电针、艾灸预处理对心肌缺血大鼠心肌细胞凋亡和自噬的影响
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摘要
目的:观察针灸预处理对心肌缺血大鼠心肌细胞凋亡指数和自噬相关蛋白LC3表达的影响,探讨针灸预处理对心肌缺血大鼠心肌细胞自噬能力的预保护作用。方法:SD大鼠随机分为假手术组、模型组、缺血预适应(IP)组、电针组、艾灸组,每组8只。电针组及艾灸组造模前分别电针或艾灸"内关"穴,每天1次,每次20min,连续7d。采用冠状动脉结扎术复制心肌缺血模型。电镜观察左心室缺血区组织病理形态学及心肌细胞自噬体的变化,TUNEL法检测心肌细胞凋亡情况及凋亡指数,Western blot法检测LC3蛋白在心肌组织中的表达情况。结果:假手术组心肌细胞形态完整,排列整齐,线粒体较完整,可见少量自噬空泡;与假手术组相比,模型组心肌细胞形态不完整,排列紊乱,肌纤维溶解,线粒体肿大,胞核边聚化,自噬空泡较多;与模型组相比,IP组、电针组、艾灸组心肌细胞损伤较轻,少量肌纤维溶解,线粒体丰富,自噬空泡及溶酶体数量增多。与假手术组比较,模型组凋亡指数升高(P<0.05);与模型组相比,IP组、电针组、艾灸组凋亡指数均降低(P<0.05);与IP组、艾灸组比较,电针组凋亡指数降低(P<0.05)。与假手术组比较,模型组心肌组织LC3-Ⅱ表达水平、LC3-Ⅱ/Ⅰ比值均升高(P<0.05);与模型组比较,IP组、电针组及艾灸组心肌组织LC3-Ⅱ表达水平及LC3-Ⅱ/Ⅰ比值均降低(P<0.05);与IP组、艾灸组比较,电针组LC3-Ⅱ的表达及LC3-Ⅱ/Ⅰ比值上调(P<0.05)。结论:针灸预处理能产生与缺血预适应相似的心脏保护作用,适度提高细胞自噬能力,减少细胞凋亡。
Objective To observe the effect of "Neiguan"(PC6)-electroacupuncture(EA)or moxibustion(Moxi)pretreatment on myocardial apoptosis and expression of autophagy related proteins light chain(LC)3-Ⅰand LC3-Ⅱin rats with myocardial ischemia/reperfusion injury(MIRI),so as to explore their mechanisms underlying improvement of MIRI.Methods Forty SD rats(half male and half female)were randomly divided into sham operation,model,ischemic preconditioning(IP),EA and Moxi groups(n=8 in each group).EA(10 Hz/50 Hz,1 mA)or Moxi(ignited moxa stick)was respectively applied to bilateral "Neiguan"(PC6)for 20 min,once daily for 7 days.The MIRI model was established by occlusion of the anterior descending branch of the left coronary artery for 40 min,followed by reperfusion for 60 min.The ultrastructural changes and autophagy of myocardial cells were observed by electron microscopy(EM),and the myocardial cellular apoptosis[apoptotic index=(number of apoptotic cells/total number of cardiomyocytes)×100%]was detected by the terminal deoxyribonucleotidyl transferase mediated dUTP nick end labelling(TUNEL)method.The expressions of LC3-Ⅰand LC3-Ⅱproteins(markers for autophagy)in myocardial tissue were detected by Western blot.Results Following MI,EM observation revealed a vague structure of cardiomyocytes and muscular horizontal grain,dissolution of myofibers,mitochondrial swelling,some autophagic vacuoles and autophagic lysosomes at different degrees and surrounded by a double membrane in the model group,these situations were apparently milder in the EA and Moxi groups.The apoptosis index,myocardial LC3-Ⅰ and LC3-Ⅱ protein expression levels,and the ratio of LC3-Ⅱ/Ⅰwere significantly increased in the model group relevant to the sham operation group(P<0.05).After the treatment,the apoptosis index,the expression level of myocardial LC3-Ⅱ protein and the ratio of LC3-Ⅱ/Ⅰ were considerably down-regulated in the IP,EA and Moxi groups in comparison with those in the model group(P<0.05).The effect of EA was obviously superior to those of IP and Moxi in down-regulating the apoptosis index(P<0.05),but obviously inferior to those of IP and Moxi in down-regulating the levels of LC3-Ⅱ and LC3-Ⅱ/Ⅰ(P<0.05).No significant changes were found in the expression of LC3-Ⅰ after IP,EA and Moxi interventions in comparison with the model group(P>0.05),and no significant differences were observed in the apoptosis index and levels of LC3-Ⅱ and LC3-Ⅱ/Ⅰ between the IP and Moxi groups(P>0.05).Conclusion Both EA and moxibustion pretreatments,similar to IP,have a positive role in reducing myocardiocyte apoptosis and regulating autophagy-related protein expression in MIRI rats,which maybe contribute to their protective effects on ischemic myocardium.
