过表达前脑啡肽原(PPENK)增强线粒体自噬减轻大鼠心肌缺血再灌注损伤
|
摘要
目的探讨前脑啡肽原-最低限度免疫定义基因表达-核定位信号(PPENK-MIDGE-NLS)基因载体后处理对大鼠心肌缺血再灌注线粒体自噬的影响。方法成年雄性SD大鼠随机分为假手术对照组(sham)组:开胸不进行冠状动脉左前降支(LAD)血流阻断及再灌注;缺血再灌注(I/R)组、PPENK-MLDGE(MIDGE)-NLS载体(PPENK)组及Control-MIDGE-NLS载体(对照)组均阻断LAD 30 min,分别于再灌注前给予生理盐水、PPENK-MIDGE-NLS 200μg及Control-MIDGE-NLS 200μg,股静脉注射各1.5 m L。再灌注24 h后取材。ELISA测定血浆心肌肌钙蛋白I(c Tn I)含量;2,3,5-氯化三苯基四氮唑(TTC)法测定心肌梗死面积;透射电镜下观察心肌细胞超微结构及线粒体自噬并分析线粒体损伤评分;Western blot法检测线粒体自噬相关蛋白微管相关蛋白1轻链3B(LC3B)、p62、线粒体外膜转位酶20(TOM20)、PTEN诱导推定激酶1(PINK1)、帕金森病蛋白(parkin)水平。结果 PPENK组血浆c Tn I含量降低,心肌梗死面积减少,线粒体损伤评分改善,心肌组织及线粒体超微结构明显改善,线粒体自噬体增多。线粒体PINK1、parkin、p62、LC3B表达增强。对照组与I/R组间无显著性差异。结论 PPENK-MIDGENLS基因载体处理后可减轻大鼠心肌I/R损伤,可能与增强线粒体自噬,保护线粒体结构完整有关。
Objective To investigate the effect of preproenkephalin-minimalistic immunologically defined gene expression-nuclear localization signal( PPENK-MIDGE-NLS) vector postconditioning on mitophagy during myocardial ischemia reperfusion in rats. Methods Forty male SD rats were randomly divided into 4 groups: sham operation group,ischemia reperfusion group,PPENK-MIDGE-NLS group and Control-MIDGE-NLS group. Myocardial ischemia reperfusion injury model was induced by ligating left anterior descending branch of coronary artery( LAD). Sham operation group was treated identically with the ischemia reperfusion group except that LAD was not tied and occluded. Ischemia reperfusion group,PPENK-MIDGE-NLS group,and Control-MIDGE-NLS group were treated with ligation and occlusion of LAD for 30 minutes followed by 24 hour-reperfusion,with 1. 5 m L saline,or 1. 5 m L PPENK-MIDGE-NLS vectors(200 μg),or 1. 5 m L ControlMIDGE-NLS vectors(200 μg) administered respectively right before reperfusion started. Serum c Tn I was assayed by ELISA and myocardial infarct size was measured by TTC. Ultrastructural changes of mitochondria and mitophagy were observed using electron microscope,and mitochondrial damage scores were analyzed. Expressions of the mitophagy-related proteins such as PINK1,parkin,p62,TOM20 and LC3 B were measured by Western blotting. Results Compared with the ischemia reperfusion group,both serum c Tn I content and myocardial infarct size in the PPENK group decreased; the expression of PINK1,parkin,p62 and LC3 B were all up-regulated. Mitophagy was enhanced and mitochondrial damages were alleviated,with conspicuous improvement in mitochondria ultrastructure. Conclusion PPENK-MIDGE-NLS vector postconditioning can mitigate myocardial ischemia reperfusion injury by promoting mitophagy in rats.
