摘要
目的:研究通络方剂和褪黑素、α-硫辛酸和金属硫蛋白等抗氧化剂对糖尿病大鼠心肌细胞和组织的抗凋亡作用及超氧化物歧化酶2(SOD2)、泛素D表达的影响及其作用机理。方法:乳鼠心肌细胞在高糖培养基(25mmo/L)中培养48h,MTT法检测细胞活力,RT-PCR检测SOD2mRNA表达;链脲佐菌素诱导大鼠糖尿病模型,电镜观察心肌组织形态,免疫组化原位检测细胞凋亡,基因芯片技术比较大鼠心肌组织中基因表达差异,RT-PCR检测泛素DmRNA表达。结果:α-硫辛酸可增强高糖培养的心肌细胞SOD2mRNA表达,金属硫蛋白、通络方剂和大剂量褪黑素减少心肌细胞凋亡,物质代谢相关基因和细胞骨架相关基因在糖尿病大鼠心肌组织明显下调,通络方剂和大剂量褪黑素可使上述基因上调。结论:褪黑素、α-硫辛酸和金属硫蛋白等抗氧化剂通过抑制氧化应激反应,抑制凋亡,对糖尿病大鼠心肌起保护作用,通络方剂对糖尿病心肌病变也有一定保护作用。
Objective: to investigate the effect of Tongluo recipe and melatonin, Metallothionein, a-lipoic acid on diabetic cardiomyopathy in rats. Method: myocardial cell were cultured in high glucose (25mmol/L) for 48h, SODmRNA were measured by RT-PCR; Rats were induced in diabetes with injection of Streptozocin, histological changes of hearts were determined by electron microscope; the difference of rats heart’s gene were determined with DNA microarray, UbDmRNA were measured by RT-PCR. Result : a-lipoic acid increase the expression of SODmRNA in rat myocardial cell, Tongluo recipe, melatonin and Metallothionein decrease the apoptosis of myocardium, energy metabolism related gene were down regulated in diabetic rat’s myocardium, melatonin up regulated the gene expression mentioned above. Conclusion: oxidative stress plays an important role in the pathogenesis of diabetic cardiomyopathy. Melatonin, Metallothionein and a-lipoic acid have protective effect on diabetic rat’s myocardium.
引文
[1] 司徒镇强.细胞培养基本知识.见:司徒镇强主编.细胞培养.西安:世界图书出版公司,2004,1-3.
[2] Park S, Sohn S, Kineman R D. Fasting-induced changes in the hypothalamic -pituitary-GH axis in the absence of GH expression: lessons from the spontaneous dwarf rat. J Endocrinol, 2004, 180:369-78.
[3] Colombrita C, Calabrese V, Stella AMG, et al. Regional rat brain distribution of Superoxide dismutase mRNA: relevance of redox homeostasis in the aging processes. Exp Biol Med, 2003, 228: 517-24.
[4] Harary L,Farley B. In vitro studies of single isolated beating heart cells. Science. 1960, 131: 1674-75.
[5]Yamamoto K, Ohki R, Lee R T, et a1. Peroxisome proliferator-activated receptor activators inhibit cardiac hypertrophy in cardiac myocytes. Circulation, 2001, 104: 1670-81.
[6] Fuller SJ, Davies EL, Gillespie BJ, et a1. Mitogen-activated protein kinase phosphataseⅠinhibits the stimulation of expression by hypertrophic agonists in cardiac myocytes. Biochem, 1997, 323(Pt 2):313-9.
[7] Pinsky D J, Aji W, Szabolcs M, et a1.Nitric oxide triggers programmed cell death (apoptosis) of adult rat ventricular myocytes in culture.Am J PhysioI, 1999,277(3 Pt 2): H1189-99.
[8] Yonemochi H, Yasunaga S, Teshima Y, et al. Mechanism of adrenergic receptor up regulation induced by ACE inhibition in cultured neonatal rat cardiac myocytes. Circ, 1998, 97: 2268-73
[9] Simpson P, Savion S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Circ Res, 1982, 50: 101-16.
[10] 刘斌.细胞培养中的研究方法.见:司徒镇强主编.细胞培养.西安:世界图书出版公司,2004,250-252.
