目的 研究利拉鲁肽对缺氧和高糖所致心肌细胞钙超载和细胞凋亡的影响。方法 采用原代培养的乳鼠心肌细胞,建立缺氧和高糖模型。实验分为:正常对照组、利拉鲁肽对照组、缺氧和高糖模型组、利拉鲁肽处理组、胰高血糖素样肽-1 (glucagon like peptide-1, GLP-1)受体抑制剂组、高渗对照组6组。采用荧光分光光度法检测心肌细胞内钙离子变化,化学荧光法检测活性氧族(reactive oxygen species, ROS)水平、利用生化方法检测Ca2+-ATP 和Na+-K+-ATP酶活性、RT-PCR技术检测钙敏感的钙蛋白酶(μ-calpain)基因表达、流式细胞仪测定细胞凋亡率、ELISA法检测细胞caspase-3活性。结果 与正常对照组相比,缺氧高糖模型组细胞内ROS水平、μ-calpain mRNA表达量、caspase-3活性均显著升高(P<0.01);Ca2+-ATP 和Na+-K+-ATP酶活性显著降低(P<0.01);游离钙离子浓度由(117.19±15.04)nmol/L增加至(508.53±26.11)nmol/L,细胞凋亡率由(3.95±0.12)%增加至(31.03±4.30)%(P<0.01)。利拉鲁肽处理组细胞较缺氧高糖模型组上述参数均明显改善,游离钙离子浓度和细胞凋亡率显著降低(P<0.01);GLP-1R抑制剂exendin(9-39)可拮抗利拉鲁肽的上述作用(P<0.05)。结论 利拉鲁肽可明显抑制缺氧和高糖所致心肌细胞钙超载及μ-calpain基因的上调,降低caspase-3水平,减少心肌细胞凋亡,对心肌细胞具有保护作用。
Abstract
Objective To investigate the effect and possible mechanism of liraglutide on hypoxia and high glucose-induced calcium overload and apoptosis in neonatal rat cardiomyocytes in vitro.Methods A model of hypoxia and high glucose was established using primarily cultured neonatal rat cardiomyocytes. Then, cells were divided into six groups: normal control group, liraglutide control group, hypoxia and high glucose model group, liraglutide treatment group, GLP-1R antagonist group, and high osmolality control group. The concentration of intracellular dissociative calcium in myocardial cells was determined with fluorospectrophotometry. The levels of Ca2+-ATPase, Na+-K+-ATPase and ROS were measured with biochemical approaches. The apoptosis rate was observed by a flow cytometer. The level of μ-calpain mRNA was examined with RT-PCR method. The level of caspase-3 was measured with ELISA.Results Compared with normal control group, the levels of expression of ROS, caspase-3, and μ-calpain mRNA were significantly increased in hypoxia and high glucose model group (P<0.01), the activity of Ca2+-ATPase and Na+-K+-ATPase decreased (P<0.01), the concentration of free Ca2+ increased from 117.19±15.04 nmol/L to 508.53±26.11 nmol/L, and the apoptosis rate increased from (3.95±0.12)% to (31.03±4.30)%. Liraglutide improved the parameters mentioned above (P<0.01). However, exendin(9-39), an antagonist of GLP-1R, attenuated the protective effect of liraglutide under hypoxia and high glucose.Conclusions Liraglutide can obviously inhibit calcium influx of myocardial cells and μ-calpain up-regulation, and decrease the level of caspase-3 and cell apoptosis.
关键词
利拉鲁肽 /
心肌细胞 /
缺氧 /
高糖 /
钙超载 /
凋亡
Key words
liraglutide /
cardiomyocyte /
hypoxia /
high glucose /
calcium overload /
apoptosis
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参考文献
[1] Gitt A K, Schiele R, Wienbergen H, et al. Intensive treatment of coronary artery disease in diabetic patients in clinical practice: results of the MITRA study [J]. Acta Diabetol, 2003, 40 (suppl 2):s343-s347.
[2] Thandavarayan R A, Giridharan V V, Watanabe K, et al. Diabetic cardiomyopathy and oxidative stress: role of antioxidants [J]. Cardiovasc Hematol Agents Med Chem, 2011, 9(4): 225-230.
[3] Xue W, Liu Y, Zhao J, et al. Activation of HIF-1 by metallothionein contributes to cardiac protection in the diabetic heart [J]. Am J Physiol Heart Circ Physiol, 2012, 302 (12): 2528-2535.
[4] Stamler J, Vaccaro O, Neaton J D, et al. Diabetes, Other Risk Factors, and 12-Yr Cardiovascular Mortality for Men Screened in the Multiple Risk Factor Intervention Trial[J]. Diabetes Care, 1993, 16(2): 434-444.
[5] Thom T, Haase N, Rosamond W, et al. Heart disease and stroke statistics-2006 update: a report from the american heart association statistics committee and stroke statistics subcommittee[J]. Circulation,2006,113(6):e85-e151.
[6] 杜燕京,封宇飞,傅得兴.利拉鲁肽的药理及临床研究进展[J].中国新药杂志,2010,19(23):2115-2119.
[7] 李建宇,李灵芝,张永亮,等.蕨麻醇提物对心肌细胞缺氧损伤的保护作用[J].中国新药杂志,2007,16(12):944-946.
[8] 龚海英,张 岭,李灵芝,等.蕨麻正丁醇部位对EAHY926内皮细胞缺氧损伤的保护作用[J]. 武警后勤学院学报(医学版),2012,21(6):401-404.
[9] Weiderpass E, Gridley G, Nyrén O, et al. Cause-specific mortality in a cohort of patients with diabetes mellitus: a population-based study in Sweden[J]. J Clin Epidemiol, 2001, 54(8): 802-809.
[10] Robertson R P. Chronic oxidative stress as a central mechanism for glucose toxicity in pancreatic islet beta cells in diabetes [J]. J Biol Chem, 2004, 279(41):42351-42354.
[11] 魏登邦,张宝琛.缺氧引起的自由基增殖及其损伤[J].生物学通报,2003,38(1):7-10.
[12] Takahashi H, Tran P O, LeRoy E, et al. D-Glyceraldehyde causes Production of intracellular Peroxide in Pancreatic islets, oxidative stress, and defective beta cell function via non-mitochondrial Pathways [J]. J Biol Chem, 2004, 279(16): 37316-37323.
[13] Haluzík M, Trachta P, Mráz M. Cardiovascular effects of GLP-1 receptor agonist treatment: focus on liraglutide [J]. Vnitr Lek, 2015, 61(7-8):635-640.
[14] Muhammad M H, Jason R R. Mechanism of pyrethroid pesticide-induced apoptosis: role of calpain and the ER stress pathway [J]. Toxicol Sci, 2011, 122(2):512-525.
[15] 陈运清,王 琳,苏陶凉,等.风湿性心房颤动患者calpain-Ⅰ/calpastatin和caspase-3与细胞凋亡的关系及其作用[J].中华心血管病杂志,2006,34(4):303-307.
[16] Carpi A, Venerando R, Miotto G, et al. Calpain and mitochondrial dysfunction in Ca2+ overloaded cardiomyocytes [J]. J Mol Cell Cardiol, 2007, 42(6):S105.
[17] Nehra S, Bhardwaj V, Saraswat D. Amlodipine protects rat ventricular cardiomyoblast H9c2 From hypoxia-induced apoptosis and restores oxidative balance by Akt-1-dependent manner [J]. J Cardiovasc Pharmacol, 2014, 64(4):375-384.
基金
湖北省知识创新专项(自然科学基金)(2016CFB198),湖北省卫计委科研基金(WJ2017Q031)
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