督脉电针调控胱氨酸/谷氨酸反向转运体改善脑卒中后肢体痉挛的作用机制

The mechanism of Du meridian electroacupuncture regulating cystine/glutamate reverse transporter to improve limb spasm after stroke

目的探讨督脉电针对大脑中动脉阻塞再灌注(MCAO/R)后肢体痉挛大鼠大脑皮质中胱氨酸/谷氨酸反向转运体[System Xc(-)]的影响,并探讨其可能的作用机制。方法选择SPF级雄性SD大鼠60只,随机分为空白组、假手术组及造模组。空白组不进行任何处理,假手术组仅分离血管,造模组采用大脑中动脉栓塞法制备MCAO/R大鼠模型,并于术后第3天进行Zea-Longa神经功能缺损评分、肌张力评定和电生理描记检测筛选肢体痉挛大鼠,造模组造模成功后随机分为模型组和督脉电针组。其中,督脉电针组于术后第3天开始治疗,每日1次,每次30 min,连续7 d。治疗结束后再次评定其神经功能及肌张力恢复情况;TTC染色测算脑梗死体积;干湿质量法检测各组大鼠脑含水量;比色法检测大脑皮质中谷氨酸(Glu)、半胱氨酸(Cys)和谷胱甘肽(GSH)浓度;蛋白免疫印迹(Western blot)法和实时荧光定量PCR(RT-qPCR)分别从蛋白和基因水平检测大脑皮质中血清溶质载体家族7成员11(SLC7A11)、溶质转运蛋白家族3成员2(SLC3A2)、γ-谷氨酰半胱氨酸合酶(γ-GCS)和谷胱甘肽合成酶(GSS)的表达。结果与模型组相比,治疗后督脉电针组大鼠神经功能缺损评分和改良Ashworth评分降低,肌电信号值增强,脑水肿程度减轻,脑梗死程度有所恢复,Cys和GSH含量显著增加,SLC7A11、SLC3A2、γ-GCS和GSS蛋白和mRNA的表达量显著提高,而Glu浓度显著降低(P<0.05)。结论督脉电针可以改善脑卒中后大鼠神经功能损伤和肢体痉挛程度,其机制可能与调控System Xc(-),改善Glu兴奋性毒性和氧化应激有关。

Objective To investigate the effect and mechanism of Du meridian electroacupuncture on cystine/glutamate reverse transporter[System Xc(-)]in cerebral cortex of middle cerebral artery occlusion/reperfusion(MCAO/R)model rats.Methods A total of 60 SPF male Sprague-Dawley(SD)rats were randomly divided into the blank group,the sham operation group and the model group.The blank group did not do any treatment,the sham operation group only separated the blood vessels and the model group was prepared by middle cerebral artery embolization for MCAO/R rat model.On the third day after operation,Zea-Longa neurological deficit score,muscle tension evaluation and electrophysiological tracing were used to screen rats with limb spasm.After successful modeling,rats was randomly divided into the model group and the Du meridian electroacupuncture group.The Du meridian electroacupuncture group began treatment on the third day after operation,once a day for 7 days.After the end of treatment,the neurological function and muscle tension recovery were evaluated again.TTC staining was used to measure the cerebral infarction volume.The brain water content of rats in each group was detected by wet and dry weight method.The concentrations of glutamic acid(Glu),cysteine(Cys)and glutathione(GSH)in cerebral cortex were detected by colorimetric assay kit.Western blot assay and quantitative real-time PCR(RTqPCR)were used to detect the expression of SLC7A11,SLC3A2,γ-glutamylcysteine synthetase(γ-GCS)and glutathione synthetase(GSS)in cerebral cortex at the protein level and gene level,respectively.Results Compared with the model group,the neurological deficit score and modified Ashworth score were decreased in the Du meridian electroacupuncture group,the EMG signal value was enhanced,the brain water content was decreased,the degree of cerebral infarction was restored,the contents of Cys and GSH were significantly increased,the expression levels of SLC7A11,SLC3A2,γ-GCS and GSS protein and mRNA were significantly increased,and the concentration of Glu was significantly decreased(P<0.05).Conclusion Du meridian electroacupuncture can improve the degree of neurological impairment and limb spasm in rats after stroke.The mechanism may be related to the regulation of System Xc(-)and improving oxidative stress.