[11] Brownlee M. The pathobiology of diabetic complications: a unifying mechanism. Diabetes. 2005;54(6):1615-25.
[12] 周庆峰,王洪新,王桂君,等.高糖对去甲肾上腺素诱导的心肌细胞肥大的促进作用.中国药理学通报,2003,19(9):1054-7.
[13]Yokota T, Ma RC, Park JY, et al. Role of protein kinase C on the expression of platelet-derived growth factor and endothelin-1 in the retina of diabetic rats and cultured retinal capillary pericytes. Diabetes. 2003 ; 52 3):838-45.
[14] Sung MJ, Kim W, Ahn SY, et al. Protective effect of alpha-lipoic acid in lipopolysaccharide -induced endothelial fractalkine expression. Circ Res. 2005, 7:880-90.
[15] Bojunga J, Nowak D, Mitron PS, et al. Antioxidative treatment prevents activation of death-receptor- and mitochondrion-dependent apoptosis in the hearts of diabetic rats. Diabetologia. 2004 , 47:2072-80.
[16] Stockklauser-Farber K, Ballhausen T, Laufer A, et al. Influence of diabetes on cardiac nitric oxide synthase expression and activity. Biochim Biophys Acta. 2000, 1535: 10-20.
[17] Ramnath S, Tritschler HJ, Eckel J. Stimulation of cardiac glucose transport by thioctic acid and insulin. Horm Metab Res. 1999,31:632-5.
[18] Strodter D, Lehmann E, Lehmann U, et al. The influence of thioctic acid on metabolism and function of the diabetic heart. Diabetes Res Clin Pract. 1995, 29: 19-26.
[19] Xie JT, Shao ZH, Vanden Hoek TL. Antioxidant effects of ginsenoside Re in cardiomyocytes. Eur J Pharmacol, 2006, 532:201-7.
[20] Cho WC, Chung WS, Lee SK, et al. Ginsenoside Re of Panax ginseng possesses significant antioxidant and antihyperlipidemic efficacies in streptozotocin-induced diabetic rats. Eur J Pharmacol, 2006, 550:173-9.
[21] Kang KS, Kim HY, Pyo JS, et al. Increase in the free radical scavenging activity of ginseng by heat-processing. Biol Pharm Bul,. 2006, 29:750-4.
[22]Shao ZH, Xie JT, Vanden Hoek TL. Antioxidant effects of American ginseng berry extract in cardiomyocytes exposed to acute oxidant stress. Biochim Biophys Acta. 2004, 1670:165-71.
[23] Liao B, Newmark H, Zhou R. Neuroprotective effects of ginseng total saponinand ginsenosides Rb1 and Rg1 on spinal cord neurons in vitro. Exp Neurol. 2002, 173: 224-34.
[24] Clark W A, Decker M L, Behnke-Barclay M, et a1. Cell contact as an independent factor modulating cardiacmyocytes hypertrophy and survival in long term primary culture. J Mol Cell Cardiol, 1998, 30(1):139-55.
[1] Hamby RI,Zoneraich S, Sherman L.Diabetic cardiomyopathy.JAMA,1974; 229(13): 1749-54.
[2] Schannwell CM, Schneppenheim M,Perings S,et a1.Left ventricular diastolic dysfunction as an early manifestation of diabetic cardiomyopathy.Cardiology,2002;98(1-2):33-9.
[3] Poirier P,Bogaty P,Garneau C,et a1.Diastolic dysfunction in normotensive men with well—controlled type 2 diabetes:importance of maneuvers in echocardiographic screening for preclinical diabetic cardiomyopathy.Diabetes Care,2001,24(1):5-10.
[4] 白永虹,田杰,江德勤,等. 大鼠心肌病模型的建立及彩色超声心动图监测的研究. 重庆医学,2005,34(2):168-70
[5] Fiordaliso F, Li B, Latini R, et a1.Myocyte death in streptozotocin -induced diabetes in rats in angiotensin II- dependent. Lab Invest 2000; 80(4): 513-27
[6]Rodrique B, Cam MC, Mcneill JH. Metabolic disturbances in diabetic cardiomyopathy. Mol Cell Biochem 1998;180(1):53-7
[7] Kockx MM,Knaapen MW.The role of apoptosis in vascular disease.J Pathol,2000,190:267-80.
