线粒体乙醛脱氢酶影响心肌缺血再灌注损伤大鼠心肌线粒体功能和室性心动过速的机制
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摘要
目的探讨线粒体乙醛脱氢酶(ALDH2)影响心肌缺血再灌注损伤(MIRI)大鼠心肌线粒体功能和室性心动过速的机制。方法选取60只健康雄性SD大鼠,随机分为3组:假手术(sham)组、MIRI model组和ALDH2组,每组20只。ALDH2组在建模之前,大鼠给予2. 5%乙醇(乙醛脱氢酶2激动剂)日常饮用,1周后乙醇调整至5%,持续8周。建立大鼠心肌缺血再灌注(MI/R)模型。采用标准肢体Ⅱ导联心电图连续监测大鼠心律失常变化;采用ELISA法测定大鼠心肌组织中超氧化物歧化酶(SOD)和丙二醛(MDA)的含量;采用流式细胞仪测定大鼠心肌线粒体膜电位的变化;采用实时定量PCR(q-PCR)法检测大鼠心肌组织中Bax的m RNA表达;采用Western blot检测大鼠心肌组织中ALDH2和Caspase-3的蛋白表达。结果心律失常测定显示,与sham组相比,model组大鼠室性心动过速的发生率升高(P <0. 01),其持续时间较长(P <0. 01);而ALDH2组大鼠室性心动过速的发生率低于model组(P <0. 01),其持续时间较短(P <0. 01)。ELISA测定显示,与sham组相比,model组大鼠心肌组织中SOD的含量降低(P <0. 01),MDA的含量升高(P <0. 01);而与model组相比,ALDH2组大鼠心肌组织中SOD的含量升高(P <0. 01),MDA的含量降低(P <0. 01)。采用阳离子绿色荧光染料罗丹明123对线粒体的膜电位进行检测,与sham组相比,model组大鼠心肌线粒体膜电位降低(P <0. 01),而ALDH2组大鼠心肌线粒体膜电位较model组升高(P <0. 01)。q-PCR测定显示,与sham组相比,model组大鼠心肌组织中Bax的m RNA表达上调(P <0. 01);而ALDH2组大鼠心肌组织中Bax的m RNA表达较model组降低(P <0. 01)。Western blot测定显示,与sham组相比,model组大鼠心肌组织中ALDH2的蛋白表达下调(P <0. 01),Caspase-3蛋白表达上调(P <0. 01);而ALDH2组大鼠心肌组织中ALDH2的蛋白表达相对于model组升高(P <0. 01),Caspase-3蛋白表达低于model组(P <0. 01)。结论增强ALDH2在MIRI大鼠心肌组织中的表达对MIRI有保护作用,其机制可能与抵抗室性心动过速、缓解氧化应激反应、提高心肌组织中线粒体膜电位及抑制细胞凋亡的发生有关。
Objective To explore the mechanism of mitochondrial acetaldehyde dehydrogenase affecting the myocardial mitochondrial function and ventricular tachycardia in rats with myocardial ischemia reperfusion injury. Methods Sixty healthy male SD rats were selected and randomly divided into 3 groups( n = 20 in each group) : sham group,MIRI model group and ALDH2 group. Before modeling,the rats in ALDH2 group were given 2. 5% ethanol( acetaldehyde dehydrogenase 2 agonist) for daily drinking,and the ethanol was adjusted to 5% after 1 week for 8 weeks. Rat model of myocardial ischemia reperfusion( MI/R) was established. A standard body Ⅱlead electrocardiogram was used to continuously monitor the heart rate in rats. The contents of superoxide dismutase( SOD) and malondialdehyde( MDA) in myocardial tissue of rats were determined by ELISA. The changes of myocardial mitochondrial membrane potential in rats were measured by flow cytometry. Real-time quantitative PCR( q-PCR) was used to detect the m RNA expression levels of Bax in myocardium tissue of rats. Western blot was used to detect the protein expression levels of ALDH22 and caspase-3 in myocardium tissue of rats. Results Compared with sham group,the incidence of arrhythmia was increased in model group( P < 0. 01) and its duration was enlarged( P < 0. 01). However,the incidence of arrhythmia in ALDH2 group was lower than that in model group( P <0. 01),and its duration was shorter( P < 0. 01). ELISA results showed that compared with sham group,the content of SOD in the myocardium tissue was decreased in model group( P < 0. 01),while the content of MDA was increased( P < 0. 01). However,the content of SOD in the myocardium tissue was higher in ALDH2 group than in model group( P < 0. 01),while the content of MDA was lower( P< 0. 01). Cationic green fluorescent dye rhodamine 123 was used to detect the membrane potential of mitochondria. Compared with sham group,the myocardial mitochondrial membrane potential of rats was decreased in model group( P < 0. 01). The myocardial mitochondrial membrane potential in ALDH2 group was higher than that in model group( P < 0. 01). The results of q-PCR showed that compared with sham group,the m RNA expression level of Bax was increased in model group( P < 0. 01). However,the m RNA expression level of Bax was lower in ALDH2 group than in model group( P < 0. 