黄芪苷Ⅳ抗阿霉素致大鼠心肌自由基损伤作用的研究
摘要
目的：研究黄芪苷Ⅳ(AST)对氧自由基(OFR)所致心肌损伤的保护作用，并探讨其作用机制。
方法：用阿霉素(ADR)制备心肌OFR损伤模型。实验分为两个部分：第一部分—整体动物实验，实验分3组，模型组(ADR组)：以大鼠腹腔注射ADR4.5mg／kg／d，共2日，建立OFR心肌损伤模型；预防组(ADR+AST组)：在注射ADR前30分钟腹腔注射AST2.0mg／kd／d，共2日：正常对照组，每日腹腔注射生理盐水1 ml／kg，共2日。给药后第5天，测定心肌组织中MDA含量和SOD活性，并取心室肌在电镜下观察心肌超微结构的改变。第二部分—细胞实验，以原代培养乳鼠心肌细胞建立OFR损伤模型，即ADR组为ADR(0.5mg／L和1.0mg／L)与乳鼠心肌细胞共同培养24小时，ADR+AST组为在加入ADR前1小时给予AST(10mg／L和20mg／L)，然后加入与ADR组浓度相同的ADR培养24小时，观察心肌细胞MTT代谢率、MDA含量及NOS活性的变化，并测定心肌细胞内游离Ca~(2+)浓度的改变。
结果：
一、整体动物实验
1．MDA含量：与正常对照组(1.39±0.25 nmol／mg protein)相比，ADR组(1.88±0.42nmol／mg protein)心肌组织的MDA含量增高(P＜0.05)，与ADR组相比，ADR+AST组(1.32±0.36nmol／mg protein)MDA值降低(P＜0.05)。
2．SOD活性：与正常对照组(33.0±3.21 U／mg protein)相比，ADR组(36.5±1.97 U／mg protein)和ADR+AST组(36.9±3.12 U／mg protein)心肌组织的SOD活性均明显增高(P＜0.05)。
天津医科大学硕士月开究生学位论文’中文摘要
3.心肌超微结构:电镜观察显示，ADR组心肌线粒体增生、扭曲、变形，
T管扩张，心肌间质水肿及微血管痉挛;而ADR+AST组上述病理改变明显减
轻。
二、原代培养心肌细胞实验
1.M，IT代谢率:与正常对照组(100士o%)比较，ADR两个剂量组(o.sm叭，
l.omg/L)的MTT代谢率(60.74士10.63%，55.99士4.04%)均明显降低(p<0.02)，
以高剂量组降低更为明显;AST大小剂量对正常心肌细胞的MTT代谢率(94.73
士9.48%，96.18士6.39%)均没有影响(P>0.05)，但可提高ADR损伤心肌细胞的
MTT代谢率，在ADR剂量相同的条件下，大剂量AST升高程度更为明显。
2.MDA含量:与正常对照组(5 .77士1.43nrn。腼9 protein)相比，ADR
组(9.65士1.43 nmo腼9 nrotein)MDA含量显著增高(p<0.05)，与ADR组相比，
ADR+AsT大小两个剂量组的MDA值(6.73士2.84 nxnol/m 9 protein，3.84士0.87
nlnof/m gProtein)均明显降低(P<0.05)，大剂量AST降低更为显著(P<0.01)。
3.NOS活性:与正常对照组(26.96士1.68U/mgProtein)比较，ADR组(38.62
士1.56U/mgprotein)NOS活性明显升高(P<0.05)，与ADR组比较，ADR+AsT
大小两个剂量组的Nos活性(22.68士5.6oU/mg protei几19.67士l.23U/mg
protein)均明显降低，高剂量组降低更为明显(P<0.01)。
4.[eaZ勺i:与正常对照组(225.14士21.91 nmoljL)比较，心R组(357.00士
46.53tnno讥)[eaZ勺i明显升高(P<0.01)，与心R组比较，AnR+AsT组(155.64
士28.05 nmol几)[eaZ+]i显著降低(P<0.01)。
结论:
1.黄茂昔W提高心肌细胞SOD的活性，降低MDA的含量。
2.黄茂昔W剂量依赖地提高ADR抑制的心肌细胞MTT代谢率。
3.黄蔑昔W降低ADR导致的NOS活性的增高和对抗可能由此产生的过
，反匀卜医耳牛大月卜月阿d匕友开，匕月匕学位论文
量NO造成的心肌线粒体损伤。
中文摘要
4.黄茂昔W降低由ADR引起的心肌细胞内C扩+超载。
OBJECTIVE: To investigate protective effects and mechanisms of astragaloside IV on myocardium injury caused by free radicals.
