"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

doi:10.12114/j.issn.1007-9572.2023.0056

中国全科医学 ›› 2023, Vol. 26 ›› Issue (24): 3050-3060.DOI: 10.12114/j.issn.1007-9572.2023.0056

"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

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^,^*()

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

收稿日期:2022-12-20 修回日期:2023-03-10 出版日期:2023-08-20 发布日期:2023-03-28
通讯作者: 韩为
作者贡献：张国庆、童婷婷、李奎武提出研究构思与实验设计，负责实验实施，观察指标的测量与数据收集；张国庆负责撰写论文初稿；李奎武负责统计学分析；吴晓晴、汪俊丽负责数据收集与统计结果审核；童婷婷、张利达、张君宇负责整体实验评估，观察指标的数据收集；韩为、王颖负责文章的质量控制及审校，对文章整体进行监督管理；所有作者确认了论文终稿。
基金资助:
国家自然科学基金面上项目(81973933); 安徽省第十三批115产业创新团队"针药结合防治脑病创新团队"项目(皖人才办[2020]4号); 安徽省名中医王颖工作室建设项目(中发展[2022]5号)

Effect and Mechanism of Tongdu Tiaoshen Electroacupuncture Pretreatment-mediated MiR-124-3p/GSK-3β/Cyp-D Signaling Pathway on Mitochondrial Permeability Transition Pore in Cerebral Cortex of Rats with Cerebral Ischemia-reperfusion Injury

ZHANG Guoqing¹, TONG Tingting², WANG Ying¹, LI Kuiwu², ZHANG Lida³, WU Xiaoqing², LI Chenglong², WANG Junli², ZHANG Junyu⁴, HAN Wei¹^,^*()

1. Department of Encephalopathy, the Second Affiliated Hospital of Anhui University of Chinese Medicine, Hefei 230061, China
2. The Second Clinical Medical College, Anhui University of Chinese Medicine, Hefei 230061, China
3. Department of TCM, Brain Hospital Affiliated to Guangzhou Medical University, Guangzhou 510370, China
4. Department of Geriatrics, the Second Affiliated Hospital of Anhui University of Chinese Medicine, Hefei 230061, China

Received:2022-12-20 Revised:2023-03-10 Published:2023-08-20 Online:2023-03-28
Contact: HAN Wei

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摘要/Abstract

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β（GSK-3β）/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6—8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7 d，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7 d；造模前24 h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（5 nmol）。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、Western blotting、实时荧光定量PCR检测各组大鼠脑皮质GSK-3β、p-GSK-3β、Cyp-D、细胞色素C（Cyt-C）、半胱氨酸天冬氨酸蛋白酶3（Caspase-3）表达；流式细胞检测MPTP开放程度，MPTP阳性细胞率越高，代表MPTP开放程度越大。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评分及相对脑梗死体积均增加，线粒体结构破坏严重，细胞凋亡指数升高，p-GSK-3β、Cyp-D阳性表达分别减弱和增强，miR-124-3p及Cyp-D、Cyt-C、Caspase-3 mRNA表达增高，p-GSK-3β/ GSK-3β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评分及相对脑梗死体积均降低，线粒体结构破坏减轻，p-GSK-3β/GSK-3β增加，Caspase-3蛋白相对表达量降低；电针+激动剂组GSK-3β mRNA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-3β、Cyp-D阳性表达分别减弱和增强，miR-124-3p及Cyp-D、Cyt-C、Caspase-3 mRNA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-3β阳性表达增强，Cyp-D阳性表达减弱，miR-124-3p及Cyp-D、Cyt-C、Caspase-3 mRNA表达降低，GSK-3β mRNA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-3β阳性表达减弱，Cyp-D阳性表达增强，miR-124-3p、Cyt-C、Caspase-3 mRNA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-3β/Cyp-D信号通路、抑制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。

关键词: 再灌注损伤, 脑缺血, 卒中, 线粒体通透性转换孔, 通督调神, 电针疗法, miR-124-3p, 穴，百会, 穴，风府, 穴，大椎

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 β (GSK-3β) /cyclin D (Cyp-D) signaling pathway and mitochondrial permeability transition pore (MPTP) of cerebral ischemia-reperfusion injury (CIRI) rats, and explore its possible mechanism of prevention and control of CIRI.

Methods

From 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 24 h 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-3β, p-GSK-3β, 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. The degree of MPTP openness was detected by flow cytometry, the higher rate of MPTP-positive cells indicated greater degress of MPTP openness.

