穴位埋线对缺血再灌注损伤高脂血症合并2型糖尿病大鼠心肌保护作用的机制研究

Mechanism of Catgut Implantation at Acupoints for the Protection of Myocardium from Ischemia-reperfusion Injury in Hyperlipidemia Rats with Type 2 Diabetes Mellitus

目的探讨穴位埋线对缺血再灌注损伤高脂血症合并2型糖尿病(ZDF)大鼠心肌保护作用的机制,为临床治疗提供理论依据。方法 2015年1月—2016年5月选取ZDF大鼠24只,随机分为对照组、缺血再灌注组、缺血预处理组和穴位埋线组,每组6只。适应性饲养1周后第7天,对照组直接开胸取材,其余3组均结扎冠状动脉左前降支,建立缺血再灌注损伤模型。缺血再灌注组缺血30 min,再灌注60 min;缺血预处理组缺血5 min、再灌注5 min,重复3次,之后操作同缺血再灌注组;穴位埋线组在适应性饲养1周后第1天,"内关""膻中""心俞"穴进行穴位埋线,之后操作同缺血再灌注组。采用透射电镜观察各组ZDF大鼠心肌组织超微结构及自噬水平,Western blotting法检测心肌内质网应激相关蛋白葡萄糖调节蛋白78(GRP78)、自噬相关蛋白Beclin-1水平,酶联免疫吸附实验(ELISA)法检测髓过氧化物酶(MPO)、丙二醛(MDA)水平。结果透射电镜下对照组线粒体和心肌细胞形态结构正常,未见明显自噬体;缺血再灌注组线粒体和心肌细胞肿胀明显,细胞膜破裂、内容物泄漏,仅见空泡或空腔及少量自噬泡;缺血预处理组、穴位埋线组与缺血再灌注组相比,心肌细胞超微结构损伤较轻,且自噬泡数量明显增多;缺血再灌注组心肌细胞超微结构破坏最为严重,且未见明显自噬泡。缺血再灌注组、缺血预处理组、穴位埋线组GRP78、Beclin-1、MPO、MDA水平高于对照组(P<0.05);缺血预处理组、穴位埋线组GRP78、Beclin-1水平高于缺血再灌注组,但MPO、MDA水平低于缺血再灌注组(P<0.05)。结论穴位埋线可能通过促进GRP78、Beclin-1水平升高,进而起到对再灌注损伤心肌的保护作用。

Objective To explore the mechanism of catgut implantation at acupoints for the protection of myocardium from ischemia- reperfusion injury in hyperlipidemia zucker diabetic fatty （ZDF） rats, models of type 2 diabetes mellitus, in order to provide a theoretical basis for clinical treatment. Methods This study was conducted during January 2015 to May 2016. Twenty -four ZDF rats were selected and randomly divided into control group, isehemia reperfusion （IR） group, ischemic pretreatment （IP） group and catgut implantation （CI） group, with 6 in each. On the 7th day after one week of adaptive feeding, myocardial tissues of the control group were taken out via thoracotomy, the other three groups were used for establishing IR models after ligation of the left anterior descending coronary artery. IR group first received myocardial ischemia treatment for 30 min, then received reperfusion for 60 min. IP group received 3 rounds of 5 - min myocardial ischemia treatment and 5 - min reperfusion, then received 30 - min myocardial ischemia treatment and 60 - min reperfusion. CI group received the same treatment given to IR group except on the first day after one week of adaptive feeding, they received catgut implantation at Neiguan, Danzhong, and Xinshu acupoints. The ultrastructure and level of autophagy of myocardium of ZDF rats were observed by transmission electron microscope. Western blotting was used to detect the levels of myocardial endoplasmic reticulum stress - related protein glucose - regulated protein 78 （GRP78） and autophagy - related protein Beclin - 1. The levels of myeloperoxidase （MPO） and malondialdehyde （MDA） were measured by enzyme -/inked immunosorbent assay i ELISA ） . Results Transmission electron microscope observation showed that, the control group demonstrated normal mitochondria and cardiomyocytes without many autophagysomes; IR group presented significantly swollen mitochondria and cardiomyocytes with ruptured cell membrane and leaked contents, leaving vacuoles or cavities and a small amount of autophagic vacuoles. Compared with IR group, the myocardial cell uhrastructural damage in IP and CI groups was lighter and the number of autophagic vacuoles was significantly increased. The myocardial cell uhrastructural damage in IR group was the most serious, even there were no many autophagic vacuoles. The levels of GRP78, Beclin - 1, MPO and MDA were significantly lower in the control group than in the other three groups （ P 〈 0. 05 ）. IR group had lower levels of GRP78 and Beclin - 1 but higher levels of MPO and MDA compared with IP and CI groups （P 〈 0. 05） . Conclusion Catgut implantation at Neiguan, Danzhong, and Xinshu acupoints may play a role in the protection of myocardium from ischemia - reperfusion injury by increasing the levels of GRP78 and Beclin - 1.