METHODS: Adriamycin (ADR) was administered intraperitoneally to establish the model of myocardium injury caused by oxygen free radicals in rats. Experiments were performed in vivo and in vitro, respectively. In vivo experiments: rats were divided into three groups randomly: the model group (ADR group) received ADR (4.5 mg/kg) intraperitoneally for 2 days; the prophylaxes group (ADR±AST group) received same dose of ADR 30 minutes after being treated with astragaloside IV (AST) (2.0 mg/kg) every day for 2 days; the control group received normal saline (1 ml/kg) intraperitoneally in stead of drugs for 2 days. On the fifth day after treatment with drugs, content of MDA and activity of SOD in myocardium tissue were measured. Ultrastructure of ventricular muscle was observed under the electronic microscope. In vitro experiments: myocytes of new-born rats were incubated with ADR (0.5mg/L and 1.0 mg/L) for 24 hour to induce oxygen free radical injury in model group; same dose of ADR was administered 1 hour after tre
atment with AST (10 mg/L and 20 mg/L) in therapeutic group and incubated for another 24 hours. Metabolic rate of MTT, content of MDA, activity of NOS, and intracellular free calcium of cardiac myocyte were measured.
RESULTS: Experiment in vivo
1. Content of MDA: Compared with that of control group (1.39±0.25 nmol/mg protein) , the content of MDA was increased in ADR group (1.88±0.42 nmol/mg
protein), (p<0.05). Compared with that of model group, the content of MDA was decreased in ADR±AST group (1.32 ±0.36 nmol/mg protein), (p<0.05).
2. Activity of SOD: Compared with that of control group (33.0 ± 3.21 U/mg protein), the activities of SOD were increased significantly both in ADR group(36.5 ±1.97 U/mg protein) and in ADR±AST group (36.39 ± 3.12 U/mg protein), QK0.05).
3. Ultrastructure of myocardium: Hyperplasia, twisting, deformation of myocardial mitochondria, T-tube dilation, interstitial edema, microvessel spasm of myocardium were showed under the electronic microscope, while the above pathologic alterations were attenuated in ADR±AST group.
In vitro experiment
1. Metabolic rate of MTT: Compared with that of control group (100.0±0%), metabolic rates of MTT was decreased in ADR 0.5 mg/L and 1.0 mg/L groups in a dose dependent manner significantly (60.74 ± 10.63%, 55. 99 ±4.04% ), (PO.01). Compared with that of control group, metabolic rates of MTT in both the small and the large dosage of AST were 94.73 ±9.48%, 96.1 8 ±6.39 respectively (p>0.05). However, the metabolic rate of MTT in cardiac myocyte injured by ADR could be increased. Under the condition that the dosages of ADR were the same, metabolic rate of MTT in the large dosage of AST was increased much more significantly.
2. Content of MDA: Compared with that of control group (5 .77 ±1.43 nmol/mg protein), the content of MDA was increased in ADR group (9.65 ±1.43 nmol/mg protein), (p<0.05). Compared with that of ADR group, the content of MDA was dose-dependently decreased in AST 10 mg/L and 20 mg/L groups significantly (6.73 ±2.84 nmol/mg protein, 3.84±0.87 nmol/mg protein), (p<0.05).