Objective To investigate the effect of preproenkephalin-minimalistic immunologically defined gene expression-nuclear localization signal( PPENK-MIDGE-NLS) vector postconditioning on mitophagy during myocardial ischemia reperfusion in rats. Methods Forty male SD rats were randomly divided into 4 groups: sham operation group,ischemia reperfusion group,PPENK-MIDGE-NLS group and Control-MIDGE-NLS group. Myocardial ischemia reperfusion injury model was induced by ligating left anterior descending branch of coronary artery( LAD). Sham operation group was treated identically with the ischemia reperfusion group except that LAD was not tied and occluded. Ischemia reperfusion group,PPENK-MIDGE-NLS group,and Control-MIDGE-NLS group were treated with ligation and occlusion of LAD for 30 minutes followed by 24 hour-reperfusion,with 1. 5 m L saline,or 1. 5 m L PPENK-MIDGE-NLS vectors(200 μg),or 1. 5 m L ControlMIDGE-NLS vectors(200 μg) administered respectively right before reperfusion started. Serum c Tn I was assayed by ELISA and myocardial infarct size was measured by TTC. Ultrastructural changes of mitochondria and mitophagy were observed using electron microscope,and mitochondrial damage scores were analyzed. Expressions of the mitophagy-related proteins such as PINK1,parkin,p62,TOM20 and LC3 B were measured by Western blotting. Results Compared with the ischemia reperfusion group,both serum c Tn I content and myocardial infarct size in the PPENK group decreased; the expression of PINK1,parkin,p62 and LC3 B were all up-regulated. Mitophagy was enhanced and mitochondrial damages were alleviated,with conspicuous improvement in mitochondria ultrastructure. Conclusion PPENK-MIDGE-NLS vector postconditioning can mitigate myocardial ischemia reperfusion injury by promoting mitophagy in rats.
引文
[1]曾萍,杨简.线粒体相关性药物在冠心病治疗中的研究进展[J].实用医学杂志,2015,31(24):4139-4140.
[2]Bartz R R,Suliman H B,Piantadosi C A.Redox mechanisms of cardiomyocyte mitochondrial protection[J/OL].Front Physiol,2015,6:291.doi:10.3389/fphys.2015.00291.e Collection 2015.
[3]Ji W,Wei S,Hao P,et al.Aldehyde dehydrogenase 2 has cardioprotective effects on myocardial ischaemia/reperfusion injury via suppressing mitophagy[J/OL].Front.Pharmacol,2016,7:101.doi:10.3389/fphar.2016.00101.
[4]Zhang W,Ren H,Xu C,et al.Hypoxic mitophagy regulates mitochondrial quality and platelet activation and determines severity of I/R heart injury[J/OL].ELife,2016,5:e21407.e21407.doi:10.7554/e Life.21407.
[5]Vásquez-Trincado C,García-Carvajal,Pennanen C,et al.Mitochondrial dynamics,mitophagy and cardiovascular disease[J].J Physiol,2016,594(3):509-525.
[6]Huang C,Andres A M,Ratliff E P,et al.Preconditioning involves selective mitophagy mediated by parkin and p62/SQSTM1[J/OL].PLo S One,2011,6(6):e20975.doi:10.1371/journal.pone.0020975.Epub 2011 Jun 8.
[7]Headrick J P,See Hoe L E,Du Toit E F,et al.Opioid receptors and cardioprotection-‘opioidergic conditioning’of the heart[J].Br J Pharmacol,2015,172(8):2026-2050.
[8]陈曦,徐学敏,彭细娟,等.PPENK-MIDGE-NLS基因载体的构建及其在活体大鼠的表达[J].生物工程学报,2015,31(2):258-268.
[9]姜静,韩瑞丽,陈曦,等.前脑啡肽原基因转染对大鼠心肌缺血再灌注损伤的影响[J].国际麻醉学与复苏杂志,2017,38(5):390-396.
[10]Flameng W,Borgers M,Daenen W,et al.Ultrastructural and cytochemical correlates of myocardial protection by cardiac hypothermia in man[J].J Thorac Cardiovasc Surg,1980,79(3):413-424.
[11]Evans T D,Sergin I,Zhang X,et al.Target acquired:selective autophagy in cardiometabolic disease[J/OL].Sci Signal,2017,10(468):eaag2298.doi:10.1126/scisignal.aag2298.
[12]Moyzis A G,Sadoshima J,GustafssonB.Mending a broken heart:the role of mitophagy in cardioprotection[J].Am J Physiol Heart Circ Physiol,2015,308(3):H183-H192.
