摘要
目的探讨电针预处理通过磷酸酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)通路对心肌缺血再灌注损伤大鼠的保护机制。方法将30只雄性大鼠随机分为假手术组、模型组、电针组、LY294002组、LY294002+电针组,每组6只。在造模前,电针组电针内关穴,LY294002组腹腔注射PI3K/Akt抑制剂LY294002,LY294002+电针组电针内关穴和腹腔注射LY294002,假手术组和模型组腹腔注射等量生理盐水,均1次/d,连续7 d。第8天,除假手术组外,其余组大鼠采用冠脉结扎法进行心肌缺血再灌注造模。再灌注1 h后,HE染色观察心肌细胞病理学形态,Western blot法检测心肌组织中p-PI3K、p-Akt、Bcl-2相互作用蛋白1(Beclin1)、微管相关蛋白1轻链3Ⅱ型(LC3Ⅱ)表达情况。结果模型组大鼠心肌细胞肿胀、排列紊乱,心肌纤维浊肿,肌原纤维不清,细胞间质有出血及水肿;电针组大鼠轻微心肌损伤,心肌细胞结构依旧清晰;LY294002组大鼠心肌损伤严重,心肌细胞肿胀,肌纤维间隙明显增宽,排列紊乱,线粒体肿胀,自噬泡存在,细胞间质可见多处出血、水肿;LY294002+电针组大鼠心肌损伤较轻,部分心肌细胞肿胀,心肌纤维排列欠整齐。与假手术组比较,模型组、LY294002组、LY294002+电针组大鼠心肌组织中Beclin 1、LC3Ⅱ蛋白相对表达量均明显升高(P均<0.05),p-PI3K、p-Akt蛋白相对表达量均明显降低(P均<0.05);与模型组、LY294002组、LY294002+电针组比较,电针组大鼠心肌组织中Beclin 1、LC3Ⅱ蛋白相对表达量均明显降低(P均<0.05),p-PI3K、p-Akt蛋白相对表达量均明显升高(P均<0.05);与LY294002组比较,LY294002+电针组大鼠心肌组织中Beclin 1、LC3Ⅱ蛋白相对表达量均明显降低(P均<0.05),p-PI3K、p-Akt蛋白相对表达量均明显升高(P均<0.05)。结论电针内关穴预处理可通过激活PI3K/Akt/mTOR信号通路,调节心肌缺血再灌注损伤大鼠的心肌自噬水平,抑制心肌细胞在缺血再灌注期间的过度自噬,从而保护心肌组织。
Objective It is to investigate the protective mechanism of electroacupuncture pretreatment in rats with myocardial ischemia-reperfusion injury(MIRI)via phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)mammalian target of rapamycin(mTOR)pathway.Methods Thirty male rats were randomly divided into sham operation group,model group,electroacupuncture group,LY294002 group,LY294002+electroacupuncture group,with 6 rats in each group.Before modeling,the electroacupuncture group was treated with electroacupuncture at Neiguan acupoints,the LY294002 group was injected intraperitoneally with PI3K/Akt inhibitor LY294002,the LY294002+electroacupuncture group was treated with electroacupuncture at Neiguan acupoints and intraperitoneal injection of LY294002,and the sham operation group and model group were injected intraperitoneally with the same amount of normal saline,all administered 1 time/d,continuously treated for 7 days.On the 8th day,the rats of all groups except for the sham operation group were treated with coronary artery ligation to establish models of myocardial ischemia-reperfusion.After 1h of reperfusion,the pathological morphology of cardiomyocytes was observed by HE staining,the expressions of p-PI3K,p-Akt,Bcl-2-interacting protein 1(Beclin1),and microtubule-associated protein 1 light chain type 3Ⅱ(LC3Ⅱ)in myocardial tissues were detected by and Western blot method.Results The myocardial injury in model group was severe,myocardial cells were swollen and arranged disordered,myocardial fibers were turbid,myofibrils were not clear,and interstitial hemorrhage and edema were present.The myocardial injury was minor in the electroacupuncture group,and the cardiomyocyte structure remained clear.The myocardial damage was serious in the LY294002 group,myocardial cells were swollen,muscle fiber space was significantly widened,arrangement was disordered,mitochondrial swelling,autophagic vesicles existed,and multiple bleeding and edema could be seen in the interstitium cells.The myocardial injury in the LY294002+electroacupuncture group was mild,some myocardial cells were swollen,and the myocardial fibers were not arranged neatly.Compared with the sham operation group,the relative expressions of Beclin 1 and LC3Ⅱproteins in myocardial tissues were increased significantly,while the relative expressions of p-PI3K and p-Akt in myocardial tissues were decreased significantly in the rats of the model group,LY294002 group,LY294002+electroacupuncture group(all P<0.05).Compared with the model group,LY294002 group,LY294002+electroacupuncture group,the relative expressions of Beclin 1 and LC3Ⅱproteins in myocardial tissues were decreased significantly,while the relative expressions of p-PI3K and p-Akt in myocardial tissues were increased significantly in the rats of the electroacupuncture group(all P<0.05).Compared with the LY294002 group,the relative expressions of Beclin 1 and LC3Ⅱproteins in myocardial tissues were decreased significantly,while the relative expressions of p-PI3K and p-Akt in myocardial tissues were increased significantly in the rats of the LY294002+electroacupuncture group(all P<0.05).Conclusion The pretreatment with electroacupuncture at Neiguan acupoint could regulate the level of myocardial autophagy and inhibit excessive autophagy in cardiomyocytes during ischemia-reperfusion by activating the PI3K/Akt/mTOR signaling pathway,thereby protecting myocardial tissues in rats with myocardial ischemia-reperfusion injury.
作者
李丹
贺春艳
张燚格
何妙嬴
柳冬冬
王超
LI Dan;HE ChunYan;ZHANG Yige;HE Miaoying;LIU Dongdong;WANG Chao(College of Acupuncture Moxibustion and Tuina,Hunan University of Chinese Medicine,Changsha 410208,Hunan,China)
出处
《现代中西医结合杂志》
CAS
2023年第22期3109-3113,3120,共6页
Modern Journal of Integrated Traditional Chinese and Western Medicine
基金
湖南省自然科学基金资助项目(2021JJ30507)
湖南省教育厅优秀青年项目(23B1113)。
