| Objective:Based on the molecular biological pathway of glutamate-induced neuronal injury,the mechanism of brain protective effect of electroacupuncture pretreatment was explored based on the injury model of experimental middle cerebral artery infarction-reperfusion in SD rats,so as to provide basis for acupuncture prevention and treatment of cerebrovascular diseases.Methods:The model of middle cerebral artery occlusion and reperfusion in SD rats was constructed,and the reperfusion was restored after middle cerebral artery was obstructed 90min.Animals were divided into sham operation group,model group and electroacupuncture pretreatment group.MCAO/R model was made 2h after the treatment of the last intervention.24h after modeled,neuronal motor function was tested by Garcia score,cerebral infarction volume was evaluated by TTC staining and protective effect of brain cells in each group was evaluated by TUNEL staining.Also we use Liquid chromatography-mass spectrometry to detected the concentration of extracellular glutamate and use Western Blot and double immunofluorescence staining to observe the expression of GluN2B,m-Calpain and p38 MAPK in hippocampus.Results:1.Garcia neurobehavioral score decreased significantly after cerebral ischemia-reperfusion injury in rats(P<0.001)while electroacupuncture pretreatment could siginificantly improve the neurological behavioral of cerebral ischemia-reperfusion injury in rats(P<0.01).TTC staining revealed that the brain of rats have obvious infarction after ischemia-reperfusion(P<0.001)and the percentage of cerebral infarction volume in the electroacupuncture pretreatment group was lower than that in rats with simple ischemia-reperfusion injury(P<0.001).After cerebral ischemia injury,apoptosis in hippocampal CA1 region of rats increased compared with sham operation group.Compared with simple ischemia-reperfusion injury model group,electroacupuncture pretreatment could significantly reduce the number of TUNEL positive cells in hippocampal CA1 region(P<0.001).2.The concentration of extracellular glutamate in hippocampal neurons was detected by liquid chromatography-mass spectrometry(LC-MS).It was found that the concentration of extracellular glutamate increased after cerebral ischemia-reperfusion injury in rats.however,the concentration of extracellular glutamate in the electroacupuncture pretreatment group was lower than that in the rats with simple ischemia-reperfusion injury,but the difference is not obvious.3.Western Blot and double immunofluorescence staining were used to detect the regulatory effect of electroacupuncture pretreatment on GluN2B receptor.It was found that the expression of GluN2B in hippocampus increased(P<0.001)and the number of GluN2B/NeuN co-located cells in hippocampal CA1 region increased after cerebral ischemia-reperfusion injury.Compared with simple ischemia-reperfusion injury,electroacupuncture pretreatment decreased the expression of GluN2B(P<0.01)and GluN2B/NeuN co-located cells in hippocampal CA1 region.4.Western Blot and double immunofluorescence staining were used to detect the expression of m-Calpain in hippocampal neurons.It was found that the m-Calpain in hippocampus was activated and the number of m-Calpain/NeuN co-located cells in hippocampal CA1 region increased after cerebral ischemia-reperfusion injury(P<0.001).Compared with simple ischemia-reperfusion injury,electroacupuncture pretreatment decreased hippocampal m-Calpain expression(P<0.001)and m-Calpain/NeuN co-localization cells.5.Western Blot was used to detect The expression of p38 MAPK in hippocampal neurons,and it was found that there was no difference in the expression of total p38 MAPK among the three groups.However,the level of p38 MAPK phosphorylation was observed by p-p38/t-p38 ratio that electroacupuncture pretreatment could significantly reduce the level of p38 MAPK phosphorylation in hippocampus compared with the model group with simple ischemia-reperfusion injury(P<0.01).Conclusion:1.Electroacupuncture pretreatment can reduce cerebral ischemia-reperfusion injury in rats and has delayed neuroprotective effect.2.The delayed neuroprotective effect of electroacupuncture pretreatment may not be achieved by regulating the concentration of extracellular glutamate.3.Electroacupuncture pretreatment can reduce the neuronal injury caused by cerebral ischemia-reperfusion by regulating the expression of GluN2B protein,and stop the excitotoxicity induced by ischemic stroke at the upstream starting point of the injury cascade.4.Electroacupuncture pretreatment can down-regulate the expression of m-Calpain and p-p38 MAPK in hippocampal neurons,playing an important role in the brain protection of electroacupuncture,which may be the downstream mechanism of electroacupuncture pretreatment in inhibiting the molecular biological pathway of neuronal apoptosis. |
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