利多卡因预处理对高糖孵育H9c2大鼠成肌细胞缺氧复氧损伤的影响
|
摘要
研究背景
在缺血基础上恢复血流后组织损伤加重,甚至发生不可逆性损伤的现象称为缺血再灌注损伤(ischemia-reperfusion injury)。现已证实,心、脑、肝、肾、肺、胃肠道、肢体及皮肤等多种组织器官都存在缺血再灌注损伤的现象。心肌缺血再灌注损伤(myocardial ischemia/repeifusion injury, MIRI)为再灌注后缺血的心肌出现舒缩功能降低、心律失常、心肌能量代谢障碍、超微结构的变化和血管无复流等现象。缺血再灌注损伤的发生机制尚未彻底阐明,既往研究证实氧自由基(oxygen free radical, OFR)的作用、白细胞激活、内皮细胞(endothelial cell,EC)功能障碍、细胞内钙超载以及细胞凋亡是引起缺血一再灌注损伤的重要原因。
糖尿病(diabetes mellitus, DM)是常见病、多发病,现已明确DM是冠状动脉粥样硬化性心脏病发病的一个重要因素。流行病学资料显示：DM人群中心绞痛、急性心肌梗死(actue myocardial inarction, AMI)、充血性心力衰竭以及严重心律失常等疾病发病率远高于非DM患者,而且当DM患者并发心肌梗死后行药物或手术(包括冠状动脉搭桥和导管介入)治疗,效果均不及非DM患者。人们将这种不良后果归咎于缺血再灌注(ischemia/repeifusion, I/R),这可能与糖尿病患者冠状动脉粥样病变严重而弥漫、内皮功能障碍、储备能力降低所造成的心肌灌注不良有关,另外DM本身的促凝和炎症状态,易形成冠状动脉内微血栓堵塞；DM心肌缺氧应激,NO活性降低,存在冠脉微循环痉挛。而日本的一项多中心研究却显示：急诊经皮冠状动脉介入治疗(percutaneous coronary intervention, PCI)住院期间患者病死率与是否患有DM无关,而与血糖增高有关,且发现无DM但伴有高血糖者的死亡率最高,该研究推测这可能与DM性冠心病梗死相关血管(infarct-related artery, IRA)再通后I/R损伤增加有关。
基础研究显示DM发生时,心肌组织的结构、功能以及代谢均发生异常,主要表现为：①心肌能量代谢异常,且高糖环境能够直接引起正常心肌细胞功能改变；②细胞内钙调控异常；③微血管病变和微循环障碍,缓激肽释放酶—缓激肽系统在高血糖状态下该系统被抑制；④高血糖时氧化应激增加,信号转导途径改变,激活细胞凋亡。国内外研究证实,DM急性期,NO代偿性合成增加,I/R对DM心肌有保护作用；而DM晚期,由于慢性期NO减少,心肌肥厚,收缩和舒张功能受损,心脏血管周围或间质纤维化, I/R后损伤后将加重心肌损害。
近来,心肌缺血再灌注损伤和心肌保护一直是基础研究和临床研究的重点。1986年Murry等首次提出缺血预处理(Ischemia preconditioning, IPC)可以减轻缺血再灌注所致的心肌损伤,随后的一系列研究则发现许多麻醉药物(包括全身麻醉药、阿片类镇痛药以及局部麻醉药)预处理能产生与经典缺血预处理同样的心肌保护作用,这为预处理心肌保护的临床应用开辟了新的途径。利多卡因在急性心梗、恶性心律失常等引起的工R损伤治疗中具有较好的效果。有关预防性应用利多卡因的临床有效性和安全性一直备受争议。既往的荟萃分析(meta-analysis)表明预防性应用利多卡因虽然可减少急性心肌梗死患者心室纤颤的发生,但却增加了患者的死亡率。然而,一项多国家多中心观察研究表明：预防性应用利多卡因并不增加急性心肌梗死患者的死亡率,预防性应用利多卡因也许是无害的。总而言之,关于利多卡因预处理在心肌缺血再灌注损伤防治中的作用目前尚不明了,更不能确定利多卡因预处理心肌保护效应的具体作用机制。