[13]Yu P,Zhang J,Yu S,et al.Protective effect of sevoflurane postconditioning against cardiac ischemia/reperfusion injury via ameliorating mitochondrial impairment,oxidative stress and rescuing autophagic clearance[J/OL].PLo S One,2015,10(8):e0134666.doi:10.1371/journal.pone.0134666.e Collection 2015.
[14]Hamacher-Brady A,Brady N R.Mitophagy programs:mechanisms and physiological implications of mitochondrial targeting by autophagy[J].Cell Mol Life Sci,2016,73(4):775-795.
[15]Kubli D A,GustafssonB.Mitochondria and mitophagy:The Yin and Yang of cell death control[J].Circ Res,2012,111(9):1208-1221.
[16]Zhu J,Wang K Z,Chu C T.After the banquet:Mitochondrial biogenesis,mitophagy,and cell survival[J].Autophagy,2013,9(11):1663-1676.
[17]Maslov L N,Khaliulin I,Oeltgen P R,et al.Prospects for creation of cardioprotective and antiarrhythmic drugs based on opioid receptor agonists[J].Med Res Rev,2016,36(5):871-923.
[18]王静,王玲,王自彬.线粒体自噬过程的超微结构分析[J].中国细胞生物学学报,2017,39(4):435-439.
[19]王自彬,王静,王玲,等.自噬小体的超微结构分析[J].南京医科大学学报,2016,36(4):426-429.
[20]Dorn G W 2nd.Parkin-dependent mitophagy in the heart[J].J Mol Cell Cardiol,2016,95:42-49.
[21]Siddall H K,Yellon D M,Ong S B,et al.Loss of PINK1 increases the heart’s vulnerability to ischemia-reperfusion injury[J/OL].PLo SOne,2013,8(4):e62400.doi:10.1371/journal.pone.0062400.Print 2013.
[22]Kubli D A,Zhang X,Lee Y,et al.Parkin protein deficiency exacerbates cardiac injury and reduces survival following myocardial infarction[J].J Biol Chem,2013,288(2):915-926.
[23]Ikeda Y,Shirakabe A,Maejima Y,et al.Endogenous Drp1 mediates mitochondrial autophagy and protects the heart against energy stress[J].Circ Res,2015,116(2):264-278.
[24]Suliman H B,Keenan J E,Piantadosi C A.Mitochondrial quality-control dysregulation in conditional HO-1-/-mice[J/OL].JCI Insight,2017,2(3):e89676.doi:10.1172/jci.insight.89676.
[25]Andres A M,Tucker K C,Thomas A,et al.Mitophagy and mitochondrial biogenesis in atrial tissue of patients undergoing heart surgery with cardiopulmonary bypass[J/OL].JCI Insight,2017,2(4):e89303.doi:org/10.1172/jci.insight.89303.
[2]Bartz R R,Suliman H B,Piantadosi C A.Redox mechanisms of cardiomyocyte mitochondrial protection[J/OL].Front Physiol,2015,6:291.doi:10.3389/fphys.2015.00291.e Collection 2015.
[3]Ji W,Wei S,Hao P,et al.Aldehyde dehydrogenase 2 has cardioprotective effects on myocardial ischaemia/reperfusion injury via suppressing mitophagy[J/OL].Front.Pharmacol,2016,7:101.doi:10.3389/fphar.2016.00101.
[4]Zhang W,Ren H,Xu C,et al.Hypoxic mitophagy regulates mitochondrial quality and platelet activation and determines severity of I/R heart injury[J/OL].ELife,2016,5:e21407.e21407.doi:10.7554/e Life.21407.
[5]Vásquez-Trincado C,García-Carvajal,Pennanen C,et al.Mitochondrial dynamics,mitophagy and cardiovascular disease[J].J Physiol,2016,594(3):509-525.
[6]Huang C,Andres A M,Ratliff E P,et al.Preconditioning involves selective mitophagy mediated by parkin and p62/SQSTM1[J/OL].PLo S One,2011,6(6):e20975.doi:10.1371/journal.pone.0020975.Epub 2011 Jun 8.