Objective To observe the effect of "Neiguan"(PC6)-electroacupuncture(EA)or moxibustion(Moxi)pretreatment on myocardial apoptosis and expression of autophagy related proteins light chain(LC)3-Ⅰand LC3-Ⅱin rats with myocardial ischemia/reperfusion injury(MIRI),so as to explore their mechanisms underlying improvement of MIRI.Methods Forty SD rats(half male and half female)were randomly divided into sham operation,model,ischemic preconditioning(IP),EA and Moxi groups(n=8 in each group).EA(10 Hz/50 Hz,1 mA)or Moxi(ignited moxa stick)was respectively applied to bilateral "Neiguan"(PC6)for 20 min,once daily for 7 days.The MIRI model was established by occlusion of the anterior descending branch of the left coronary artery for 40 min,followed by reperfusion for 60 min.The ultrastructural changes and autophagy of myocardial cells were observed by electron microscopy(EM),and the myocardial cellular apoptosis[apoptotic index=(number of apoptotic cells/total number of cardiomyocytes)×100%]was detected by the terminal deoxyribonucleotidyl transferase mediated dUTP nick end labelling(TUNEL)method.The expressions of LC3-Ⅰand LC3-Ⅱproteins(markers for autophagy)in myocardial tissue were detected by Western blot.Results Following MI,EM observation revealed a vague structure of cardiomyocytes and muscular horizontal grain,dissolution of myofibers,mitochondrial swelling,some autophagic vacuoles and autophagic lysosomes at different degrees and surrounded by a double membrane in the model group,these situations were apparently milder in the EA and Moxi groups.The apoptosis index,myocardial LC3-Ⅰ and LC3-Ⅱ protein expression levels,and the ratio of LC3-Ⅱ/Ⅰwere significantly increased in the model group relevant to the sham operation group(P<0.05).After the treatment,the apoptosis index,the expression level of myocardial LC3-Ⅱ protein and the ratio of LC3-Ⅱ/Ⅰ were considerably down-regulated in the IP,EA and Moxi groups in comparison with those in the model group(P<0.05).The effect of EA was obviously superior to those of IP and Moxi in down-regulating the apoptosis index(P<0.05),but obviously inferior to those of IP and Moxi in down-regulating the levels of LC3-Ⅱ and LC3-Ⅱ/Ⅰ(P<0.05).No significant changes were found in the expression of LC3-Ⅰ after IP,EA and Moxi interventions in comparison with the model group(P>0.05),and no significant differences were observed in the apoptosis index and levels of LC3-Ⅱ and LC3-Ⅱ/Ⅰ between the IP and Moxi groups(P>0.05).Conclusion Both EA and moxibustion pretreatments,similar to IP,have a positive role in reducing myocardiocyte apoptosis and regulating autophagy-related protein expression in MIRI rats,which maybe contribute to their protective effects on ischemic myocardium.
引文
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[14]HAMACHER-BRADY A,BRADYN R,GOTTIEB R A,et al.Autophagy as a protective response to Bnip3-mediated apoptotic signaling in the heart[J].Autophagy,2006,2(4):307-309.
[15]YAN L,VATNER D E,KIM S J,et al.Autophagy in chronically ischemic myocardium[J].Proc Natl Acad Sci USA,2005,102(39):13807-13812.
[16]TAKAGI H,MATSUI Y,SADOSHIMA J.The role of autophagy in mediating cell survival and death during ischemia and reperfusion in the heart[J].Antioxid Redox Signal,2007,9(9):1373-1381.
[17]孙瑶.九龙藤总黄酮对心肌缺血/再灌注损伤细胞自噬及凋亡的作用[D].桂林:桂林医学院,2015.
[18]JIMENEZ R E,KUBLI D A,GUSTAFSSON A B.Autophagy and mitophagy in the myocardium:therapeutic potential and concerns[J].Br J Pharmacol,2014,171(8):1907-1916.
[19]KABEYA Y,MIZUSHIMA N,UENO T,et al.LC3,a mammalian homologue of yeast Apg8p,is localized in autophagosome membranes after processing[J].EMBO J,2000,19(21):5720-5728.
