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浙江大学学报（医学版）
浙江大学学报（医学版）  2018, Vol. 47 Issue (5): 507-513    DOI: 10.3785/j.issn.1008-9292.2018.10.10
原著     
参麦注射液保护氧化损伤心肌细胞线粒体的机制研究
赵誉1(),张凤1,赵筱萍2,袁玮3,张金华3,王毅1,*()
1. 浙江大学药物信息学研究所, 浙江 杭州 310058
2. 浙江中医药大学基础医学院, 浙江 杭州 310053
3. 云南大理药业股份有限公司, 云南 大理 671000
Shenmai injection protects mitochondria from oxidative injury in myocardial cells and its mechanism
ZHAO Yu1(),ZHANG Feng1,ZHAO Xiaoping2,YUAN Wei3,ZHANG Jinhua3,WANG Yi1,*()
1. Institute of Pharmaceutical Informatics, Zhejiang University, Hangzhou 310058, China
2. School of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
3. Dali Pharmaceutical Co., Ltd., Dali 671000, Yunnan Province, China
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摘要：

目的: 探究参麦注射液对氧化损伤心肌细胞的保护作用及其对线粒体能量代谢过程的影响。方法: 采用叔丁基过氧化氢（t-BHP）诱导构建H9c2心肌细胞氧化损伤模型。分别采用MTT法和化学发光法检测参麦注射液对细胞存活率和ATP水平的影响；采用Clark氧电极检测参麦注射液对细胞有氧呼吸速率的调节作用；采用ELISA法检测丙酮酸及丙酮酸脱氢酶（PDH）的含量及活性；采用蛋白质印迹法检测丙酮酸脱氢酶亚基（PDHA1）含量；采用实时定量RT-PCR检测丙酮酸脱氢酶激酶1（PDK1）mRNA的表达。结果: 与氧化损伤模型比较，参麦注射液可有效提高心肌细胞存活率和ATP水平（均P < 0.01），提高细胞氧呼吸速率，减少丙酮酸含量，并提高PDH活性（P < 0.05或P < 0.01）。在分子水平上，参麦注射液可提高PDHA1蛋白表达（P < 0.05），并具有降低PDK1 mRNA表达的趋势（P>0.05）。结论: 参麦注射液可有效保护氧化损伤心肌细胞，其机制可能与提高PDH活性、减少丙酮酸堆积，从而维持线粒体功能稳定和细胞能量代谢稳定有关。
关键词： 肌细胞, 心脏/药物作用;  肌细胞, 心脏/病理学;  线粒体, 心脏/病理学;  氧/代谢;  丙酮酸脱氢酶(硫辛酰胺);  麦冬/药理学;  人参/药理学    
Abstract:

Objective: To investigate the effect of Shenmai injection on myocardial cells with oxidative injury and the underlying mechanisms. Methods: Tert-butyl hydroperoxide (t-BHP) was used to induce the oxidative stress in H9c2 myocardial cells. The cell viability and ATP level were evaluated using MTT-colorimetric method and CellTiter-Glo luminescent cell viability assay. The oxygen respiration rate was examined by Clark oxygen electrode. Pyruvate and pyruvate dehydrogenase (PDH) levels were evaluated by ELISA kit. Western blot and quantitative real-time RT-PCR were employed to evaluate the expression of pyruvate dehydrogenase alpha 1(PDHA1) and pyruvate dehydrogenase kinase 1(PDK1). Results: Shenmai injection significantly improved viability and respiration of H9c2 myocardial cells after t-BHP injury (P < 0.05 or P < 0.01). It increased ATP contents by consuming pyruvate and increasing PDH level (P < 0.05 or P < 0.01). Furthermore, Shenmai injection had the tendency to increase protein expression of PDHA1(P < 0.05) and decrease mRNA expression of PDK1 (P>0.05). Conclusion: Shenmai injection protects mitochondria from oxidative stress by increasing PDH level, which indicates that it may improve energy metabolism of myocardial cells.
Key words: Myocytes, cardiac/drug effects    Myocytes, cardiac/pathology    Mitochondria, heart/pathology    Oxygen/metabolism    Pyruvate dehydrogenase(lipoamide)    Ophiopogon japonicus/pharmacology    Ginseng/pharmacology
收稿日期: 2018-03-08 出版日期: 2019-01-23
:  R285  
基金资助: 国家自然科学基金(81822047);浙江省自然科学基金(LR16H280001);浙江大学基本科研业务费专项资金(2017FZA7015);浙江大学曹光彪高科技发展基金(2017RC016)
通讯作者: 王毅     E-mail: zy-joy-zy@hotmail.com;zjuwangyi@zju.edu.cn
作者简介: 赵誉(1994-), 女, 博士研究生, 主要从事中药药效物质研究; E-mail:zy-joy-zy@hotmail.com; https://orcid.org/0000-0002-8436-5386
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引用本文:

赵誉,张凤,赵筱萍,袁玮,张金华,王毅. 参麦注射液保护氧化损伤心肌细胞线粒体的机制研究[J]. 浙江大学学报（医学版）, 2018, 47(5): 507-513.

ZHAO Yu,ZHANG Feng,ZHAO Xiaoping,YUAN Wei,ZHANG Jinhua,WANG Yi. Shenmai injection protects mitochondria from oxidative injury in myocardial cells and its mechanism. J Zhejiang Univ (Med Sci), 2018, 47(5): 507-513.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2018.10.10    或    http://www.zjujournals.com/med/CN/Y2018/V47/I5/507

图 1  不同浓度参麦注射液细胞毒性比较(n=3)
图 2  各组氧呼吸曲线比较
图 3  各组PDHA1蛋白和PDK1 mRNA相对表达量比较(n=3)
图 4  参麦注射液调控氧化损伤心肌细胞中的能量代谢过程示意图
1 隋辉, 陈伟伟, 王文 . 《中国心血管病报告2015》要点解读[J]. 中国心血管杂志, 2016, 21 (4): 259- 261
SUI Hui , CHEN Weiwei , WANG Wen . Interpretation of Report on cardiovascular diseases in China (2015)[J]. Chinese Journal of Cardiovascular Medicine, 2016, 21 (4): 259- 261
doi: 10.3969/j.issn.1007-5410.2016.04.001
2 陈昕, 赵雷, 韩蕴琪 et al. 维生素E对心肌氧化损伤的保护作用研究[J]. 中国实验诊断学, 2012, 16 (6): 975- 977
CHEN Xin , ZHAO Lei , HAN Yunqi et al. Effect of vitamin E on myocardium injury caused by free radicals[J]. Chinese Journal of Laboratory Diagnosis, 2012, 16 (6): 975- 977
doi: 10.3969/j.issn.1007-4287.2012.06.008
3 李爱群, 王绮雯 . 熊果酸对过氧化氢诱导大鼠心肌细胞氧化损伤的保护作用[J]. 血栓与止血学, 2017, 23 (6): 908- 911
LI Aiqun , WANG Qiwen . Protective effect of ursolic acid on oxidative injury induced by hydrogen peroxide in cultured rat cardiomyocytes[J]. Chinese Journal of Thrombosis and Hemostasis, 2017, 23 (6): 908- 911
doi: 10.3969/j.issn.1009-6213.2017.06.003
4 李傅冬, 沈毅 . 参麦注射液治疗冠心病疗效与安全性的Meta分析[J]. 中国中药杂志, 2012, 37 (23): 3651- 3658
LI Fudong , SHEN Yi . Meta-analysis on efficacy and safety of Shenmai injection in treatment of coronary heart disease[J]. China Journal of Chinese Materia Medica, 2012, 37 (23): 3651- 3658
5 宋建丽 . 参麦注射液治疗冠心病的临床分析[J]. 药学进展, 1999, 23 (1): 44- 46
SONG Jianli . Clinical analysis of Shenmai injection in the treatment of coronary heart disease[J]. Progress in Pharmaceutical Sciences, 1999, 23 (1): 44- 46
6 苏萍, 吕书峰, 范雪梅 et al. 参麦注射液及其有效组分对H2O2诱导心肌细胞损伤的保护作用[J]. 中成药, 2011, 33 (12): 2150- 2153
SU Ping , LYU Shufeng , FAN Xuemei et al. Protective effect of Shenmai injection and its components on H2O2 induced oxidative injury of cardiomyocytes[J]. Chinese Traditional Patent Medicine, 2011, 33 (12): 2150- 2153
doi: 10.3969/j.issn.1001-1528.2011.12.035
7 许浩, 葛亚坤, 邓同乐 et al. 人参皂苷Rb1对H2O2诱导新生大鼠心肌细胞凋亡的保护作用[J]. 中国药理学通报, 2005, 21 (7): 803- 806
XU Hao , GE Yakun , DENG Tongle et al. Protective effect of ginsenoside Rb1 against H2O2-induced apoptosis in neonatal rat cardiomyocytes[J]. Chinese Pharmacological Bulletin, 2005, 21 (7): 803- 806