01). Western blot results showed that compared with sham group,the protein expression level of ALDH2 was decreased in model group( P < 0. 01),while that of Caspase-3 was increased( P < 0. 01).However,the protein expression of ALDH2 was higher in ALDH2 group than in model group( P < 0. 01),while the protein expression of Caspase-3 was lower( P < 0. 01). Conclusion Enhanced ALDH2 expression in myocardial tissue of MIRI rats has a significant protective effect against MIRI by resisting ventricular tachycardia,relieving oxidative stress,increasing mitochondrial membrane potential in cardiac tissues and inhibiting apoptosis.
Objective To explore the mechanism of mitochondrial acetaldehyde dehydrogenase affecting the myocardial mitochondrial function and ventricular tachycardia in rats with myocardial ischemia reperfusion injury. Methods Sixty healthy male SD rats were selected and randomly divided into 3 groups( n = 20 in each group) : sham group,MIRI model group and ALDH2 group. Before modeling,the rats in ALDH2 group were given 2. 5% ethanol( acetaldehyde dehydrogenase 2 agonist) for daily drinking,and the ethanol was adjusted to 5% after 1 week for 8 weeks. Rat model of myocardial ischemia reperfusion( MI/R) was established. A standard body Ⅱlead electrocardiogram was used to continuously monitor the heart rate in rats. The contents of superoxide dismutase( SOD) and malondialdehyde( MDA) in myocardial tissue of rats were determined by ELISA. The changes of myocardial mitochondrial membrane potential in rats were measured by flow cytometry. Real-time quantitative PCR( q-PCR) was used to detect the m RNA expression levels of Bax in myocardium tissue of rats. Western blot was used to detect the protein expression levels of ALDH22 and caspase-3 in myocardium tissue of rats. Results Compared with sham group,the incidence of arrhythmia was increased in model group( P < 0. 01) and its duration was enlarged( P < 0. 01). However,the incidence of arrhythmia in ALDH2 group was lower than that in model group( P <0. 01),and its duration was shorter( P < 0. 01). ELISA results showed that compared with sham group,the content of SOD in the myocardium tissue was decreased in model group( P < 0. 01),while the content of MDA was increased( P < 0. 01). However,the content of SOD in the myocardium tissue was higher in ALDH2 group than in model group( P < 0. 01),while the content of MDA was lower( P< 0. 01). Cationic green fluorescent dye rhodamine 123 was used to detect the membrane potential of mitochondria. Compared with sham group,the myocardial mitochondrial membrane potential of rats was decreased in model group( P < 0. 01). The myocardial mitochondrial membrane potential in ALDH2 group was higher than that in model group( P < 0. 01). The results of q-PCR showed that compared with sham group,the m RNA expression level of Bax was increased in model group( P < 0. 01). However,the m RNA expression level of Bax was lower in ALDH2 group than in model group( P < 0. 01). Western blot results showed that compared with sham group,the protein expression level of ALDH2 was decreased in model group( P < 0. 01),while that of Caspase-3 was increased( P < 0. 01).However,the protein expression of ALDH2 was higher in ALDH2 group than in model group( P < 0. 01),while the protein expression of Caspase-3 was lower( P < 0. 01). Conclusion Enhanced ALDH2 expression in myocardial tissue of MIRI rats has a significant protective effect against MIRI by resisting ventricular tachycardia,relieving oxidative stress,increasing mitochondrial membrane potential in cardiac tissues and inhibiting apoptosis.