[7]Headrick J P,See Hoe L E,Du Toit E F,et al.Opioid receptors and cardioprotection-‘opioidergic conditioning’of the heart[J].Br J Pharmacol,2015,172(8):2026-2050.
[8]陈曦,徐学敏,彭细娟,等.PPENK-MIDGE-NLS基因载体的构建及其在活体大鼠的表达[J].生物工程学报,2015,31(2):258-268.
[9]姜静,韩瑞丽,陈曦,等.前脑啡肽原基因转染对大鼠心肌缺血再灌注损伤的影响[J].国际麻醉学与复苏杂志,2017,38(5):390-396.
[10]Flameng W,Borgers M,Daenen W,et al.Ultrastructural and cytochemical correlates of myocardial protection by cardiac hypothermia in man[J].J Thorac Cardiovasc Surg,1980,79(3):413-424.
[11]Evans T D,Sergin I,Zhang X,et al.Target acquired:selective autophagy in cardiometabolic disease[J/OL].Sci Signal,2017,10(468):eaag2298.doi:10.1126/scisignal.aag2298.
[12]Moyzis A G,Sadoshima J,GustafssonB.Mending a broken heart:the role of mitophagy in cardioprotection[J].Am J Physiol Heart Circ Physiol,2015,308(3):H183-H192.
[13]Yu P,Zhang J,Yu S,et al.Protective effect of sevoflurane postconditioning against cardiac ischemia/reperfusion injury via ameliorating mitochondrial impairment,oxidative stress and rescuing autophagic clearance[J/OL].PLo S One,2015,10(8):e0134666.doi:10.1371/journal.pone.0134666.e Collection 2015.
[14]Hamacher-Brady A,Brady N R.Mitophagy programs:mechanisms and physiological implications of mitochondrial targeting by autophagy[J].Cell Mol Life Sci,2016,73(4):775-795.
[15]Kubli D A,GustafssonB.Mitochondria and mitophagy:The Yin and Yang of cell death control[J].Circ Res,2012,111(9):1208-1221.
[16]Zhu J,Wang K Z,Chu C T.After the banquet:Mitochondrial biogenesis,mitophagy,and cell survival[J].Autophagy,2013,9(11):1663-1676.
[17]Maslov L N,Khaliulin I,Oeltgen P R,et al.Prospects for creation of cardioprotective and antiarrhythmic drugs based on opioid receptor agonists[J].Med Res Rev,2016,36(5):871-923.
[18]王静,王玲,王自彬.线粒体自噬过程的超微结构分析[J].中国细胞生物学学报,2017,39(4):435-439.
[19]王自彬,王静,王玲,等.自噬小体的超微结构分析[J].南京医科大学学报,2016,36(4):426-429.
[20]Dorn G W 2nd.Parkin-dependent mitophagy in the heart[J].J Mol Cell Cardiol,2016,95:42-49.
[21]Siddall H K,Yellon D M,Ong S B,et al.Loss of PINK1 increases the heart’s vulnerability to ischemia-reperfusion injury[J/OL].PLo SOne,2013,8(4):e62400.doi:10.1371/journal.pone.0062400.Print 2013.
[22]Kubli D A,Zhang X,Lee Y,et al.Parkin protein deficiency exacerbates cardiac injury and reduces survival following myocardial infarction[J].J Biol Chem,2013,288(2):915-926.
[23]Ikeda Y,Shirakabe A,Maejima Y,et al.Endogenous Drp1 mediates mitochondrial autophagy and protects the heart against energy stress[J].Circ Res,2015,116(2):264-278.
[24]Suliman H B,Keenan J E,Piantadosi C A.Mitochondrial quality-control dysregulation in conditional HO-1-/-mice[J/OL].JCI Insight,2017,2(3):e89676.doi:10.1172/jci.insight.89676.
[25]Andres A M,Tucker K C,Thomas A,et al.Mitophagy and mitochondrial biogenesis in atrial tissue of patients undergoing heart surgery with cardiopulmonary bypass[J/OL].JCI Insight,2017,2(4):e89303.doi:org/10.1172/jci.insight.89303.