基于谷胱甘肽抗氧化系统探讨督脉电针对脑卒中后肢体痉挛大鼠氧化应激的影响

Effects of governor vessel electroacupuncture on oxidative stress in rats with post-stroke limb spasm based on the glutathione antioxidant system

目的通过观察谷胱甘肽抗氧化系统相关因子的变化,评估督脉电针对脑卒中后肢体痉挛的作用机制。方法60只SD大鼠任意分为正常组(12只)、假手术组(12只)和造模组(36只)。造模组采用线栓法制备大脑中动脉阻塞模型,选取造模成功的24只大鼠任意分为模型组和电针组,每组12只。造模后第3天,电针组选取督脉穴位“大椎”“脊中”“后会”行电针治疗,每次30 min,每日1次,共治疗7 d。采用Zea Longa神经功能缺损评分评估大鼠神经功能,采用改良Ashworth肌张力分级和电生理描记法检测大鼠肌张力,采用干湿重法测定大鼠脑组织含水量,TTC染色法检测大鼠脑梗死体积,比色法检测大鼠皮质谷胱甘肽(GSH)、过氧化氢酶(CAT)、氧化型谷胱甘肽(GSSG)、超氧化物歧化酶(SOD)、丙二醛(MDA)含量,蛋白质印迹法和实时荧光PCR法检测大鼠大脑皮层谷胱甘肽还原酶(GR)、谷氨酰半胱氨酸连接酶(GCL)C、GCLM、谷胱甘肽过氧化物酶4(GPX4)蛋白和mRNA表达。结果与正常组和假手术组大鼠比较,模型组大鼠Zea Longa神经功能缺损评分、改良Ashworth肌张力分级、脑梗死体积、脑组织含水量及大脑皮层GSSG、MDA含量增加,张力信号值及大脑皮层GR、GCLC、GCLM、GPX4蛋白和mRNA表达降低,大脑皮层GSH、CAT、SOD含量降低(P<0.05)。与模型组比较,电针组大鼠Zea Longa神经功能缺损评分、改良Ashworth肌张力分级、脑梗死体积、脑组织含水量及大脑皮层GSSG、MDA含量降低,张力信号值及大脑皮层GR、GCLC、GCLM、GPX4蛋白和mRNA表达增加,大脑皮层GSH、CAT、SOD含量增加(P<0.05)。结论督脉电针可改善缺血性脑卒中后大鼠的肢体痉挛严重程度,其机制可能与调节大脑皮层谷胱甘肽抗氧化系统有关。

Objective To evaluate the mechanism of governor vessel electroacupuncture in rats with post-stroke limb spasm by observing the changes of glutathione antioxidant system-related factors.Methods A total of 60 SD rats were randomly divided into the normal group(n=12),sham operation group(n=12)and modeling group(n=36).The middle cerebral artery obstruction model was prepared by thread approach method in the modeling group,and 24 rats with successful modeling were randomly divided into the model group and the electroacupuncture group,with 12 rats in each group.At the 3rd day after modeling,the electroacupuncture group was treated with electroacupuncture at three acupoints of the governor vessel,namely,"Dazhui"(GV14),"Jizhong"(GV6)and"Houhui"(anteromedial of the transverse process of the sixth lumbar vertebra),for 30 min each time,once a day for 7 days.The neurological function of rats was assessed by Zea Longa neurological deficit score.The muscle tension of rats was detected by modified Ashworth dystonia rating and electrophysiological tracing method.The brain tissue water content was measured by the dry-wet weight method.The volume of cerebral infarction of rats was measured by the TTC staining method.The contents of glutathione(GSH),catalase(CAT),oxidized glutathione(GSSG),superoxide dismutase(SOD),and malondialdehyde(MDA)in the cortex of rats were detected by colorimetry.The protein and mRNA expressions of glutathione reductase(GR),glutamate cysteine ligase(GCL)C,GCLM,and glutathione peroxidase 4(GPX4)in the cortex of rats were measured by Western blotting and real-time PCR,respectively.Results Compared with rats in the normal and sham operation groups,the Zea Longa neurological deficit score,modified Ashworth dystonia rating,the volume of cerebral infarction,brain tissue water content,and GSSG and MDA contents in cortex were increased in the model group,the tension signal value and the proteins and mRNA expressions of GR,GCLC,GCLM,and GPX4 in cortex were decreased,and the contents of GSH,CAT,and SOD in cortex were decreased(P<0.05).Compared with the model group,the Zea Longa neurological deficit score,modified Ashworth dystonia rating,the volume of cerebral infarction,brain tissue water content,and GSSG and MDA contents in cortex were decreased in the electroacupuncture group,the tension signal value and the proteins and mRNA expressions of GR,GCLC,GCLM,and GPX4 in cortex were increased,and the contents of GSH,CAT,and SOD in cortex were increased(P<0.05).Conclusion Governor vessel electroacupuncture can improve the severity of post-stroke limb spasm in rats,and its mechanism may be related to the regulation of glutathione antioxidant system in cerebral cortex.