doi: 10.3321/j.issn:1001-1978.2005.07.009
8 刘俊伟, 任冶龙, 刘旭玲 et al. 人参皂苷Rb1对大鼠局灶性脑缺血再灌注损伤后脑梗死体积及脑组织和血清IL-1β的影响[J]. 中国中西医结合杂志, 2013, 33 (12): 1696- 1700
LIU Junwei , REN Yelong , LIU Xuling et al. Effect of ginsenoside Rb1 on cerebral infarction volume and IL-1β in the brain tissue and sera of focal cerebral ischemia/reperfusion injury model rats[J]. Chinese Journal of Integrated Traditional and Western Medicine, 2013, 33 (12): 1696- 1700
9 SU X , MA Y , HUANG R et al. Effects of shenmai injection on blood SOD activity and MDA level in senile patients with coronary heart disease[J]. J Tradit Chin Med, 2005, 25 (1): 50- 53
10 李丽, 黄启福 . 参麦注射液对大鼠急性心肌缺血再灌注损伤的影响[J]. 中国病理生理杂志, 2003, 19 (11): 1472- 1475
LI Li , HUANG Qifu . Effects of Shenmai injection on acute myocardial ischemia/reperfusion injury in rats[J]. Chinese Journal of Pathophysiology, 2003, 19 (11): 1472- 1475
doi: 10.3321/j.issn:1000-4718.2003.11.009
11 余健, 辛艳飞, 宣尧仙 . 参麦注射液药理作用的物质基础研究进展[J]. 医药导报, 2013, 32 (4): 497- 500
YU Jian , XIN Yanfei , XUAN Yaoxian . Progress in pharmacological basis of Shenmai injection[J]. Herald of Medicine, 2013, 32 (4): 497- 500
doi: 10.3870/yydb.2013.04.027
12 LAHERA V , DE LAS H N , LOPEZ-FARRE A et al. Role of mitochondrial dysfunction in hypertension and obesity[J]. Curr Hypertens Rep, 2017, 19 (2): 11
doi: 10.1007/s11906-017-0710-9
13 GRAHAM B H , WAYMIRE K G , COTTRELL B et al. A mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the heart/muscle isoform of the adenine nucleotide translocator[J]. Nat Genet, 1997, 16 (3): 226- 234
doi: 10.1038/ng0797-226
14 KANAI A J , PEARCE L L , CLEMENS P R et al. Identification of a neuronal nitric oxide synthase in isolated cardiac mitochondria using electrochemical detection[J]. Proc Natl Acad Sci U S A, 2001, 98 (24): 14126- 14131
doi: 10.1073/pnas.241380298
15 CERNIGLIA G J , DEY S , GALLAGHER-COLOMBO S M et al. The PI3K/Akt pathway regulates oxygen metabolism via pyruvate dehydrogenase (PDH)-E1alpha phosphorylation[J]. Mol Cancer Ther, 2015, 14 (8): 1928- 1938
doi: 10.1158/1535-7163.MCT-14-0888
16 BRUN S , AUBRY C , LIMA O et al. Relationships between respiration and susceptibility to azole antifungals in Candida glabrata[J]. Antimicrob Agents Chemother, 2003, 47 (3): 847- 853
doi: 10.1128/AAC.47.3.847-853.2003
17 STANLEY W C , RECCHIA F A , LOPASCHUK G D . Myocardial substrate metabolism in the normal and failing heart[J]. Physiol Rev, 2005, 85 (3): 1093- 1129
doi: 10.1152/physrev.00006.2004
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