方法：用阿霉素(ADR)制备心肌OFR损伤模型。实验分为两个部分：第一部分—整体动物实验，实验分3组，模型组(ADR组)：以大鼠腹腔注射ADR4.5mg／kg／d，共2日，建立OFR心肌损伤模型；预防组(ADR+AST组)：在注射ADR前30分钟腹腔注射AST2.0mg／kd／d，共2日：正常对照组，每日腹腔注射生理盐水1 ml／kg，共2日。给药后第5天，测定心肌组织中MDA含量和SOD活性，并取心室肌在电镜下观察心肌超微结构的改变。第二部分—细胞实验，以原代培养乳鼠心肌细胞建立OFR损伤模型，即ADR组为ADR(0.5mg／L和1.0mg／L)与乳鼠心肌细胞共同培养24小时，ADR+AST组为在加入ADR前1小时给予AST(10mg／L和20mg／L)，然后加入与ADR组浓度相同的ADR培养24小时，观察心肌细胞MTT代谢率、MDA含量及NOS活性的变化，并测定心肌细胞内游离Ca~(2+)浓度的改变。
结果：
一、整体动物实验
1．MDA含量：与正常对照组(1.39±0.25 nmol／mg protein)相比，ADR组(1.88±0.42nmol／mg protein)心肌组织的MDA含量增高(P＜0.05)，与ADR组相比，ADR+AST组(1.32±0.36nmol／mg protein)MDA值降低(P＜0.05)。
2．SOD活性：与正常对照组(33.0±3.21 U／mg protein)相比，ADR组(36.5±1.97 U／mg protein)和ADR+AST组(36.9±3.12 U／mg protein)心肌组织的SOD活性均明显增高(P＜0.05)。
天津医科大学硕士月开究生学位论文’中文摘要
3.心肌超微结构:电镜观察显示，ADR组心肌线粒体增生、扭曲、变形，
T管扩张，心肌间质水肿及微血管痉挛;而ADR+AST组上述病理改变明显减
轻。
二、原代培养心肌细胞实验
1.M，IT代谢率:与正常对照组(100士o%)比较，ADR两个剂量组(o.sm叭，
l.omg/L)的MTT代谢率(60.74士10.63%，55.99士4.04%)均明显降低(p<0.02)，
以高剂量组降低更为明显;AST大小剂量对正常心肌细胞的MTT代谢率(94.73
士9.48%，96.18士6.39%)均没有影响(P>0.05)，但可提高ADR损伤心肌细胞的
MTT代谢率，在ADR剂量相同的条件下，大剂量AST升高程度更为明显。
2.MDA含量:与正常对照组(5 .77士1.43nrn。腼9 protein)相比，ADR
组(9.65士1.43 nmo腼9 nrotein)MDA含量显著增高(p<0.05)，与ADR组相比，
ADR+AsT大小两个剂量组的MDA值(6.73士2.84 nxnol/m 9 protein，3.84士0.87
nlnof/m gProtein)均明显降低(P<0.05)，大剂量AST降低更为显著(P<0.01)。
3.NOS活性:与正常对照组(26.96士1.68U/mgProtein)比较，ADR组(38.62
士1.56U/mgprotein)NOS活性明显升高(P<0.05)，与ADR组比较，ADR+AsT
大小两个剂量组的Nos活性(22.68士5.6oU/mg protei几19.67士l.23U/mg
protein)均明显降低，高剂量组降低更为明显(P<0.01)。
4.[eaZ勺i:与正常对照组(225.14士21.91 nmoljL)比较，心R组(357.00士
46.53tnno讥)[eaZ勺i明显升高(P<0.01)，与心R组比较，AnR+AsT组(155.64
士28.05 nmol几)[eaZ+]i显著降低(P<0.01)。
结论:
1.黄茂昔W提高心肌细胞SOD的活性，降低MDA的含量。
2.黄茂昔W剂量依赖地提高ADR抑制的心肌细胞MTT代谢率。
3.黄蔑昔W降低ADR导致的NOS活性的增高和对抗可能由此产生的过
，反匀卜医耳牛大月卜月阿d匕友开，匕月匕学位论文
量NO造成的心肌线粒体损伤。
中文摘要
4.黄茂昔W降低由ADR引起的心肌细胞内C扩+超载。
OBJECTIVE: To investigate protective effects and mechanisms of astragaloside IV on myocardium injury caused by free radicals.
METHODS: Adriamycin (ADR) was administered intraperitoneally to establish the model of myocardium injury caused by oxygen free radicals in rats. Experiments were performed in vivo and in vitro, respectively. In vivo experiments: rats were divided into three groups randomly: the model group (ADR group) received ADR (4.5 mg/kg) intraperitoneally for 2 days; the prophylaxes group (ADR±AST group) received same dose of ADR 30 minutes after being treated with astragaloside IV (AST) (2.0 mg/kg) every day for 2 days; the control group received normal saline (1 ml/kg) intraperitoneally in stead of drugs for 2 days. On the fifth day after treatment with drugs, content of MDA and activity of SOD in myocardium tissue were measured. Ultrastructure of ventricular muscle was observed under the electronic microscope. In vitro experiments: myocytes of new-born rats were incubated with ADR (0.5mg/L and 1.0 mg/L) for 24 hour to induce oxygen free radical injury in model group; same dose of ADR was administered 1 hour after tre
atment with AST (10 mg/L and 20 mg/L) in therapeutic group and incubated for another 24 hours. Metabolic rate of MTT, content of MDA, activity of NOS, and intracellular free calcium of cardiac myocyte were measured.