引文
[1] Calvert JW,Gundewar S,Yamakuchi M,et al. Inhibition of Nethylmaleimide-sensitive factor protects against myocardial ischemia/reperfusion injury[J]. Circ Res,2016,101(12):1247-1254.
[2] He B,Zhao Y,Xu L,et al. The nuclear melatonin receptor ROR±is a novel endogenous defender against myocardial ischemia/reperfusion injury[J]. J Pineal Res,2016,60(3):313-326.
[3] Song M,Lan H. GW27-e1017 Beneficial effects of a polysaccharide from Salvia miltiorrhiza on myocardial ischemia-reperfusion injury in rats[J]. J Am Coll Cardiol,2016,68(16):C65-C65.
[4] Fang SJ,Li PY,Wang CM,et al. Inhibition of endoplasmic reticulum stress by neuregulin-1 protects against myocardial ischemia/reperfusion injury[J]. Peptides,2017,88:196.
[5] Tianxin HU,Guo W,Miaomiao XI,et al. Synergistic cardioprotective effects of Danshensu and hydroxysafflor yellow A against myocardial ischemia-reperfusion injury are mediated through the Akt/Nrf2/HO-1 pathway[J]. Int J Mol Med,2016,38(1):83-94.
[6] Wang Z,Sun Y,Li X,et al. A novel acetaldehyde dehydrogenase with salicylaldehyde dehydrogenase activity from Rhodococcus ruber strain OA1[J]. Curr Microbiol,2017,74(12):1404-1410.
[7] Mcclerklin SA,Lee SG,Harper CP,et al. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC3000[J]. PLo S Pathog,2018,14(1):e1006811.
[8] Xu Y,Lee J,Yang HS,et al. A computational integrating kinetic study on the flexible active site of human acetaldehyde dehydrogenase 1[J]. Process Biochem,2016,51(6):725-733.
[9] Yeh JZ,Byington KH. Interactions of acetaldehyde,ethyl alcohol and oxybarbiturates affecting mitochondrial functions[J].Biochem Pharmacol,2016,22(16):2045-2057.
[10] Nene A,Chen CH,Disatnik MH,et al. Aldehyde dehydrogenase 2 activation and coevolution of itsεPKC-mediated phosphorylation sites[J]. J Biomed Sci,2017,24(1):3.
[11] Bertsch J,Siemund AL,Kremp F,et al. A novel route for ethanol oxidation in the acetogenic bacterium Acetobacterium woodii:the acetaldehyde/ethanol dehydrogenase pathway[J]. Environ Microbiol,2016,18(9):2913-2922.
[12] Chen Y,Ba L,Huang W,et al. Role of carvacrol in cardioprotection against myocardial ischemia/reperfusion injury in rats through activation of MAPK/ERK and Akt/e NOS signaling pathways[J]. Eur J Pharmacol,2017,796:90-100.
[13] Zhai M,Li B,Duan W,et al. Melatonin ameliorates myocardial ischemia reperfusion injury through SIRT3-dependent regulation of oxidative stress and apoptosis[J]. J Pineal Res,2017,63(2):10.
[14] Yang Q,Wang HC,Liu Y,et al. Resveratrol cardioprotection against myocardial ischemia/reperfusion injury involves upregulation of adiponectin levels and multimerization in type 2 diabetic mice[J]. J Cardiovasc Pharmacol,2016,68(4):304.
[15] Li R,Zhao Z,Sun M,et al. ALDH2 gene polymorphism in different types of cancers and its clinical significance[J]. Life Sci,2016,147:59-66.
[16] Mali VR,Deshpande M,Pan G,et al. Impaired ALDH2 activity decreases the mitochondrial respiration in H9C2 cardiomyocytes[J]. Cell Signal,2016,28(2):1-6.
[17] Zhou YH,Yu JP,Liu YF,et al. Effects of Ginkgo biloba extract on inflammatory mediators(SOD,MDA,TNF-alpha,NF-kappaBp65,IL-6)in TNBS-induced colitis in rats[J]. Mediators Inflamm,2017,2006(5):1-9.
