乳大鼠心肌细胞糖氧剥夺-营养恢复模型模拟在体缺血/再灌注损伤的可行性研究
|
摘要
目的乳大鼠心肌细胞糖氧剥夺-营养恢复(OGD-NR)模型是最常用的模拟心肌缺血-再灌注损伤(myocardial ischemiareperfusion injury,MIRI)的离体建模方法之一。但是,至今尚无研究对该模型的可行性进行评估。该实验旨在评价乳大鼠心肌细胞OGD-NR离体心肌细胞模型模拟在体MIRI的可行性。方法将乳大鼠心肌细胞随机分成对照(C)组、模拟缺血(SI)组和模拟缺血再灌注(SIR)组。实验结束时检测细胞形态学、乳酸脱氢酶(LDH)释放情况、三磷酸腺苷(ATP)水平、活性氧(ROS)水平、线粒体膜电位(MMP)水平以及炎症因子水平,以评价各组细胞损伤的表型和特点。结果与C组相比,SI组的心肌细胞形态学出现损伤、LDH释放显著增加(P<0.05)、ATP明显降低(P<0.05)、ROS生成增加(P<0.05)、MMP水平降低(P<0.05)。与SI组相比,SIR组的心肌细胞形态学并未进一步恶化,LDH释放明显降低(P<0.05)、ATP水平明显增高(P<0.05)。与SI组相比,相应的SIR组未发生大量ROS生成、MMP坍塌以及过度的炎症反应。结论乳大鼠心肌细胞OGD-NR不能成功模拟在体MIRI的特征,即对发生SI损伤的心肌细胞实施SIR处理后,形态学上未出现进一步的损伤、LDH释放明显降低、ATP明显增高、无大量ROS生成、无MMP坍塌且没有过度炎症反应发生。
Objective Oxygen glucose deprivation nutrition resumption(OGD-NR) model on neonatal rat cardiomyocytes(NRCs) is commonly used in vitro model of simulated myocardial ischemia-reperfusion injury(MIRI), but there has been no study to assess whether this model can effectively imitate the characteristics of MIRI in vivo. This experiment was designed to assess the feasibility of this model simulating MIRI.Methods NRCs were randomly divided into control(C), simulated ischemia(SI) and simulated ischemia-reperfusion(SIR) groups, and cell morphology, lactate dehydrogenase(LDH) release, adenosine triphosphate(ATP) levels, reactive oxygen species(ROS), mitochondrial membrane potential(MMP) and inflammatory cytokines were examined to evaluate the phenotype and characteristics of cell injury. Results Compared with the C group, the cells suffered from morphological damage, LDH release significantly increased(P<0.05), ATP evidently decreased(P<0.05), ROS production significantly increased(P<0.05) and MMP levels obviously decreased in the SI group(P<0.05). Compared with the SI group, however,cellular morphology did not worsen, LDH release significantly decreased(P<0.05), and ATP level evidently increased(P<0.05) in the SIR group.In addition, large generation of ROS, MMP collapse, and over-inflammatory response did not occur in the SIR group compared with the SI group.Conclusion OGD-NR of neonatal rat cardiomyocytes could not successfully simulate the characteristics of MIRI in vivo, as demonstrated by no further damage in morphology, significant decrease in LDH release, significant increase in ATP, no large generation of ROS, no MMP collapse, and no over-inflammatory response by SIR treatment after SI treatment.
Objective Oxygen glucose deprivation nutrition resumption(OGD-NR) model on neonatal rat cardiomyocytes(NRCs) is commonly used in vitro model of simulated myocardial ischemia-reperfusion injury(MIRI), but there has been no study to assess whether this model can effectively imitate the characteristics of MIRI in vivo. This experiment was designed to assess the feasibility of this model simulating MIRI.Methods NRCs were randomly divided into control(C), simulated ischemia(SI) and simulated ischemia-reperfusion(SIR) groups, and cell morphology, lactate dehydrogenase(LDH) release, adenosine triphosphate(ATP) levels, reactive oxygen species(ROS), mitochondrial membrane potential(MMP) and inflammatory cytokines were examined to evaluate the phenotype and characteristics of cell injury. Results Compared with the C group, the cells suffered from morphological damage, LDH release significantly increased(P<0.05), ATP evidently decreased(P<0.05), ROS production significantly increased(P<0.05) and MMP levels obviously decreased in the SI group(P<0.05). Compared with the SI group, however,cellular morphology did not worsen, LDH release significantly decreased(P<0.05), and ATP level evidently increased(P<0.05) in the SIR group.In addition, large generation of ROS, MMP collapse, and over-inflammatory response did not occur in the SIR group compared with the SI group.Conclusion OGD-NR of neonatal rat cardiomyocytes could not successfully simulate the characteristics of MIRI in vivo, as demonstrated by no further damage in morphology, significant decrease in LDH release, significant increase in ATP, no large generation of ROS, no MMP collapse, and no over-inflammatory response by SIR treatment after SI treatment.