RESULTS: Experiment in vivo
1. Content of MDA: Compared with that of control group (1.39±0.25 nmol/mg protein) , the content of MDA was increased in ADR group (1.88±0.42 nmol/mg
protein), (p<0.05). Compared with that of model group, the content of MDA was decreased in ADR±AST group (1.32 ±0.36 nmol/mg protein), (p<0.05).
2. Activity of SOD: Compared with that of control group (33.0 ± 3.21 U/mg protein), the activities of SOD were increased significantly both in ADR group(36.5 ±1.97 U/mg protein) and in ADR±AST group (36.39 ± 3.12 U/mg protein), QK0.05).
3. Ultrastructure of myocardium: Hyperplasia, twisting, deformation of myocardial mitochondria, T-tube dilation, interstitial edema, microvessel spasm of myocardium were showed under the electronic microscope, while the above pathologic alterations were attenuated in ADR±AST group.
In vitro experiment
1. Metabolic rate of MTT: Compared with that of control group (100.0±0%), metabolic rates of MTT was decreased in ADR 0.5 mg/L and 1.0 mg/L groups in a dose dependent manner significantly (60.74 ± 10.63%, 55. 99 ±4.04% ), (PO.01). Compared with that of control group, metabolic rates of MTT in both the small and the large dosage of AST were 94.73 ±9.48%, 96.1 8 ±6.39 respectively (p>0.05). However, the metabolic rate of MTT in cardiac myocyte injured by ADR could be increased. Under the condition that the dosages of ADR were the same, metabolic rate of MTT in the large dosage of AST was increased much more significantly.
2. Content of MDA: Compared with that of control group (5 .77 ±1.43 nmol/mg protein), the content of MDA was increased in ADR group (9.65 ±1.43 nmol/mg protein), (p<0.05). Compared with that of ADR group, the content of MDA was dose-dependently decreased in AST 10 mg/L and 20 mg/L groups significantly (6.73 ±2.84 nmol/mg protein, 3.84±0.87 nmol/mg protein), (p<0.05).
引文
1. Prasad K, Kalra J. Oxygen free radicals and heart failure. Angiology, 1988, 39(5): 417-20
2. Prasad K, Kalra J, Massey KL, et al. Increased production of oxygen free radicals by polymorphonuclear leukocytes in heart failure due to aortis stenosis. Angiology, 1989, 45 (5): 472-75
3. Hori M, Gotoch K, Kitakaze M, et al. Role of oxygen-derived free radicals in myocardial edema and ischemia in coronary microvascular embolization. Circulation. 1991, 84(2): 828
4．杨成明，刘光濯，牟永方等．脂质过氧化与充血性心力衰竭．中国循环杂志，1991，6(5)：376
5. Mcmurray J, Mclay J, Chopra M, et al. Evidence for enhanced free radical activity in chronic congestive heart failure secondary to coronary artery disease. Am J Cardiol, 1990, 15(5):1261
6. Hill MF, Singal PK. Antioxidant and oxidative stress changes during heart failure subsequent to myocardial infarction in rats. Am J Pathol, 1996, 148(1): 291-300
7．陈瑗，周玫主编．自由基医学基础与病理生理．北京：人民卫生出版社，2002年第一版
8．李中言，赵连友．一氧化氮与心血管疾病．中华心血管病杂志，1996，24：73-76
9. Oyama JI, Shimokawa H, Momal H, et al. Role of nitric oxide and peroxynitrite in the cytokine-induced sustained myocardial dysfunction in dogs in rive. J Clin Invest, 1998, 101 (2):207-14