[18] Liu D,Yu S,Zhou B,et al. GW27-e0523 The effect of Ginsenoside Rb1 on the expression of SOD and MDA in HUVECs injured by homocysteine[J]. J Am Coll Cardiol,2016,68(16):C21.
[2] He B,Zhao Y,Xu L,et al. The nuclear melatonin receptor ROR±is a novel endogenous defender against myocardial ischemia/reperfusion injury[J]. J Pineal Res,2016,60(3):313-326.
[3] Song M,Lan H. GW27-e1017 Beneficial effects of a polysaccharide from Salvia miltiorrhiza on myocardial ischemia-reperfusion injury in rats[J]. J Am Coll Cardiol,2016,68(16):C65-C65.
[4] Fang SJ,Li PY,Wang CM,et al. Inhibition of endoplasmic reticulum stress by neuregulin-1 protects against myocardial ischemia/reperfusion injury[J]. Peptides,2017,88:196.
[5] Tianxin HU,Guo W,Miaomiao XI,et al. Synergistic cardioprotective effects of Danshensu and hydroxysafflor yellow A against myocardial ischemia-reperfusion injury are mediated through the Akt/Nrf2/HO-1 pathway[J]. Int J Mol Med,2016,38(1):83-94.
[6] Wang Z,Sun Y,Li X,et al. A novel acetaldehyde dehydrogenase with salicylaldehyde dehydrogenase activity from Rhodococcus ruber strain OA1[J]. Curr Microbiol,2017,74(12):1404-1410.
[7] Mcclerklin SA,Lee SG,Harper CP,et al. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC3000[J]. PLo S Pathog,2018,14(1):e1006811.
[8] Xu Y,Lee J,Yang HS,et al. A computational integrating kinetic study on the flexible active site of human acetaldehyde dehydrogenase 1[J]. Process Biochem,2016,51(6):725-733.
[9] Yeh JZ,Byington KH. Interactions of acetaldehyde,ethyl alcohol and oxybarbiturates affecting mitochondrial functions[J].Biochem Pharmacol,2016,22(16):2045-2057.
[10] Nene A,Chen CH,Disatnik MH,et al. Aldehyde dehydrogenase 2 activation and coevolution of itsεPKC-mediated phosphorylation sites[J]. J Biomed Sci,2017,24(1):3.
[11] Bertsch J,Siemund AL,Kremp F,et al. A novel route for ethanol oxidation in the acetogenic bacterium Acetobacterium woodii:the acetaldehyde/ethanol dehydrogenase pathway[J]. Environ Microbiol,2016,18(9):2913-2922.
[12] Chen Y,Ba L,Huang W,et al. Role of carvacrol in cardioprotection against myocardial ischemia/reperfusion injury in rats through activation of MAPK/ERK and Akt/e NOS signaling pathways[J]. Eur J Pharmacol,2017,796:90-100.
[13] Zhai M,Li B,Duan W,et al. Melatonin ameliorates myocardial ischemia reperfusion injury through SIRT3-dependent regulation of oxidative stress and apoptosis[J]. J Pineal Res,2017,63(2):10.
[14] Yang Q,Wang HC,Liu Y,et al. Resveratrol cardioprotection against myocardial ischemia/reperfusion injury involves upregulation of adiponectin levels and multimerization in type 2 diabetic mice[J]. J Cardiovasc Pharmacol,2016,68(4):304.
[15] Li R,Zhao Z,Sun M,et al. ALDH2 gene polymorphism in different types of cancers and its clinical significance[J]. Life Sci,2016,147:59-66.
[16] Mali VR,Deshpande M,Pan G,et al. Impaired ALDH2 activity decreases the mitochondrial respiration in H9C2 cardiomyocytes[J]. Cell Signal,2016,28(2):1-6.
[17] Zhou YH,Yu JP,Liu YF,et al. Effects of Ginkgo biloba extract on inflammatory mediators(SOD,MDA,TNF-alpha,NF-kappaBp65,IL-6)in TNBS-induced colitis in rats[J]. Mediators Inflamm,2017,2006(5):1-9.
[18] Liu D,Yu S,Zhou B,et al. GW27-e0523 The effect of Ginsenoside Rb1 on the expression of SOD and MDA in HUVECs injured by homocysteine[J]. J Am Coll Cardiol,2016,68(16):C21.