引文
[1] Yellon DM, Hausenloy DJ. Myocardial reperfusion injury. N Engl J Med, 2007,357(11):1121-1135.
[2] Vidavalur R, Swarnakar S, Thirunavukkarasu M, et al. Ex vivo and in vivo approaches to study mechanisms of cardioprotection targeting ischemia/reperfusion(i/r)injury:useful techniques for cardiovascular drug discovery. Curr Drug Discov Technol, 2008,5(4):269-278.
[3] Boccalini G, Sassoli C, Formigli L, et al. Relaxin protects cardiac muscle cells from hypoxia/reoxygenation injury:involvement of the Notch-1 pathway. FASEB J, 2015,29(1):239-249.
[4] Chen C, Jia KY, Zhang HL, et al. MiR-195 enhances cardiomyocyte apoptosis induced by hypoxia/reoxygenation injury via downregulating c-myb. Eur Rev Med Pharmacol Sci, 2016,20(16):3410-3416.
[5] Habener A, Chowdhury A, Echtermeyer F, et al. MitoNEETProtects HL-1 Cardiomyocytes from Oxidative Stress Mediated Apoptosis in an In Vitro Model of Hypoxia and Reoxygenation.PLoS One, 2016,11(5):e0156054.
[6] Fernández-Jimenez R, Galan-Arriola C, Sanchez-Gonzalez J, et al. Effect of Ischemia Duration and Protective Interventions on the Temporal Dynamics of Tissue Composition After Myocardial
[7] Infarction. Circ Res, 2017,121(4):439-450.Becker LC, Jeremy RW, Schaper J, et al. Ultrastructural assessment of myocardial necrosis occurring during ischemia and 3-h reperfusion in the dog. Am J Physiol, 1999, 277(1 Pt 2):H243-H252.
[8] Vandergriff A C, Hensley M T, Cheng K. Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes. J Vis Exp,2015,117(98):161-169.
[9] Lu Y, Feng Y, Liu D, et al. Thymoquinone Attenuates Myocardial Ischemia/Reperfusion Injury Through Activation of SIRT1Signaling. Cell Physiol Biochem, 2018,47(3):1193-1206.
[10] Pasque MK, Wechsler AS. Metabolic intervention to affect myocardial recovery following ischemia. Ann Surg, 1984,200(1):1-12.
[11] Bagheri F, Khori V, Alizadeh AM, et al. Reactive oxygen speciesmediated cardiac-reperfusion injury:Mechanisms and therapies.Life Sci, 2016,165:43-55.
[12] Ong SB, Samangouei P, Kalkhoran SB, et al. The mitochondrial permeability transition pore and its role in myocardial ischemia reperfusion injury. J Mol Cell Cardiol, 2015,78:22-34.
[13] Steffens S, Montecucco F, Mach F. The inflammatory response as a target to reduce myocardial ischaemia and reperfusion injury.Thromb Haemost,2009,102(2):240-247.
[14] Altschuld RA, Hostetler JR, Brierley GP. Response of isolated rat heart cells to hypoxia, re-oxygenation, and acidosis. Circ Res, 1981,49(2):307-316.
[15] Hearse DJ, Humphrey SM, Chain EB. Abrupt reoxygenation of the anoxic potassium-arrested perfused rat heart:a study of myocardial enzyme release. J Mol Cell Cardiol,1973,5(4):395-407.
[16] Siegmund B, Koop A, Klietz T, et al. Sarcolemmal integrity and metabolic competence of cardiomyocytes under anoxiareoxygenation. Am J Physiol, 1990,258(2 Pt 2):H285-H291.
[17] Piper HM, Abdallah Y, Schafer C. The first minutes of reperfusion:a window of opportunity for cardioprotection. Cardiovasc Res,2004,61(3):365-371.
[18] Avkiran M, Marber MS. Na+/H+exchange inhibitors for cardioprotective therapy:progress, problems and prospects. J Am Coll Cardiol, 2002, 39(5):747-753.
[19] Befroy DE, Powell T, Radda GK, et al. Osmotic shock:modulation of contractile function, pHi, and ischemic damage in perfused guinea pig heart. Am J Physiol, 1999,276(4 Pt 2):H1236-H1244.
[20] Raedschelders K, Ansley DM, Chen DD. The cellular and molecular origin of reactive oxygen species generation during myocardial ischemia and reperfusion. Pharmacol Ther, 2012,133(2):230-255.
[21] Bonaventura A, Montecucco F, Dallegri F. Cellular recruitment in myocardial ischaemia/reperfusion injury. Eur J Clin Invest, 2016,46(6):590-601.
[22] Garcia-Dorado D, Théroux P, Desco M, et al. Cell-to-cell interaction:a mechanism to explain wave-front progression of myocardial necrosis. Am J Physiol, 1989, 256(5 Pt 2):H1266-H1273.
[23] Gross GJ, Auchampach JA. Reperfusion injury:does it exist? J Mol Cell Cardiol, 2007,42(1):12-18.
[24] De Groot H. Isolated cells in the study of the molecular mechanisms of reperfusion injury. Toxicol Lett, 1992,63(2):111-125.
