the expression of Bcl-2 in hippocampus and cortex of CIRI mice,down regulate the expression of Bax,and inhibit the expression of Caspase-3 and the expression of NSE and AIF in neurons of hippocampus and cortex of CIRI mice.