10. Liu P, Hock CE, Nagele R, et al. Formation of nitric oxide, superoxide, and peroxynitrite in myocardial ischemia-reperfusion injury in rat. Am J Physiol, 1997, 272(52): H2 327-336
11. Zingarelli B, Cuzzocrea S, Zsengeller Z, et al. Protection against myocardial ischemia and reperfusion injury by 3-aminobenzamide, an inhibitor of poly (ADP-ribose) systhetase. Cardiovasc Res, 1997, 36:205-15
12. Ma XL, Loper BL, Liu GL, et al. Peroxynitrite aggravates myocardial reperfusion injury in the isolated perfuse rat heart. Cardiovase Res, 1997, 36:195-204
13．杨藻宸主编．药理学和药物治疗学．北京：人民卫生出版社，2000年第一版p1780-81
14. Singal PK, Iliskovic N. Doxorubicin-induced cardiomyopathy. N Engl J Med, 1998, 339: 900-5
15. Gewirtz DA. A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem Pharmacol, 1999, 57:727-41
16. Papadopoulou LC, Theophilidis G, Thomopoulous GN, et al. Structural and functional impairment of mitochondria in adriamycin-induced cardiomyopathy in mice: suppression of cytochrome c oxidase Ⅱ gene expression. Biochem Pharmacol, 1999, 57:481-9
17. Dealtey SM, Aksenov MY, Aksenova MV, et al. Adriamycin-induced changes of creatine kinase activity in vivo and in cardiomyocyte culture. Toxicology, 1999, 134:51-62
18. Serrano J, Palmeira CM, Kuehl DW, et al. Cardioselective and cumulative oxidation of mitochondrial DNA following subchronic doxorubicin administration. Biochem Biophys Acta, 1999, 1411:201-5
19．国家药典委员会．中华人民共和国药典．北京：化学工业出版社．2000，249-250
20．刘道芳，袁敏．黄芪研究进展．中医药信息，1998，(2)：13-15
21．韩玲，陈可冀．黄芪对心血管系统作用的实验药理学研究进展．中国中西医结合杂志，2000，20(3)：234-37
22．张燕燕，颜敏，文学明．黄芪抗脂质过氧化作用的研究．医药导报，2002，21(9)：542-43
23. Li ZP, Cao Q. Effects of astragaloside Ⅳ on myocardial calcium transport and cardiac function in ischemic rats. Acta Pharmacol Sin, 2002. 23(10): 898-904
24．刘恭鑫，杨英珍，顾全宝，等．黄芪对感染病毒大鼠心肌细胞钙通道及钠钙交换载体的效应．中国病理生理杂志，1999，15(7)：591-93
25．刘星楷，江明华，俞正坤．黄芪有效成分研究．黄芪中清除超氧阴离子成分的分离和检测．天然产物研究与开发，1991，3(4)：1-6
26．刘艳霞，刘在萍，焦建杰，等．黄芪苷Ⅳ对正常和心功能受抑制大鼠左心室心肌力学的影响．中草药，2001，32(4)：332-34
27．MDA测定试剂盒说明书．南京建成生物工程研究所
28．SOD测定试剂盒说明书．南京建成生物工程研究所
29．汪谦主编．现代医学实验方法．北京：人民卫生出版社，1997年第一版：P183-5，505-6
30．李会强，姚智，赵瑾莹，等．IL-2、IL-6及TNFαMTT检测方法的建立．天津第二医学院学报，1993，9：4-5
31．NOS测定试剂盒说明书．南京建成生物工程研究所
32．林秀珍，马德禄，崔荣芬．大黄素及番泻甙和大黄多糖对培养大肝细胞内游离钙浓度的影响．中国中西医结合杂志，1995，15(7)：419-21
33. Ito H, Torii M, Suzuki T. Decreased superoxide dismutase activity and increased superoxide anion production in cardiac hypertrophy of spontaneously hypertensive rats. Clin Exp Hypertens, 1995, 17(5): 803-16
34. Miller FJ Jr, Gutterman DD, Rios CD, et al. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res, 1998, 82(12):1298-1305
35. Iliskovic N, Li T, Khaper N, et al. Modulation of adriamycin-induced changes in serum free fatty acids, albumin and cardiac oxidative stress. Mol Cell Biochem, 1998, 188(1-2):161-66
36. Campo GM, Squadrito F, Campo S, et al. Beneficial effect of raxofellast, an hydrophilic vitamin E analogue, in the rat heart after ischemia and reperfusion injury. J Mol Cell Cardiol, 1998, 30(8): 1493-1503
