线粒体自噬相关蛋白在阿霉素诱导的心脏毒性中的表达及意义
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摘要
目的探讨线粒体自噬相关蛋白在阿霉素诱导的急性心肌损伤中的表达及意义。方法将40只8～10周龄的雄性野生型C57小鼠随机分为对照组和阿霉素组,每组各20只,阿霉素组小鼠腹腔注射大剂量阿霉素(15mg/kg)诱导急性心肌损伤模型,对照组小鼠给予腹腔注射等量生理盐水。各组于第6天进行心功能检测并取材。分别采用RT-PCR和免疫印迹法检测PTEN诱导性激酶蛋白1(PINK1)、Parkin、自噬标记轻链3(LC3)、Bax、Bcl-2、半胱氨酸天冬氨酸特异性蛋白酶3(C-caspase3)mRNA和蛋白表达水平。苏木精-伊红染色检测各组心肌损伤,TUNEL染色检测各组凋亡心肌细胞数量。结果与对照组比较,阿霉素组左心室舒张末期内径[(4.32±0.50)mmvs (3.85±0.30)mm]、左心室收缩末期内径[(3.34±0.40)mmvs (2.56±0.19)mm]和舒张末期室间隔厚度增加[(0.76±0.12)mmvs (0.48±0.09)mm,P<0.05],左心室短轴缩短率降低[(26.0±2.7)%vs (37.0±4.0)%,P<0.05]。阿霉素组小鼠心肌PINK1、LC3、Bax和C-caspase3mRNA和蛋白表达水平均增加(P<0.05),Parkin和Bcl-2mRNA和蛋白表达水平均降低(P<0.05)。苏木精-伊红染色显示,阿霉素组心肌细胞排列紊乱,并出现细胞质空泡化现象。TUNEL染色显示,阿霉素组细胞核呈致密浓染、凋亡心肌细胞较对照组明显增多(P<0.05)。结论线粒体自噬介导细胞凋亡参与阿霉素诱导的急性心肌损伤的过程。
Objective To study the expression of mitochondrial autophagy-associated protein and its significance in doxorubicin-induced acute myocardial injury.Methods Forty male wild C57 mice aged 8-10 weeks were divided into control group(n=20)and doxorubicin treatment group(n=20).A mouse acute myocardial injury model was induced by intraperitoneal injection with doxorubicin(15 mg/kg).The animals in control group received intraperitoneal injection with normal saline.Their cardiac function was tested with myocardial tissue sampled.Expressions of PINK1,Parkin,LC3,Bax,Bcl-2 and C-caspase 3 mRNA and protein in two groups were detected by RT-PCR and Western blot respectively.Myocardial injury was detected with HE staining,and apoptotic myocytes were calculated with TUNEL staining.Results The LVEDD and LVESD were significantly longer(4.32±0.50 mmvs 3.85±0.30 mm,3.34±0.40 mmvs 2.56±0.19 mm,P<0.05),the end-diastolic interventricular septum thickness was significantly thicker while the the shortened rate of left ventricular minor axis was lower in doxorubicin treatment group than in control group(0.76±0.12 mmvs 0.48±0.09 mm,P<0.05,26.0±2.7%vs 37.0±4.0%,P<0.05).The expression levels of PINK1,LC3,Bax and C-caspase 3 mRNA and protein were significantly higher while those of Parkin and Bcl-2 mRNA and protein were significantly lower in doxorubicin treatment group than in control group(P<0.05).HE staining showed that the myocardial cells were deranged with vacuolization of cytoplasm and TUNEL staining revealed that the number of densely stained apoptotic myocytes was greater in doxorubicin treatment group than in control group(P<0.05).Conclusion Mitochondrial autophagy-mediated apoptosis of myocytes is involved in doxorubicin-induced acute myocardial injury.
Objective To study the expression of mitochondrial autophagy-associated protein and its significance in doxorubicin-induced acute myocardial injury.Methods Forty male wild C57 mice aged 8-10 weeks were divided into control group(n=20)and doxorubicin treatment group(n=20).A mouse acute myocardial injury model was induced by intraperitoneal injection with doxorubicin(15 mg/kg).The animals in control group received intraperitoneal injection with normal saline.Their cardiac function was tested with myocardial tissue sampled.Expressions of PINK1,Parkin,LC3,Bax,Bcl-2 and C-caspase 3 mRNA and protein in two groups were detected by RT-PCR and Western blot respectively.Myocardial injury was detected with HE staining,and apoptotic myocytes were calculated with TUNEL staining.Results The LVEDD and LVESD were significantly longer(4.32±0.50 mmvs 3.85±0.30 mm,3.34±0.40 mmvs 2.56±0.19 mm,P<0.05),the end-diastolic interventricular septum thickness was significantly thicker while the the shortened rate of left ventricular minor axis was lower in doxorubicin treatment group than in control group(0.76±0.12 mmvs 0.48±0.09 mm,P<0.05,26.0±2.7%vs 37.0±4.0%,P<0.05).The expression levels of PINK1,LC3,Bax and C-caspase 3 mRNA and protein were significantly higher while those of Parkin and Bcl-2 mRNA and protein were significantly lower in doxorubicin treatment group than in control group(P<0.05).HE staining showed that the myocardial cells were deranged with vacuolization of cytoplasm and TUNEL staining revealed that the number of densely stained apoptotic myocytes was greater in doxorubicin treatment group than in control group(P<0.05).Conclusion Mitochondrial autophagy-mediated apoptosis of myocytes is involved in doxorubicin-induced acute myocardial injury.