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-3β 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-3β/ GSK-3β 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-3β/GSK-3β ratio was increased, and relative expression of Caspase-3 protein was decreased in the electroacupuncture group and inhibitor group; the mRNA expression of GSK-3βwas increased in electroacupuncture + agonist group (P<0.05) ; apoptosis index was decreased, positive expression of p-GSK-3β 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-3β 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-3β 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-3β 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-3β/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.

Key words: Reperfusion injury, Brain ischemia, Stroke, Mitochondrial permeability transition pore, Tongdu Tiaoshen, Electroacupuncture therapy, miR-124-3p, Point GV20 (Baihui), Point GV16 (Fengfu), Point GV14 (Dazhui)

张国庆,童婷婷,王颖,等. "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[J]. 中国全科医学, 2023, 26(24): 3050-3060. DOI: 10.12114/j.issn.1007-9572.2023.0056.
ZHANG Guoqing,TONG Tingting,WANG Ying, et al. Effect and Mechanism of Tongdu Tiaoshen Electroacupuncture Pretreatment-mediated MiR-124-3p/GSK-3β/Cyp-D Signaling Pathway on Mitochondrial Permeability Transition Pore in Cerebral Cortex of Rats with Cerebral Ischemia-reperfusion Injury[J]. Chinese General Practice, 2023, 26(24): 3050-3060. DOI: 10.12114/j.issn.1007-9572.2023.0056.

图/表 12

表1 目的基因及内参基因的引物序列

Table 1 Primer sequence of target gene and internal reference gene

基因名称	扩增长度（bp）	上游引物（5'→3'）	下游引物（5'→3'）
β-actin	150	CCCATCTATGAGGGTTACGC	TTTAATGTCACGCACGATTTC
U6	94	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT
Cyp-D	101	ATCCTTAGCAAGTGTCTCGG	CTGGTCAAGGAAAAAGGCAG
GSK-3β	113	ACTTTGGAAGTGCAAAGCAG	ACTAGACGTGTAATCGGTGG
Cyt-C	86	TAAGACTGGACCAAACCTCC	TGTTCTTGTTGGCATCTGTG
Caspase-3	138	ATGTCGATGCAGCTAACCTC	AACTGCTCCTTTTGCTGTGA
miR-124-3p	64	ACACTCCAGCTGGGTAAGGCACGCGGTG	TGGTGTCGTGGAGTCG
miR-124-3p-RT		CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGCATT

图1 各组大鼠脑组织相对梗死体积（TTC染色）

Figure 1 Relative infarct volume of brain tissue of rats in each group

表2 各组大鼠mNSS评分及相对脑梗死体积比较

Table 2 Comparison of mNSS score and infarct volume of brain tissue of rats in each group

组别	只数	mNSS评分〔M（P₂₅，P₇₅），分〕	只数	相对脑梗死体积（±s，% ）
假手术组	20	0（0，0）	5	0
模型组	20	10（10，11）^a	5	34.95±3.80^a
电针组	20	6（6，7）^b	5	22.37±3.73^b
抑制剂组	20	6（5，7）^b	5	17.88±1.90^b
电针+激动剂组	20	10（9，10）^c	5	30.85±1.56^c
F（H）值		86.638		135.041^e
P值		<0.001		<0.001

图2 各组大鼠脑皮质神经细胞超微结构（透射电镜）注：图中红色箭头所示细胞膜，蓝色箭头所示细胞核，绿色箭头所示线粒体。

Figure 2 Ultrastructure of neurons in cerebral cortex of rats in each group

图3 各组大鼠脑皮质神经细胞凋亡情况（TUNEL染色，×400）注：蓝色荧光标记细胞核（DAPI），绿色荧光为TUNEL阳性细胞。

Figure 3 Apoptosis of neurons in cerebral cortex of rats in each group

表3 各组大鼠脑皮质AI、GSK-3β、p-GSK-3β、Cyp-D蛋白阳性表达比较（±s）

Table 3 Comparison of AI，GSK-3β，p-GSK-3β，Cyp-D protein positive expression in cerebral cortex of rats in each group

组别	只数	AI（%）	GSK-3β	p-GSK-3β	Cyp-D
假手术组	5	15.68±2.07	0.59±0.02	0.61±0.03	0.14±0.01
模型组	5	60.14±2.70^a	0.60±0.04	0.16±0.02^a	0.61±0.04^a
电针组	5	37.14±1.91^b	0.59±0.02	0.37±0.04^b	0.37±0.02^b
抑制剂组	5	28.98±1.96^bc	0.59±0.01	0.50±0.03^bc	0.28±0.02^bc
电针+激动剂组	5	41.28±2.75^bc	0.60±0.05	0.27±0.03^bc	0.46±0.04^bc
F值		251.206	0.205	157.005	193.968
P值		<0.001	0.932	<0.001	<0.001

