电针对脑缺血再灌注小鼠脑组织NOX2/gp91phox表达的影响

Effect of Electroacupunture on NOX2/gp91phox Expression in Brain Tissue of Mice with Cerebral Ischemia-Reperfusion

目的:探讨电针对脑缺血再灌注(I/R)损伤后的神经保护机制,明确电针对脑损伤后氧化应激的抑制作用。方法:采用小鼠大脑中动脉闭塞模型制备脑局灶性缺血1 h再灌注3、24、48 h模型。每组又分为模型组、电针组、药物组、gp91phox基因敲除组(下文简称基因组),另设同时段假手术组。以神经功能缺损评分和局灶脑血流(rCBF)变化为造模成功和效应指标,并RT-PCR检测NOX2/gp91phox基因在小鼠脑组织的表达情况,光泽精增强发光法检测NOX活性。结果:①神经行为学评分:造模各组均有不同程度的缺损表现,再灌注即刻缺损情况较再灌注干预后严重,基因组灌注即刻评分明显低于模型组(P<0.05),与模型组比较,灌注后3、24、48 h基因组、药物组、电针组神经行为学评分均明显降低(P<0.05)。②rCBF:各造模组rCBF明显减少,灌注后3、24、48 h各组rCBF均有增加,药物、电针组比模型组升高明显(P<0.05)。③NOX活性:模型组各时相之间无明显差异,基因组NOX活性无明显表达(P<0.01),药物组、电针组比模型组的同时相均有明显下降(P<0.01),药物和电针组的组间比较无明显差异。④NOX2/gp91phox基因表达:模型组各时相之间无明显差异,基因组无蛋白表达,与各组比较均有显著差异(P<0.01),药物组、电针组比模型组的同时相均有明显下降(P<0.01),药物组和电针组的组间比较无明显差异。结论:通过与敲除gp91phox基因组的小鼠相比较,电针“百会”“大椎”穴位可以通过抑制NOX2/gp91phox表达,达到抑制I/R后小鼠的氧化应激损伤效果,发挥保护脑组织的作用。

Objective:To explore the mechanism of neuroprotective effects of Electroacupunture on cerebral injury after ischemia-reperfusion(I/R),so as to identify the effects of Electroacupuncture in inhibiting oxidative stress after brain injury.Methods:The model of focal cerebral I/R was established by the occlusion of middle cerebral artery for 60 min further by the reperfusion.The models were randomly divided into three groups according to reperfusion time(3,24,48 h),each of which was further divided into model group,Electroacupuncture group,drug group,gp91phox gene knockout group(abbreviated as gene group),sham group on each phase was set.The neurological deficit score and regional cerebellar blood flow(rCBF)were used for neurological deficit assessment and effect,and the expression of gp91phox gene and the activity of NOX in the brain of models were detected.Results:①Neurobehavioral score:Compared with the model group,the neurobehavioral scores of the gene group,drug group and Electroacupuncture group were significantly reduced at 3,24,and 48 h after reperfusion(P<0.05),and the immediate score of gene group reperfusion was significantly lower than that of the model group(P<0.05).②rCBF:The rCBF of each model was significantly reduced,and the rCBF of each group increased at 3,24,and 48 h after reperfusion.rCBF in the drug group and Electroacupuncture group were significantly higher than that of the model group(P<0.05).③NOX activity:there was no significant difference at each phase in the model group,and the NOX activity in gene group was none(P<0.01).Compared with the model group,there was a significant decrease in the drug group and the Electroacupuncture group at the same phase(P<0.01).There was no notable difference between the drug group and the electroacupuncture group.④NOX2/gp91phox:the model group showed no significant difference at various phase.None protein expression was detected in the gene group,which was significantly different from the other groups(P<0.01).The gp91phox gene expression in the drug group and the Electroacupuncture group were prominently lower than that in the model group at the same phase(P<0.01),while there was no significant difference between the drug group and the Electroacupuncture group.Conclusion:By comparing with the gene group with gp91phox gene knockout,Electroacupuncture on“Baihui”and“Dazhui”can inhibit expression of NOX2/gp91phox to restrain the oxidative stress after acute I/R,and reduce the production of large amounts of reactive oxygen species(ROS)and free radicals during reperfusion,thus exerting effects on protecting brain tissue.