“通督调神”电针预处理介导miR-124-3p/GSK-3β/Cyp-D信号通路对脑缺血再灌注损伤大鼠脑皮质线粒体通透性转换孔的影响及机制探讨

• 作者： 张国庆 ¹ 童婷婷 ² 王颖 ¹ 李奎武 ² 张利达 ³ 吴晓晴 ² 李成龙 ² 汪俊丽 ² 张君宇 ⁴ 韩为 ¹
• 作者单位：
  1.  230061 安徽省合肥市，安徽中医药大学第二附属医院脑病科

  2.  230061 安徽省合肥市，安徽中医药大学第二临床医学院

  3.  510370 广东省广州市，广州医科大学附属脑科医院中医科

  4.  230061 安徽省合肥市，安徽中医药大学第二附属医院老年病科
• 提交时间：2023-03-28

摘要: 背景 缺血性脑卒中发病率、死亡率、致残率均较高，溶栓后的再灌注损伤对患者影响较大，针刺是治疗本病的特色疗法，但作用机制尚不明确。目的 探讨通督调神电针预处理对脑缺血再灌注损伤（CIRI）大鼠miR-124-3p/糖原合成酶激酶（GSK-3）/亲环素D（Cyp-D）信号通路及线粒体膜通透性转换孔（MPTP）的影响，探讨其防治CIRI的可能机制。方法 2022年68月，将100只清洁级SD大鼠随机分为假手术组、模型组、电针组、抑制剂组和电针+激动剂组，每组20只。造模前干预7d，电针组、电针+激动剂组选取通督调神穴组：百会、风府、大椎穴进行电针干预，1次/d，连续7d；造模前24h电针+激动剂组和抑制剂组分别侧脑室注射miR-124激动剂和抑制剂（5nmol）。除假手术组，余组采用改良线栓法制备大鼠右侧脑缺血再灌注模型；造模成功后，取大鼠右侧脑皮质，采用改良神经功能损伤评分量表（mNSS）、TTC染色观察各组大鼠神经功能损伤程度，TUNEL染色以及透射电镜观察神经细胞损伤情况；免疫荧光染色、Westernblotting、实时荧光定量PCR检测各组大鼠脑皮质GSK-3、p-GSK-3、Cyp-D、细胞色素C（Cyt-C）、Caspase-3表达。结果 除假手术组，各组大鼠均造模成功。与假手术组比较，模型组大鼠mNSS评分及脑组织相对梗死体积均增加，线粒体结构破坏严重，细胞凋亡指数升高，p-GSK-3、Cyp-D阳性表达分别减弱和增强，miR-124-3p及Cyp-D、Cyt-C、Caspase-3mRNA表达增高，p-GSK-3/GSK-3值降低，Cyp-D、Cyt-C、Caspase-3蛋白相对表达量增高，MPTP开放程度增加（P<0.05）。与模型组比较，电针组、抑制剂组mNSS评分及相对梗死体积均降低，线粒体结构破坏减轻，p-GSK-3/GSK-3增加，Caspase-3蛋白相对表达量降低；电针+激动剂组GSK-3mRNA表达增高（P<0.05）；电针组、抑制剂组、电针+激动剂组细胞凋亡指数降低，p-GSK-3、Cyp-D阳性表达分别减弱和增强，miR-124-3p及Cyp-D、Cyt-C、Caspase-3mRNA表达降低，Cyp-D、Cyt-C蛋白相对表达量降低，MPTP开放程度降低（P<0.05）。与电针组比较，抑制剂组p-GSK-3阳性表达增强，Cyp-D阳性表达减弱，miR-124-3p及Cyp-D、Cyt-C、Caspase-3mRNA表达降低，GSK-3mRNA表达增高，Cyp-D、Cyt-C蛋白相对表达量降低，MPTP开放程度降低（P<0.05）；电针+激动剂组细胞凋亡指数增高，p-GSK-3阳性表达减弱，Cyp-D阳性表达增强，miR-124-3p、Cyt-C、Caspase-3mRNA表达增高，Cyp-D、Cyt-C蛋白相对表达量增高，MPTP开放程度增加（P<0.05）。结论 通督调神电针预处理可减轻CIRI大鼠神经损伤，其机制可能与介导miR-124-3p/GSK-3/Cyp-D信号通路、抑制线粒体MPTP开放进而减轻细胞损伤相关。这一研究结果从机制上进一步验证了通督调神针法治疗CIRI的疗效，同时为中医治未病提供新的科学依据，促进临床应用。