[20]CHERRA S J,KULICH S M,UECHI G.Regulation of the autophagy protein LC3by phosphorylation[J].J Cell Biol,2010,190(4):533-539.
[21]MATSUI Y,TAKAGI H,QU X,et al.Distinct roles autophagy in the heart during ischemia and reperfusion[J].Circ Res,2007,100(6):914-922.
[22]VLEZ D E,HERMANN R,BARREDA FRANK M.Effects of wortmannin on cardioprotection exerted by ischemic preconditioning in rat hearts subjected to ischemia-reperfusion[J].J Physiol Biochem,2016,72(1):83-91.
[2]SZEKERE L.Drug-induced delayed cardiac protection against the effects of myocardial ischemia[J].Pharmacol Ther,2005,108(3):269-280.
[3]OU J J,JIN Z X,GAO F.Remote preconditioning in the heart[J].Prog Physiol,2015,36(3):227-229.
[4]PRZYKLENK K,DARLING C E,DICKSON E W,et al.Cardioprotection‘outside the box’-the evoving paradigm of remote preconditioning[J].Basic Cardiol,2013,98(3):149-157.
[5]阳晶晶,严洁,王超,等.针灸内关预处理对心肌缺血再灌注损伤兔血清NO、NOS及腺苷含量的影响[J].中国中医急症,2014,23(7):1209-1211.
[6]王超,谢文娟,刘密,等.针灸预处理对不同时间心肌缺血再灌注损伤兔血浆内皮素、血清肌酸激酶和心肌组织热休克蛋白70表达的影响[J].针刺研究,2014,39(5):372-376.
[7]谭成富,严洁,王超,等.针灸预处理对心肌缺血再灌注损伤兔不同时间热休克蛋白27、70、90表达的影响[J].针刺研究,2017,42(1):31-38.
[9]KANAMORI H,TAKEMURA G,GOTO K,et al.The role of autophagy emerging in post infarction cardiac remodelling[J].Cardiovascul Res,2011,91(2),330-339.
[10]李忠仁.实验针灸学[M].2版,北京:中国中医药出版社,2007:255-257.
[11]唐春林,谭平,吴文峰.EGFR在大鼠呼吸机肺损伤中的作用研究[J].中国实验诊断学,2012,16(10):1783-1787.
[12]王砚青,江时森,刘平,等.大鼠心肌缺血再灌注模型心电图变化分析[J].医学研究生学报,2006,19(8):700-702.
[13]KROEMER G,LEVINE B.Autophagic cell death:the story of a misnomer[J].Nat Rev Mol Cell Biol,2008,9(12):1004-1010.
[14]HAMACHER-BRADY A,BRADYN R,GOTTIEB R A,et al.Autophagy as a protective response to Bnip3-mediated apoptotic signaling in the heart[J].Autophagy,2006,2(4):307-309.
[15]YAN L,VATNER D E,KIM S J,et al.Autophagy in chronically ischemic myocardium[J].Proc Natl Acad Sci USA,2005,102(39):13807-13812.
[16]TAKAGI H,MATSUI Y,SADOSHIMA J.The role of autophagy in mediating cell survival and death during ischemia and reperfusion in the heart[J].Antioxid Redox Signal,2007,9(9):1373-1381.
[17]孙瑶.九龙藤总黄酮对心肌缺血/再灌注损伤细胞自噬及凋亡的作用[D].桂林:桂林医学院,2015.
[18]JIMENEZ R E,KUBLI D A,GUSTAFSSON A B.Autophagy and mitophagy in the myocardium:therapeutic potential and concerns[J].Br J Pharmacol,2014,171(8):1907-1916.
[19]KABEYA Y,MIZUSHIMA N,UENO T,et al.LC3,a mammalian homologue of yeast Apg8p,is localized in autophagosome membranes after processing[J].EMBO J,2000,19(21):5720-5728.
[20]CHERRA S J,KULICH S M,UECHI G.Regulation of the autophagy protein LC3by phosphorylation[J].J Cell Biol,2010,190(4):533-539.
[21]MATSUI Y,TAKAGI H,QU X,et al.Distinct roles autophagy in the heart during ischemia and reperfusion[J].Circ Res,2007,100(6):914-922.
[22]VLEZ D E,HERMANN R,BARREDA FRANK M.Effects of wortmannin on cardioprotection exerted by ischemic preconditioning in rat hearts subjected to ischemia-reperfusion[J].J Physiol Biochem,2016,72(1):83-91.