引文
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[18]Przyklenk K,Dong Y,Undyala VV,et al.Autophagy as a therapeutic target for ischaemia/reperfusion injury?Concepts,controversies,and challenges[J].Cardiovasc Res,2012,94(2):197-205.DOI:10.1093/cvr/cvr358.
[2]Zhou T,Chuang CC,Zuo L.Molecular characterization of reactive oxygen species in myocardial ischemia-reperfusion injury[J].Biomed Res Int,2015,2015(3):864946.DOI:10.1155/2015/864946.
[3]Nakamura S,Yoshimori T.Autophagy and longevity[J].Mol Cells,2018,41(1):65-72.DOI:10.14348/molcells.2018.2333.
[4]Randow F,Youle RJ.Self and nonself:how autophagy targets mitochondria and bacteria[J].Cell Host Microbe,2014,15(4):403-411.DOI:10.1016/j.chom.2014.03.012.
[5]Sekine S,Youle RJ.PINK1import regulation;a fine system to convey mitochondrial stress to the cytosol[J].BMC Biol,2018,16(1):2.DOI:10.1186/s12915-017-0470-7.
[6]Kang C,Badr MA,Kyrychenko V,et al.Deficit in PINK1/PARKIN-mediated mitochondrial autophagy at late stages of dystrophic cardiomyopathy[J].Cardiovasc Res,2018,114(1):90-102.DOI:10.1093/cvr/cvx201.
[7]Yang F,Li T,Dong Z,et al.MicroRNA-410is involved in mitophagy after cardiac ischemia/reperfusion injury by targeting high-mobility group box 1protein[J].J Cell Biochem,2018,119(2):2427-2439.DOI:10.1002/jcb.26405.
[8]Wang S,Zhao Z,Fan Y,et al.Mst1inhibits Sirt3expression and contributes to diabetic cardiomyopathy through inhibiting Parkin-dependent mitophagy[J].Biochim Biophys Acta Mol Basis Dis,2018,1864(9):4439.DOI:10.1016/j.bbadis.2018.04.009.
[9]Oka SI.Protective role of Pink1-mediated mitophagy in angiotensinⅡ-induced cardiac injury[J].Int J Cardiol,2018,266:218-219.DOI:10.1016/j.ijcard.2018.04.039.
[10]Cao Y,Ruan Y,Shen T,et al.Astragalus polysaccharide suppresses doxorubicin-induced cardiotoxicity by regulating the PI3k/Akt and p38MAPK pathways[J].Oxid Med Cell Longev,2014,2014(2):674219.DOI:10.1155/2014/674219.
[11]Nitiss KC,Nitiss JL.Twisting and ironing:doxorubicin cardiotoxicity by mitochondrial DNA damage[J].Clin Cancer Res,2014,20(18):4737-4739.DOI:10.1158/1078-0432.CCR-14-0821.
[12]Morton AB,Mor Huertas A,Hinkley JM,et al.Mitochondrial accumulation of doxorubicin in cardiac and diaphragm muscle following exercise preconditioning[J].Mitochondrion,2018,38(2):25-30.DOI:10.1016/j.mito.2018.02.005.
[13]Zhang YW,Shi J,Li YJ,et al.Cardiomyocyte death in doxorubicin-induced cardiotoxicity[J].Arch Immunol Ther Exp(Warsz),2009,57(6):435-445.DOI:10.1007/s00005-009-0051-8.
[14]Yamaguchi O,Murakawa T,Nishida K,et al.Receptor-mediated mitophagy[J].J Mol Cell Cardiol,2016,95:50-56.DOI:10.1016/j.yjmcc.2016.03.010.
[15]Moyzis AG,Sadoshima J,Gustafsson AB.Mending a broken heart:the role of mitophagy in cardioprotection[J].Am JPhysiol Heart Circ Physiol,2015,308(3):H183-H192.DOI:10.1152/ajpheart.00708.2014.
[16]Jin SM,Youle RJ.The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria[J].Autophagy,2013,9(11):1750-1757.DOI:10.4161/auto.26122.
[17]赵品,高金鉴,姜静,等.脂多糖诱导脓毒症小鼠心肌细胞及线粒体自噬[J].细胞与分子免疫学杂志,2016,32(2):177-181.DOI:10.3760/cma.j.issn.2095-4352.2018.02.019.
[18]Przyklenk K,Dong Y,Undyala VV,et al.Autophagy as a therapeutic target for ischaemia/reperfusion injury?Concepts,controversies,and challenges[J].Cardiovasc Res,2012,94(2):197-205.DOI:10.1093/cvr/cvr358.