[8] Hengartner M.The biochemistry of apoptosis.Nature,2000,407:770 -76.
[9] Cai L, Li W, Wang GW, et al. Hyperglycemia induced apoptosis in mouse myocardium. Diabetes,2002,51:1938-48
[10]张超英,党寅虎,王 辉,等.硒缺乏对大鼠心肌细胞凋亡的影响及褪黑素的干预作用. 西安交通大学学报(医学版),2004,25(6):542-44
[11] Sailaja D M,Suresh Y,Das.Preservation of the antioxidant status in chemically—induced diabetes mellitus by melatonin.J Pineal Res.2000.29:108-l5.
[12] Abdel-Wahab MM,Abd-Allah AR. Possible Protective effect of melatonin and/ or desferrioxamine against streptozotocin—induced hyperglycemia in mice. Pharmacol Res. 2000,41:533-37.
[13] Burikhanov RB, Wakame K, Igarashi Y, et al. Suppressive effect of active hexosecorrelated compound (AHCC) on thymic apoptosis induced by dexamethasone in the rat. Endocr Regul, 2000, 34(4):181-8.
[14] Kaneko S , Okumura K, Numaguchi Y, et al . Melatonin scavenges hydroxyl radical and protects isolated rat hearts from ischemic reperfusion injury. Life Sci , 2000 , 67 (2) :101- 12. [ 15] Reiter RJ , Tan D X. Melatonin : a novel protective agent against oxidative injury of the ischemic/ reperfused heart J . Cardiovasc Res, 2003 , 58 (1) :10-9.
[16] Dobsak P , Siegelova J , Eicher J C , et al . Melatonin protects against ischemia reperfusion injury and inhibits apoptosis in isolated working rat heart. Pathophysiology, 2003 , 9 (3) : 179-87.
[17]Wang JX, Song Y, Elsherif M, et al. Cardiac Metallothionein induction plays the majoy role in the prevention of diabetic cardiomyopathy by zinc supplementation. Circulation, 2006,113:544-54.
[18] Song Y, Wang JX, Li Y, et al. Cardiac Metallothionein synthesis in Streptozocin induced diabetic mice and its protection against diabetes induced cardiac injury. Am J Pathol, 2005,167:17-26.
[19] Baird SK, Kurz T, Brunk UT. Metallothionein protects against oxidative stress induced lysosomal destabilization. Biochem, 2006,394:275-83.
[20] Cai L, Wang JX, Li Y, et al. Inhibition of Superoxide generation and association nitrosative damage is involved in Metallothionein prevention of diabetic cardiomyopathy. Diabetes, 2005,54:1829-37.
[21] Bojunga J, Nowak D, Mitron PS, et al. Antioxidative treatment prevents activation of death-receptor- and mitochondrion-dependent apoptosis in the hearts of diabetic rats. Diabetologia. 2004 , 47:2072-80.
[22] Berryman AM, Maritim AC, Sanders RA. Influence of treatment of diabetic rats with combinations of pycnogenol, beta-carotene, and alpha-lipoic acid on parameters of oxidative stress. J Biochem Mol Toxicol. 2004;18(6):345-52.
[23] Maritim AC, Berryman AM, Watkins JB. Effects of alpha-lipoic acid on biomarkers of oxidative stress in streptozotocin-induced diabetic rats. J Nutr Biochem. 2003 May;14(5):288-94.
[24] Diesel B, Kulhanek-Heinze S, Holtje M, et al. Alpha-lipoic acid as a directly binding activator of the insulin receptor: protection from hepatocyte apoptosis. Biochemistry, 2007,46(8): 2146-55.
[1] Lopez-Avalos M, Duvivier-Kali V, Xu G, et al. Evidence for a role of the ubiquitin-proteasome pathway in pancreatic islets. Diabetes,2006,55:1223-31.
[2] 孙啸,王晔,何农跃,等.生物信息学在基因芯片中的应用.生物生理学报,2001,17(1):27-34.
[3] Susztak K, Bottinger E, Novetsky A, et al. Molecular profiling of diabetic mouse kidney reveals novel genes linked to glomerular disease. Diabetes. 2004, 53(3): 784-94.
