电针对脑缺血大鼠大脑皮层γ-谷氨酰半胱氨酸合成酶蛋白及基因表达的影响

Effect of Electroacupuncture on Expression of γ-glutamylcysteine Synthetase Protein and mRNA in Cerebral Cortex in Rats with Focal Cerebral Ischemia-reperfusion

目的:观察电针"百会""大椎"穴对局灶性脑缺血再灌注大鼠大脑皮层γ-谷氨酰半胱氨酸合成酶(GCS)蛋白及其mRNA表达的影响,进一步探讨电针提高脑缺血再灌注大鼠抗氧应激能力的分子生物学机制。方法:SD大鼠随机分为假手术组、缺血再灌注组(模型组)、缺血再灌注+电针组(电针组),每组10只。用改良Longa线栓法制备脑缺血再灌注模型,电针组取"百会""大椎"穴,电针刺激30min。运用免疫组织化学法和RT-PCR技术分别检测大鼠大脑皮层γ-GCS蛋白及其mRNA的表达。结果:模型组与假手术组相比,大脑皮层锥体细胞层的γ-GCS蛋白阳性细胞数表达差异无统计学意义(P>0.05);而电针组与模型组相比,可见到大量的γ-GCS蛋白阳性细胞表达(P<0.01),且平均吸光度显著升高(P<0.01)。电针组的GCS重亚单位(GCSh)mRNA和GCS轻亚单位(GCSl)mRNA表达量亦明显高于模型组(P<0.05)。结论:电针"百会""大椎"穴可明显增加局灶性脑缺血再灌注大鼠大脑皮层γ-GCS蛋白及其mRNA表达,提示电针可促进机体谷胱甘肽系统清除受损神经元的氧自由基紊乱,保护大脑神经细胞。

Objective To observe the effect of electroacupuncture（EA） of ＂Baihui＂（GV 20） and ＂Dazhui＂（GV 14） on the expression of gamma-glutamylcysteine synthetase（γ-GCS） protein and gene in the cerebral cortex in rats with cerebral ischemia-reperfusion（CI/R） injury,so as to explore its molecular biological mechanism underlying anti-oxidative stress.Methods A total of 30 male Sprague-Dawley rats were randomized into sham operation（sham,n=10）,model（n=10）,and EA（n=10） groups.CI/R model was established by right middle cerebral artery occlusion（modified Longa＇s thread occlusion method） for 2 hours and reperfusion for 24 hours.EA（3 Hz,1-3 mA） was applied to ＂Dazhui＂（GV 14） and ＂Baihui＂（GV 20） for 30 min.γ-GCS protein expression of the parietotemporal region of cerebral cortex was detected by immunohistochemistry and γ-GCS heavy subunit（γ-GCSh） mRNA and γ-GCS light subunit（γ-GCSI） mRNA expression levels were assayed by real-time quantitative polymerase chain reaction（RT-PCR）.Results Compared with the sham group,the expression levels of γ-GCS protein in the pyramidal cell layer of the cerebral cortical parietotemporal region,and γ-GCSh mRNA and γ-GCSl mRNA,and the number of γ-GCS immuno-reaction positive cells had no remarkable changes in the model group（P〉0.05）,while in comparison with the mo-del group,the expression levels of cerebral cortical γ-GCS protein,and γ-GCSh mRNA and γ-GCSl mRNA,and the number of γ-GCS immuno-reaction positive cells were increased considerably in the EA group（P0.01,P〈0.05）.Conclusion EA of GV 20 and GV 14 can upregulate expression levels of γ-GCS protein,γ-GCSh mRNA and γ-GCSl mRNA of the cerebral cortical parietotemporal region in CI/R rats,which may contribute to its effect in protecting cerebral cortical cells from injury by clearing away excessive oxygen free radicals.