主 办:北 京 中 医 药 大 学
ISSN 1006-2157 CN 11-3574/R

北京中医药大学学报 ›› 2020, Vol. 43 ›› Issue (2): 163-170.doi: 10.3969/j.issn.1006-2157.2020.02.010

• 针灸 • 上一篇    下一篇

电针预处理内关穴调控心肌缺血再灌注大鼠细胞自噬*

韩永丽, 陈松#, 张艳琳, 陈子琴   

  1. 1 湖北中医药大学针灸骨伤学院 针灸治未病湖北省协同创新中心 湖北 430061
  • 收稿日期:2019-09-07 发布日期:2020-03-24
  • 通讯作者: 陈松, 男, 博士, 讲师, 研究方向:针灸效应的基础研究, E-mail:chensong19860221@hbtcm.edu.cn
  • 作者简介:韩永丽, 女, 在读硕士生
  • 基金资助:
    *国家自然科学基金面上项目(No.81574065、No.81774420),湖北省教育厅科学技术研究计划青年人才项目(No.Q20192012)

Electroacupuncture at PC 6 (nei guan) regulates cell autophagy in rats with myocardial ischemia and reperfusion*

Han Yongli, Chen Song#, Zhang Yanlin, Chen Ziqin   

  1. College of Acupuncture and Orthopedics, Hubei University of Chinese Medicine, Hubei 430061, China; Hubei Provincial Collaborative Innovation Center of Preventive Treatment by Acupuncture and Moxibustion, Hubei 430061, China)
  • Received:2019-09-07 Published:2020-03-24
  • Contact: Chen Song, Ph.D., Lecturer. Hubei University of Chinese Medicine, Hubei 430061, China. E-mail: chensong19860221@hbtcm.edu.cn
  • Supported by:
    National Natural Science Foundation of China (No. 81574065, No.81774420); Hubei Provincial Department of Education Science and Technology Research Program for Youth Talents (No. Q20192012)Ethical Review: Ethics Review Committee of the Laboratory Animal Center of Hubei Academy of Medical Sciences (No. SXDX2019050A)

摘要: 目的 观察电针预处理内关穴对心肌缺血再灌注大鼠细胞自噬的影响,阐明其作用及潜在机制。方法 将50只大鼠随机分为5组:假手术组、模型组、电针组、抑制剂组和电针+抑制剂组。假手术组大鼠开胸后暴露心脏不做其他处理,模型组大鼠行在体心肌缺血再灌注术,电针组大鼠于造模前1周行电针内关穴治疗,抑制剂组大鼠于造模前1周注射磷脂酰肌醇3-激酶(PI3K)抑制剂渥曼青霉素,第8天行心肌缺血再灌注术,电针+抑制剂组同时进行电针和抑制剂预处理。治疗结束后处死取材,紫外分光光度法检测血清肌酸激酶同工酶(CK-MB)、诱导型一氧化氮合酶(iNOS)、血清总抗氧化能力(T-AOC)表达。石蜡切片免疫荧光检测自噬相关蛋白13抗体(ATG13) 、哺乳动物雷帕霉素靶蛋白(mTOR)、PI3K在心肌细胞中的表达和定位。HE染色实验观察缺血再灌注大鼠心肌细胞中自噬引起的病理变化,透射电镜观察缺血再灌注大鼠心肌细胞超微结构变化。结果 与假手术组相比,模型组大鼠血清中CK-MB和iNOS浓度显著增加,T-AOC浓度显著降低(P<0.05);与模型组相比,电针组、电针+抑制剂组和抑制剂组大鼠CK-MB和iNOS浓度降低,T-AOC浓度升高(P<0.05),其中以电针组的改变效果最显著。与假手术组比较,模型组、电针组、抑制剂组和电针+抑制剂组的mTOR、ATG13 、PI3K在心肌细胞中的表达均升高;与模型组相比,电针组、抑制剂组和电针+抑制剂组的mTOR、ATG13 、PI3K在心肌组织中的表达下降。HE染色结果显示:与假手术组比较,模型组心肌肌纤维出现溶解断裂甚至坏死,肿胀明显;电针组、抑制剂组及电针+抑制剂组心肌纤维间隙轻度增宽,坏死灶减轻,心肌细胞轻微肿胀,其中电针组变化最为明显。透射电镜观察结果显示:与假手术组相比,模型组心肌肌纤维出现溶解断裂甚至坏死,线粒体嵴不规则、缺失。电针组、抑制剂组及电针+抑制剂组心肌肌纤维间隙轻度增宽,细胞核及线粒体结构基本完整,其中以电针组变化最为明显。结论 电针预处理可以促进自噬通量,通过促进自噬来减轻心肌缺血再灌注损伤,减少心肌细胞自噬体的形成,PI3K抑制剂渥曼青霉素可逆转这一保护作用。