[2] Vidavalur R, Swarnakar S, Thirunavukkarasu M, et al. Ex vivo and in vivo approaches to study mechanisms of cardioprotection targeting ischemia/reperfusion(i/r)injury:useful techniques for cardiovascular drug discovery. Curr Drug Discov Technol, 2008,5(4):269-278.
[3] Boccalini G, Sassoli C, Formigli L, et al. Relaxin protects cardiac muscle cells from hypoxia/reoxygenation injury:involvement of the Notch-1 pathway. FASEB J, 2015,29(1):239-249.
[4] Chen C, Jia KY, Zhang HL, et al. MiR-195 enhances cardiomyocyte apoptosis induced by hypoxia/reoxygenation injury via downregulating c-myb. Eur Rev Med Pharmacol Sci, 2016,20(16):3410-3416.
[5] Habener A, Chowdhury A, Echtermeyer F, et al. MitoNEETProtects HL-1 Cardiomyocytes from Oxidative Stress Mediated Apoptosis in an In Vitro Model of Hypoxia and Reoxygenation.PLoS One, 2016,11(5):e0156054.
[6] Fernández-Jimenez R, Galan-Arriola C, Sanchez-Gonzalez J, et al. Effect of Ischemia Duration and Protective Interventions on the Temporal Dynamics of Tissue Composition After Myocardial
[7] Infarction. Circ Res, 2017,121(4):439-450.Becker LC, Jeremy RW, Schaper J, et al. Ultrastructural assessment of myocardial necrosis occurring during ischemia and 3-h reperfusion in the dog. Am J Physiol, 1999, 277(1 Pt 2):H243-H252.
[8] Vandergriff A C, Hensley M T, Cheng K. Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes. J Vis Exp,2015,117(98):161-169.
[9] Lu Y, Feng Y, Liu D, et al. Thymoquinone Attenuates Myocardial Ischemia/Reperfusion Injury Through Activation of SIRT1Signaling. Cell Physiol Biochem, 2018,47(3):1193-1206.
[10] Pasque MK, Wechsler AS. Metabolic intervention to affect myocardial recovery following ischemia. Ann Surg, 1984,200(1):1-12.
[11] Bagheri F, Khori V, Alizadeh AM, et al. Reactive oxygen speciesmediated cardiac-reperfusion injury:Mechanisms and therapies.Life Sci, 2016,165:43-55.
[12] Ong SB, Samangouei P, Kalkhoran SB, et al. The mitochondrial permeability transition pore and its role in myocardial ischemia reperfusion injury. J Mol Cell Cardiol, 2015,78:22-34.
[13] Steffens S, Montecucco F, Mach F. The inflammatory response as a target to reduce myocardial ischaemia and reperfusion injury.Thromb Haemost,2009,102(2):240-247.
[14] Altschuld RA, Hostetler JR, Brierley GP. Response of isolated rat heart cells to hypoxia, re-oxygenation, and acidosis. Circ Res, 1981,49(2):307-316.
[15] Hearse DJ, Humphrey SM, Chain EB. Abrupt reoxygenation of the anoxic potassium-arrested perfused rat heart:a study of myocardial enzyme release. J Mol Cell Cardiol,1973,5(4):395-407.
[16] Siegmund B, Koop A, Klietz T, et al. Sarcolemmal integrity and metabolic competence of cardiomyocytes under anoxiareoxygenation. Am J Physiol, 1990,258(2 Pt 2):H285-H291.
[17] Piper HM, Abdallah Y, Schafer C. The first minutes of reperfusion:a window of opportunity for cardioprotection. Cardiovasc Res,2004,61(3):365-371.
[18] Avkiran M, Marber MS. Na+/H+exchange inhibitors for cardioprotective therapy:progress, problems and prospects. J Am Coll Cardiol, 2002, 39(5):747-753.
[19] Befroy DE, Powell T, Radda GK, et al. Osmotic shock:modulation of contractile function, pHi, and ischemic damage in perfused guinea pig heart. Am J Physiol, 1999,276(4 Pt 2):H1236-H1244.
[20] Raedschelders K, Ansley DM, Chen DD. The cellular and molecular origin of reactive oxygen species generation during myocardial ischemia and reperfusion. Pharmacol Ther, 2012,133(2):230-255.
[21] Bonaventura A, Montecucco F, Dallegri F. Cellular recruitment in myocardial ischaemia/reperfusion injury. Eur J Clin Invest, 2016,46(6):590-601.
[22] Garcia-Dorado D, Théroux P, Desco M, et al. Cell-to-cell interaction:a mechanism to explain wave-front progression of myocardial necrosis. Am J Physiol, 1989, 256(5 Pt 2):H1266-H1273.
[23] Gross GJ, Auchampach JA. Reperfusion injury:does it exist? J Mol Cell Cardiol, 2007,42(1):12-18.
[24] De Groot H. Isolated cells in the study of the molecular mechanisms of reperfusion injury. Toxicol Lett, 1992,63(2):111-125.