37. Doroshow JH. Doxorubicin-induced cardiac toxity. N Engl J Med, 1991, 324: 843Dealtey SM, Aksenov MY, Aksenova MV, et al. Adriamycin-induced changes of creatine kinase activity in vivo and in cardiomyocyte culture. Toxicology, 1999, 134:51-62
38. Doroshow JH. Anthracycline-enhanced, cardiac oxygen radical metabolism. In: Singal PK ed. Oxygen Radicals in the Pathophysiology of Heart Disease. Boston: Kluwer Academic publishers, 1988: 323-32
39. Nohl H. Identification of the site of adriamycin activation in the heart cell. Biochem Pharmacol, 1988, 37:2633
40. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J Immuno Methods, 1983, 65:55-63
41．万影，陈玉石．黄芪对超氧阴离子自由基的清除作用．数理医药学杂志，1998；11(1)：61-62
42．孙成文，钟国赣，江岩，等．黄芪多糖抗氧化损伤作用的研究．中国药理学通报，1996：12(2)：161-63
43．李靖，朱建华，黄崇勤，等．黄芪皂甙对培养心肌细胞损伤的影响．中国现代医学杂志，1998；8(8)：7-9
44. Kelly RA, Balligand JL, Smith TW. Nitric oxide and cardiac function. Circ Res, 1996, 79(3): 363-80
45. Haywood GA, Taso PS, Legen HE, et al. Expression of inducible nitric oxide synthase in human heart failure. Circulation, 1996, 93(6):1087
46．亢晓东，陈河，黄先玫，等．阿霉素对兔心功能及心肌一氧化氮合酶表达的影响．实用癌症杂志．2002，16(4)：377-78
47. Packer MA, et al. Biochem Mol Biol Int, 1996, 40 (3): 527-34
48. Szabo C, Salzman AL. Biochem Biophys Res Commun, 1995, 209(2): 739-43
49．陈国荣，刘毅，毛孙忠，等．黄芪对糖尿病性肾病大鼠心肌脂质过氧化作用及一氧化氮水平的影响．中国应用生理学杂志，2001，17(2)：186-88
50．裴林，刘唤荣，张少丹，等．黄芪对缺氧缺血性脑损伤幼鼠一氧化氮、丙二醛含量的影响．中国中西医结合急救杂志，2001，8(4)：222-24
51．祁中华，林善锬，黄宇锋．黄芪改善糖尿病早期肾血流动力学异常的研究．中国糖尿病杂志，1999，7(3)：147-49
52. Marks AR. Intracellular calcium-release channels: regulators of cell life and death. Am J Physiol, 1997, 272: 597-605
53. Maeda A, Honda M, Kuramochi T, et al. Doxorubicin cardiotoxicity: diastolic cardiac myocyte dysfunction as a result of imparied calcium bonding in isolated cardiac myocytes. Jpn Circ J, 1998,62(7):505
54．黄先玫，康曼丽，周卸来，等．阿霉素对实验兔心肌细胞内游离钙浓度的影响．中国医院药学杂志，2001，21(2)：73-5
55．张亚臣，荣烨之，赵美华，等．阿霉素中毒心肌细胞脂质过氧化及其导致的钙超负荷的研究．中国病理生理杂志，1998，14(6)：688-90
56. Olso RD, Mushilin PD. Doxorubicin cardiotoxicity: analysis of prevailing hypotheses. FASEB J, 1990, 4:3076
57. Kim DH, Enrhol B. Activation of calcium release channels from camine cardiac sarcoplasmic reticulum by doxorubicin or caffeine. Circulation, 1988, 78: 141
58．陈伟，朱世军，刘立志，等．阿霉素致大鼠心肌损伤及抑制蛋白基因表达分析．中国地方病杂志，2001，20(4)：265-7
59．吴立玲主编．心血管病理生理学．北京：北京医科大学出版社，2000，P97-102
60．陆曙，张寄南，杨笛，等．黄芪总皂甙对病毒性心肌炎小鼠心肌损伤及肌浆网钙泵的影响．中国中西医结合杂志，1999，19(11)：672-74
2. Prasad K, Kalra J, Massey KL, et al. Increased production of oxygen free radicals by polymorphonuclear leukocytes in heart failure due to aortis stenosis. Angiology, 1989, 45 (5): 472-75
3. Hori M, Gotoch K, Kitakaze M, et al. Role of oxygen-derived free radicals in myocardial edema and ischemia in coronary microvascular embolization. Circulation. 1991, 84(2): 828
4．杨成明，刘光濯，牟永方等．脂质过氧化与充血性心力衰竭．中国循环杂志，1991，6(5)：376