Abstract: Background The incidence，mortality and disability rates of ischemic stroke are high，and the reperfusion injury after thrombolytic therapy has a great impact on patients. Acupuncture is a characteristic therapy for the treatment of the disease，but the action mechanism remains unclear. Objective To observe the effect of Tongdu Tiaoshen electroacupuncture （EA） pretreatment on miR-124-3p/glycogen synthase kinase （GSK-3）/cyclin D （Cyp-D）signaling pathway and mitochondrial permeability transition pore of cerebral ischemia-reperfusion injury （CIRI） rats，and explore its possible mechanism of prevention and control of CIRI. Methods June to August 2022，a total of 100 clean SD rats were randomly divided into the sham operation group，model group，EA group，agonist group and EA + inhibitor group，with 20 rats in each group. For 7 d of intervention before modeling，in the EA group and EA + inhibitor group，Baihui（GV 20），Fengfu（GV 16）and Dazhui（GV 14）were selected to perform electroacupuncture 1 time a day for 7 days. For 24h before modeling，miR-124 agonist and inhibitor （5 nmol） were injected into the lateral ventricles in the EA + agonist group and inhibitor group. Except for the sham operation group，the right cerebral ischemia-reperfusion model of rats was prepared by the modified suture method in the rest groups. The right cerebral cortex of rats was taken，the degree of neurological impairment in each group was observed using mNSS scale and TTC staining，the nerve cell injury was observed by TUNEL staining and transmission electron microscopy. The expressions of GSK-3，p-GSK-3，Cyp-D，Cyt-C and Caspase-3 in cerebral cortex of each group were detected by immunofluorescence staining，Western blot and real-time quantitative PCR. Results Except for the sham operation group，all rest groups of rats were successfully modeled. Compared with the sham operation group，mNSS score and infarct volume of brain tissue were increased，mitochondrial structure was seriously damaged，cell apoptosis index was increased，p-GSK-3 positive expression was reduced，Cyp-D positive expression was enhanced，miR-124-3p，Cyp-D，Cyt-C and Caspase-3 mRNA expressions were increased，p-GSK-3/ GSK-3 ratio was decreased，relative Cyp-D，Cyt-C，and Caspase-3 protein expressions were increased，and MPTP openness degree was increased in the model group（P<0.05）.Compared with the model group，mNSS score and relative infarct volume were decreased，mitochondrial structure destruction was relieved，p-GSK-3/GSK-3 ratio was increased，and relative expression of Caspase-3 protein was decreased in the electroacupuncture group and inhibitor group；the mRNA expression of GSK-3was increased in electroacupuncture + agonist group（P<0.05）；apoptosis index was decreased，positive expression of p-GSK-3 was reduced and positive expression of Cyp-D was enhanced respectively，miR-124-3p，Cyp-D，Cyt-C and Caspase-3 mRNA expressions were decreased，Cyp-D and Cyt-C protein expressions were decreased，the degree of MPTP openness was decreased in the electroacupuncture group，inhibitor group and electroacupuncture + agonist group（P<0.05）. Compared with the electroacupuncture group，p-GSK-3 positive expression was enhanced，Cyp-D positive expression was reduced，miR-124-3p and Cyp-D，Cyt-C，Caspase-3 mRNA expressions were decreased，GSK-3 mRNA expression was increased，Cyp-D and Cyt-C protein expressions were decreased，and MPTP openness degree was reduced in the agonist group（P<0.05）；the apoptosis index was increased，the positive expression of P-GSK-3 was reduced，the positive expression of Cyp-D was enhanced，the expressions of miR-124-3p，Cyt-C and Caspase-3 mRNA were increased，the expressions of Cyp-D and Cyt-C protein were increased，and the openness degree of MPTP was increased in the electroacupuncture + agonist group（P<0.05）. Conclusion Tongdu Tiaoshen electroacupuncture pretreatment can alleviate neurological impairment in cerebral ischemia-reperfusion rats，the mechanism may be related to mediating miR-124-3p/GSK-3/Cyp-D signaling pathway，inhibiting MPTP openness and thus reducing cell injury. The results of this study further verified the therapeutic effect of Tongdu Tiaoshen electroacupuncture in the treatment of CIRI from the mechanism，providing a new scientific basis for preventive treatment of disease of TCM and promote its clinical application.

• 分类： 医学、药学 >> 预防医学与公共卫生学
• 引用： ChinaXiv:202303.10317 (或此版本 ChinaXiv:202303.10317V1)
  DOI:10.12114/j.issn.1007-9572.2023.0056
  CSTR:32003.36.ChinaXiv.202303.10317.V1
• 推荐引用方式： 张国庆,童婷婷,王颖,李奎武,张利达,吴晓晴,李成龙,汪俊丽,张君宇,韩为.(2023).“通督调神”电针预处理介导miR-124-3p/GSK-3β/Cyp-D信号通路对脑缺血再灌注损伤大鼠脑皮质线粒体通透性转换孔的影响及机制探讨.中国全科医学.doi:10.12114/j.issn.1007-9572.2023.0056 (点此复制)