[4] Albrektsen T, Frederiksen KS, Holmes WE, et al. Novel genes regulated by the insulin sensitizer rosiglitazone during adipocyte differentiation. Diabetes. 2002 , 51(4): 1042-51.
[5] 郭清华,尹玲,陆菊明,等. Kkay小鼠2型糖尿病相关基因的表达谱研究.第 二军医大学学报,2001,22(7):631-35.
[6] 崔春黎,李素基因芯片研究糖尿病肾病大鼠肾皮质基因表达、同济大学学报.2001, 22(2):13-17.
[7] Vogel H. Mitochondrial myopathies and the role of the pathologist in the molecular era. J Neuropathol Exp Neurol. 2001, 60(3):217-27.
[8]Yechoor VK, Patti M, Saccone R, et al. Coordinated patterns of gene expression for substrate and energy metabolism in skeletal muscle of diabetic mice. Proc Natl Acad Sci USA, 2002, 99(16): 10587-92.
[9] Massa L, Baltrusch S, Okar DA, et al. Interaction of 6-phosphofructo -2-kinase/ fructose-2,6-bisphosphatase (PFK-2/FBPase-2) with glucokinase activates glucose phosphorylation and glucose metabolism in insulin-producing cells. Diabetes. 2004 ,53(4):1020-9.
[10] von Eckardstein A, Nofer JR, Assmann G. High density lipoproteins and arteriosclerosis. Role of cholesterol efflux and reverse cholesterol transport. Arterioscler Thromb Vasc Biol. 2001 Jan;21(1):13-27.
[11] Hiyoshi H, Yanagimachi M, Ito M, et al. Squalene synthase inhibitors reduce plasma triglyceride through a low-density lipoprotein receptor-independentmechanism. Eur J Pharmacol, 2001, 431(3):345-52.
[12]Lyons TJ, Li W, Wells- knecht MC, et al. Toxicity of mildly modified low-density lipoproteins to cultured retinal capillary endothelial cells and pericytes.. Diabetes. 1994, 40:1090-5
[13] Nie H, Wu JL, Zhang M, et al. Endothelial nitric oxide synthase-dependent tyrosine nitration of prostacyclin synthase in diabetes in vivo. Diabetes. 2006 , 55(11):3133-41.
[14] Ceriello A, Morocutti A, Mercuri F, et al. Defective intracellular antioxidant enzyme production in type 1 diabetic patients with nephropathy. Diabetes, 2000, 49:2170-78.
[15] Mari M, Cederbaum AI. Induction of catalase, alpha, and microsomal glutathione S-transferase in CYP2E1 overexpressing HepG2 cells and protection against short-term oxidative stress. Hepatology. 2001,33(3):652-61.
[16]Cheng A, Dube N, Gu F, et al. Coordinated action of protein tyrosine phosphatase in insulin signal transduction. Eur J Biochem. 2002, 269:1050-9.
[17]Stephan M, Michael S. Pathophysiology and pharmacological treament of insulin resistance. Endocrin Reviews. 2000, 21 (6):585-618.
[18] Wang Z, Hall SD, Maya JF, et al. Diabetes mellitus increases the in vivo activity of cytochrome P450 2E1 in humans. Br J Clin Pharmacol. 2003 Jan;55(1):77-85.
[19]陈刚,林丽香,庄维特等.用基因芯片技术研究糖尿病性心肌病大鼠心肌组 织的部分基因表达谱,中华心血管病杂志,2002,30(8):493-95.
[20]Kubasiak LA, Hernaddez OM, Bishopric NH, et al. Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3.Proc Natl Acad Sci U S A. 2002 , 99(20):12825-30.
[21]Regula KM, Ens K, Kirshenbaum LA, et al. Inducible expression of BINP3 provokes mitochondrial defect and hypoxia-mediated cell death of ventricular myocytes. Circ Res. 2002, 91(3):226-31.
[22]Desagher S, Osen-sand A, Nichols A, et a1.Bid induced conformational change of Bax in responsible for mitochondrial cytochrome C release during apoptosis. Cell Biol. 1999, 144(3):891-901.