5. Mcmurray J, Mclay J, Chopra M, et al. Evidence for enhanced free radical activity in chronic congestive heart failure secondary to coronary artery disease. Am J Cardiol, 1990, 15(5):1261
6. Hill MF, Singal PK. Antioxidant and oxidative stress changes during heart failure subsequent to myocardial infarction in rats. Am J Pathol, 1996, 148(1): 291-300
7．陈瑗，周玫主编．自由基医学基础与病理生理．北京：人民卫生出版社，2002年第一版
8．李中言，赵连友．一氧化氮与心血管疾病．中华心血管病杂志，1996，24：73-76
9. Oyama JI, Shimokawa H, Momal H, et al. Role of nitric oxide and peroxynitrite in the cytokine-induced sustained myocardial dysfunction in dogs in rive. J Clin Invest, 1998, 101 (2):207-14
10. Liu P, Hock CE, Nagele R, et al. Formation of nitric oxide, superoxide, and peroxynitrite in myocardial ischemia-reperfusion injury in rat. Am J Physiol, 1997, 272(52): H2 327-336
11. Zingarelli B, Cuzzocrea S, Zsengeller Z, et al. Protection against myocardial ischemia and reperfusion injury by 3-aminobenzamide, an inhibitor of poly (ADP-ribose) systhetase. Cardiovasc Res, 1997, 36:205-15
12. Ma XL, Loper BL, Liu GL, et al. Peroxynitrite aggravates myocardial reperfusion injury in the isolated perfuse rat heart. Cardiovase Res, 1997, 36:195-204
13．杨藻宸主编．药理学和药物治疗学．北京：人民卫生出版社，2000年第一版p1780-81
14. Singal PK, Iliskovic N. Doxorubicin-induced cardiomyopathy. N Engl J Med, 1998, 339: 900-5
15. Gewirtz DA. A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem Pharmacol, 1999, 57:727-41
16. Papadopoulou LC, Theophilidis G, Thomopoulous GN, et al. Structural and functional impairment of mitochondria in adriamycin-induced cardiomyopathy in mice: suppression of cytochrome c oxidase Ⅱ gene expression. Biochem Pharmacol, 1999, 57:481-9
17. Dealtey SM, Aksenov MY, Aksenova MV, et al. Adriamycin-induced changes of creatine kinase activity in vivo and in cardiomyocyte culture. Toxicology, 1999, 134:51-62
18. Serrano J, Palmeira CM, Kuehl DW, et al. Cardioselective and cumulative oxidation of mitochondrial DNA following subchronic doxorubicin administration. Biochem Biophys Acta, 1999, 1411:201-5
19．国家药典委员会．中华人民共和国药典．北京：化学工业出版社．2000，249-250
20．刘道芳，袁敏．黄芪研究进展．中医药信息，1998，(2)：13-15
21．韩玲，陈可冀．黄芪对心血管系统作用的实验药理学研究进展．中国中西医结合杂志，2000，20(3)：234-37
22．张燕燕，颜敏，文学明．黄芪抗脂质过氧化作用的研究．医药导报，2002，21(9)：542-43
23. Li ZP, Cao Q. Effects of astragaloside Ⅳ on myocardial calcium transport and cardiac function in ischemic rats. Acta Pharmacol Sin, 2002. 23(10): 898-904
24．刘恭鑫，杨英珍，顾全宝，等．黄芪对感染病毒大鼠心肌细胞钙通道及钠钙交换载体的效应．中国病理生理杂志，1999，15(7)：591-93
25．刘星楷，江明华，俞正坤．黄芪有效成分研究．黄芪中清除超氧阴离子成分的分离和检测．天然产物研究与开发，1991，3(4)：1-6
26．刘艳霞，刘在萍，焦建杰，等．黄芪苷Ⅳ对正常和心功能受抑制大鼠左心室心肌力学的影响．中草药，2001，32(4)：332-34
27．MDA测定试剂盒说明书．南京建成生物工程研究所
28．SOD测定试剂盒说明书．南京建成生物工程研究所
29．汪谦主编．现代医学实验方法．北京：人民卫生出版社，1997年第一版：P183-5，505-6
30．李会强，姚智，赵瑾莹，等．IL-2、IL-6及TNFαMTT检测方法的建立．天津第二医学院学报，1993，9：4-5
31．NOS测定试剂盒说明书．南京建成生物工程研究所
32．林秀珍，马德禄，崔荣芬．大黄素及番泻甙和大黄多糖对培养大肝细胞内游离钙浓度的影响．中国中西医结合杂志，1995，15(7)：419-21
33. Ito H, Torii M, Suzuki T. Decreased superoxide dismutase activity and increased superoxide anion production in cardiac hypertrophy of spontaneously hypertensive rats. Clin Exp Hypertens, 1995, 17(5): 803-16