图4 各组大鼠脑皮质GSK-3β、p-GSK-3β、Cyp-D表达情况（免疫荧光染色，×400）注：蓝色荧光标记细胞核（DAPI），绿色荧光分别标记糖原合成酶激酶（GSK-3β）、p-GSK-3β、亲环素D（Cyp-D）表达，Merge为合图；A为GSK-3β，B为p-GSK-3β，C为Cyp-D。

Figure 4 Expression of GSK-3β，p-GSK-3β and Cyp-D in cerebral cortex of rats in each group

表4 各组大鼠脑皮质miR-124-3p及GSK-3β、Cyp-D、Cyt-C、Caspase-3 mRNA表达比较（±s）

Table 4 Comparison of miR-124-3p，GSK-3β，Cyp-D，Cyt-C，Caspase-3 mRNA expressions in cerebral cortex of rats in each group

组别	只数	miR-124-3p	GSK-3β	Cyp-D	Cyt-C	Caspase-3
假手术组	6	1.00±0.05	1.02±0.03	0.91±0.14	0.97±0.08	0.98±0.05
模型组	6	1.94±0.10^a	0.48±0.11^a	2.32±0.11^a	2.78±0.04^a	1.92±0.11^a
电针组	6	1.32±0.05^b	0.79±0.08^b	1.88±0.24^b	2.09±0.14^b	1.38±0.03^b
抑制剂组	6	0.16±0.01^bc	0.94±0.05^bc	1.40±0.16^bc	1.59±0.05^bc	1.24±0.05^bc
电针+激动剂组	6	1.46±0.06^bc	0.68±0.05^c	2.06±0.08^b	2.42±0.05^bc	1.59±0.08^bc
F值		664.668	57.980	78.919	487.748	162.193
P值		<0.001	<0.001	<0.001	<0.001	<0.001

图5 各组大鼠脑皮质p-GSK-3β/ GSK-3β、Cyp-D、Cyt-C、Caspase-3蛋白表达比较

Figure 5 Comparison of p-GSK-3β/ GSK-3β protein expression ratio and GSK-3β，Cyp-D，Cyt-C，Caspase-3 protein expressions in cerebral cortex of rats in each group

表5 各组大鼠脑皮质p-GSK-3β/ GSK-3β、Cyp-D、Cyt-C、Caspase-3蛋白表达比较（±s）

Table 5 Comparison of p-GSK-3β/ GSK-3β protein expression ratio and GSK-3β，Cyp-D，Cyt-C，Caspase-3 protein expressions in cerebral cortex of rats in each group

组别	只数	p-GSK-3β/ GSK-3β	Cyp-D	Cyt-C	Caspase-3
假手术组	6	0.95±0.18	0.25±0.06	0.08±0.05	0.10±0.02
模型组	6	0.37±0.13^a	0.95±0.12^a	0.82±0.10^a	0.79±0.14^a
电针组	6	0.62±0.13^b	0.51±0.09^b	0.43±0.12^b	0.46±0.13^bc
抑制剂组	6	0.73±0.10^b	0.36±0.03^bc	0.10±0.06^bc	0.31±0.08^b
电针+激动剂组	6	0.47±0.11^bc	0.75±0.11^bc	0.60±0.09^bc	0.62±0.09^bc
F值		17.343	65.481	80.363	41.303
P值		<0.001	<0.001	<0.001	<0.001

图6 各组大鼠脑皮质神经细胞线粒体通透性转换孔开放程度比较（流式细胞技术）注：SSC=侧向散射光，FITC-A=FITC通道的荧光信号脉冲面积。

Figure 6 Comparison of openness degree of MPTP in cerebral cortex neurons of rats in each group

表6 各组大鼠脑皮质神经细胞线粒体通透性转换孔开放程度比较（±s）

Table 6 Comparison of openness degree of MPTP in cerebral cortex neurons of rats in each group

组别	只数	MPTP阳性细胞率（%）
假手术组	4	20.575±2.085
模型组	4	0.865±0.207^a
电针组	4	6.753±0.076^b
抑制剂组	4	16.350±0.493^bc
电针+激动剂组	4	2.690±0.338^bc
F值		313.385
P值		<0.001

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"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

Effect and Mechanism of Tongdu Tiaoshen Electroacupuncture Pretreatment-mediated MiR-124-3p/GSK-3β/Cyp-D Signaling Pathway on Mitochondrial Permeability Transition Pore in Cerebral Cortex of Rats with Cerebral Ischemia-reperfusion Injury

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