[23] Baek KH. Conjugation and deconjugation of ubiquitin regulating the destiny of proteins. Exp Mol Med, 2003, 35(1):1-7.
[24]Yang Y, Yu X.Regulation of apoptosis: the ubiquitous way. FASEB J. 2003 May;17(8):790-9.
[25] 王晓,李果,李纪平,等.泛素在高糖诱导的血管内皮细胞凋亡中的表达.上海第二医科大学学报,2005,25:36-38.
[26]Knoll KE, Pietrusz JL, Liang M. Tissue-specific transcriptome responses in rats with early streptozotocin-induced diabetes. Physiol Genomics. 2005,21(2):222-9.
[27]Miyazaki H, Oka N, Koga A, et al. Comparison of gene expression profiling in pressure and volume overload-induced myocardial hypertrophies in rats. Hypertens Res. 2006,29(12):1029-45.
[1]Way KJ,Isshiki K,Suzuma K,et a1.Expression of connective tissue growth factor is increased in injured myocardium associated with proteinkinase C beta2 activation and diabetes.Diabetes. 2002; 51(9): 2709-18.
[2] Schannwell CM, Schneppenheim M,Perings S,et a1.Left ventricular diastolic dysfunction as an early manifestation of diabetic cardiomyopathy.Cardiology,2002;98(1-2):33-9.
[3] Poirier P,Bogaty P,Garneau C,et a1.Diastolic dysfunction in normotensive men with well—controlled type 2 diabetes:importance of maneuvers in echocardiographic screening for preclinical diabetic cardiomyopathy.Diabetes Care,2001,24(1):5-10.
[4] Brownlee M. The pathobiology of diabetic complications: a unifying mechanism. Diabetes. 2005;54(6):1615-25.
[5] Hattori Y, Matsuda N, Kimura J,et a1.Diminished function and expression of the cardiac Na+-Ca2+ exchanger in diabetic rats: implication in Ca2+ overload. J Physiol. 2000;527:85-94
[6] Teshima Y, Takahashi N, Saikawa T, et a1.Diminished expression of sarcoplasmic reticulum Ca(2+)-ATPase and ryanodine sensitive Ca(2+) Channel mRNA in streptozotocin-induced diabetic rat heart. J Mol Cell Cardiol 2000; 32(4): 655-64
[7] Kajstura J, Fiordaliso F, Andreoli AM, et a1.IGF-1 overexpression inhibits the development of diabetic cardiomyopathy and angiotensin II-mediated oxidative stress. Diabetes 2001; 50(6): 1414-1424
[8] Fiordaliso F, Li B, Latini R, et a1.Myocyte death in streptozotocin -induced diabetes in rats in angiotensin II- dependent. Lab Invest 2000; 80(4): 513-27
[9] 尤莉,陆福明,杨海春,等. 血管紧张素Ⅱ对大鼠系膜细胞葡萄糖转运蛋白1表达的影响.复旦大学学报(医学版),2002,29(6):433-36
[10] Kim H,Sasaki T, Maeda, K, et al. Protein kinase C beta selective inhibitor LY333531 attenuates diabetic hyperalgesia through ameliorating cGMP level of dorsal root ganglion neurons.Diabetes. 2003 ; 52(8): 2102 -9.
[11]Yokota T, Ma RC, Park JY, et al. Role of protein kinase C on the expression ofplatelet-derived growth factor and endothelin-1 in the retina of diabetic rats and cultured retinal capillary pericytes. Diabetes. 2003 ; 52 3):838-45.
[12] 马丽,朱邦豪,陈健义等,灯盏花素对糖尿病大鼠肾脏氧化应激的影响.中国药理学通报,2004;20(9):1030-3
[13]Du X, Matsumura T, Edelstein D, et a1. Inhibition of GAPDH activity by poly(ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells. J. Clin. Invest. 2003. 112: 1049-57.
[14]Rodrique B, Cam MC, Mcneill JH. Metabolic disturbances in diabetic cardiomyopathy. Mol Cell Biochem 1998;180(1):53-7
[15] Couture R, Girolami JP. Putative roles of kinin receptors in the therapeutic effects of angiotensin 1-converting enzyme inhibitors in diabetes mellitus. Eur J Pharmacol 2004;500(1-3):467-85