34. Miller FJ Jr, Gutterman DD, Rios CD, et al. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res, 1998, 82(12):1298-1305
35. Iliskovic N, Li T, Khaper N, et al. Modulation of adriamycin-induced changes in serum free fatty acids, albumin and cardiac oxidative stress. Mol Cell Biochem, 1998, 188(1-2):161-66
36. Campo GM, Squadrito F, Campo S, et al. Beneficial effect of raxofellast, an hydrophilic vitamin E analogue, in the rat heart after ischemia and reperfusion injury. J Mol Cell Cardiol, 1998, 30(8): 1493-1503
37. Doroshow JH. Doxorubicin-induced cardiac toxity. N Engl J Med, 1991, 324: 843Dealtey SM, Aksenov MY, Aksenova MV, et al. Adriamycin-induced changes of creatine kinase activity in vivo and in cardiomyocyte culture. Toxicology, 1999, 134:51-62
38. Doroshow JH. Anthracycline-enhanced, cardiac oxygen radical metabolism. In: Singal PK ed. Oxygen Radicals in the Pathophysiology of Heart Disease. Boston: Kluwer Academic publishers, 1988: 323-32
39. Nohl H. Identification of the site of adriamycin activation in the heart cell. Biochem Pharmacol, 1988, 37:2633
40. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J Immuno Methods, 1983, 65:55-63
41．万影，陈玉石．黄芪对超氧阴离子自由基的清除作用．数理医药学杂志，1998；11(1)：61-62
42．孙成文，钟国赣，江岩，等．黄芪多糖抗氧化损伤作用的研究．中国药理学通报，1996：12(2)：161-63
43．李靖，朱建华，黄崇勤，等．黄芪皂甙对培养心肌细胞损伤的影响．中国现代医学杂志，1998；8(8)：7-9
44. Kelly RA, Balligand JL, Smith TW. Nitric oxide and cardiac function. Circ Res, 1996, 79(3): 363-80
45. Haywood GA, Taso PS, Legen HE, et al. Expression of inducible nitric oxide synthase in human heart failure. Circulation, 1996, 93(6):1087
46．亢晓东，陈河，黄先玫，等．阿霉素对兔心功能及心肌一氧化氮合酶表达的影响．实用癌症杂志．2002，16(4)：377-78
47. Packer MA, et al. Biochem Mol Biol Int, 1996, 40 (3): 527-34
48. Szabo C, Salzman AL. Biochem Biophys Res Commun, 1995, 209(2): 739-43
49．陈国荣，刘毅，毛孙忠，等．黄芪对糖尿病性肾病大鼠心肌脂质过氧化作用及一氧化氮水平的影响．中国应用生理学杂志，2001，17(2)：186-88
50．裴林，刘唤荣，张少丹，等．黄芪对缺氧缺血性脑损伤幼鼠一氧化氮、丙二醛含量的影响．中国中西医结合急救杂志，2001，8(4)：222-24
51．祁中华，林善锬，黄宇锋．黄芪改善糖尿病早期肾血流动力学异常的研究．中国糖尿病杂志，1999，7(3)：147-49
52. Marks AR. Intracellular calcium-release channels: regulators of cell life and death. Am J Physiol, 1997, 272: 597-605
53. Maeda A, Honda M, Kuramochi T, et al. Doxorubicin cardiotoxicity: diastolic cardiac myocyte dysfunction as a result of imparied calcium bonding in isolated cardiac myocytes. Jpn Circ J, 1998,62(7):505
54．黄先玫，康曼丽，周卸来，等．阿霉素对实验兔心肌细胞内游离钙浓度的影响．中国医院药学杂志，2001，21(2)：73-5
55．张亚臣，荣烨之，赵美华，等．阿霉素中毒心肌细胞脂质过氧化及其导致的钙超负荷的研究．中国病理生理杂志，1998，14(6)：688-90
56. Olso RD, Mushilin PD. Doxorubicin cardiotoxicity: analysis of prevailing hypotheses. FASEB J, 1990, 4:3076
57. Kim DH, Enrhol B. Activation of calcium release channels from camine cardiac sarcoplasmic reticulum by doxorubicin or caffeine. Circulation, 1988, 78: 141
58．陈伟，朱世军，刘立志，等．阿霉素致大鼠心肌损伤及抑制蛋白基因表达分析．中国地方病杂志，2001，20(4)：265-7
59．吴立玲主编．心血管病理生理学．北京：北京医科大学出版社，2000，P97-102
60．陆曙，张寄南，杨笛，等．黄芪总皂甙对病毒性心肌炎小鼠心肌损伤及肌浆网钙泵的影响．中国中西医结合杂志，1999，19(11)：672-74