关键词: 心肌缺血, 电针, 预处理, 细胞自噬, PI3K-Akt-mTOR通路, 大鼠

Abstract: Objective To observe the effect of pretreatment with electroacupuncture at PC 6 (nei guan) on cell autophagy in myocardial ischemia-reperfusion (I/R) rats, and to clarify its underlying mechanism. Methods 50 rats were randomly divided into 5 groups: sham operated group, model group, electroacupuncture pretreatment group (EA group), inhibitor pretreatment group (inhibitor group), and electroacupuncture + inhibitor pretreatment group (EA + inhibitor group) (n=10 in each group). The rats in the sham operated group were directly exposed after thoracotomy without other treatments. The rats in the model group underwent in-vivo myocardial ischemia-reperfusion. Rats in the EA group received electroacupuncture treatment on PC 6 (nei guan) 7 days prior to model establishment. Rats in the inhibitor group were injected with wortmannin (PI3K inhibitor) a week before the model was established, and received myocardial ischemia-reperfusion on the 8th day. Rats in the EA + inhibitor group received both forms of pretreatment. All rats were killed after treatment. The serum CK-MB, iNOS, and T-AOC expressions were detected by ultraviolet spectrophotometry. The expression and localization of ATG13, mTOR, and PI3K in myocardial cells were detected by immunofluorescence on paraffin sections. The pathological changes caused by cardiomyocyte autophagy in the control group were observed by HE staining experiments. Ultrastructural changes of cardiomyocytes in the control group were observed by transmission electron microscopy. Results Compared with the sham operated group, the serum CK-MB and the iNOS concentration was significantly increased, T-AOC concentrations were significantly reduced in the control group (P<0.05). Compared with the control group, in the three pretreatment groups, the CK-MB and the iNOS concentration was decreased, T-AOC concentrations were increased (P<0.05); among them, the effect in the EA group was the most significant. Compared with the sham operated group, the expressions of mTOR, ATG13 and PI3K in cardiomyocytes in the other four groups were all increased Compared with the model group, in the three pretreatment groups, the expression of mTOR, ATG13, and PI3K in myocardial tissues was decreased. HE staining results showed that compared with the sham operated group, in the model group, myocardial muscle fibers showed lytic rupture or even necrosis, and obvious swelling. In the three pretreatment groups, the myocardial fiber space was slightly widened, the necrotic lesions were reduced, and the myocardial cells were slightly swollen; among them the EA group showed the most obvious changes. The transmission electron microscope observation results showed that compared with the sham operated group, in the model group, the myocardial muscle fibers showed lytic rupture or even necrosis, and there were irregular and missing mitochondria. While in the three pretreatment groups, the myocardial muscle fiber gaps were slightly widened, and the nucleus and mitochondrial structure were basically complete. Among them, there was most significant change in the EA group. Conclusion Electroacupuncture pretreatment could possibly reduce myocardial I/R damage and reduce the formation of autophagosomes by promoting autophagy. While wortmannin (PI3K inhibitor) seems to reverse such a protective effect.

Key words: myocardial ischemia, electroacupuncture, pretreatment, autophagy, PI3K-Akt-mTOR pathway, rats

中图分类号: 

  • R224.1