目的
本研究通过在体外细胞水平以缺氧复氧处理H9c2大鼠成肌细胞建立心肌缺血再灌注损伤模型,初步探讨：①不同梯度浓度利多卡因预处理对缺氧复氧引起的心肌细胞损伤是否具有保护作用；②利多卡因预处理对缺氧复氧引起的H9c2心肌细胞凋亡的影响及其可能机制。③利多卡因预处理对高糖孵育下H9c2心肌细胞缺氧复氧损伤是否具有保护作用及与高糖孵育的关系。
’方法
第一部分：研究不同浓度利多卡因预处理对H9c2大鼠成肌细胞缺氧复氧损伤的影响。实验随机分为7组：正常对照组(NC组)、缺氧复氧损伤对照组(HR组)；利多卡因预处理组(LP组),根据利多卡因的不同浓度对分为LP1(1μmol/L)、LP2(2.5μmol/L)、LP3(5μmol/L)、LP4(10μmol/L)、LP5(20μmol/L)组,每组6孔。心肌细胞损伤程度以心肌细胞活力和乳酸脱氢酶(LDH)活性来表示,同时检测心肌细胞超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。
第二部分：研究利多卡因预处理对H9c2大鼠成肌细胞缺氧复氧损伤后caspase-3活性、钙超载的影响。实验随机分为3组：①正常对照组(NC组)；②缺氧复氧组(H/R组)；③利多卡因预处理组(LPC组)。心肌细胞损伤程度以心肌细胞活力和乳酸脱氢酶(LDH)活性来表示。用流式细胞术检测心肌细胞凋亡率。通过免疫细胞化学技术检测心肌细胞胞浆caspase-3的表达。荧光分光光度法检测细胞内钙离子浓度。
第三部分：研究利多卡因预处理对高糖孵育下H9c2心肌细胞缺氧复氧性损伤是否具有保护作用。实验随机分为二组,即正常糖浓度组(N组)和高糖浓度组(H组),每组又分为正常对照组(NC组和HC组)、缺氧复氧损伤组(NHR组和HHR组)和利多卡因预处理组(NLP组和HLP组)。心肌细胞损伤程度以H9c2细胞活力和培养基中乳酸脱氢酶(LDH)释放量来表示,同时检测H9c2细胞超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。
结果
第一部分：与NC组比较,HR组细胞活力显著减弱,LDH释放量显著增加,并且MDA含量增多,SOD活性降低；与HR组比较,L2-5组细胞活力显著增强,LDH释放量显著降低；并且MDA含量下降,SOD活性增强；且Pearson积差相关分析显示,细胞活力强弱的变化与LDH释放量(r=-0.870,P=0.000)、MDA含量(r=-0.924,P=0.000)呈负相关,与SOD活性呈正相关(r=0.894,P=0.000)。细胞活力的增强与利多卡因浓度的增加呈正相关(r=0.976,P=0.001),与LDH释放量(r=-0.912,P=0.011)和MDA含量(r=-0.969,P=0.001)呈负相关。
第二部分：与NC组比较,缺氧复氧处理细胞后可使其LDH释放量显著增高(P<0.01),细胞活力明显下降(P<0.01),流式细胞仪检测到大量心肌细胞凋亡,凋亡率为20.12±2.19%。利多卡因预处理细胞可显著提高心肌细胞活力(P<0.01),降低LDH释放量和细胞凋亡率(P<0.01)。免疫细胞化学技术检测结果表明,缺氧复氧损伤后心肌细胞中caspase-3活性增强；而利多卡因预处理则可降低caspase-3活性。荧光风光光度法检测显示,缺氧复氧损伤后细胞内钙离子浓度显著升高；而利多卡因预处理则可降低细胞内钙离子浓度。
第三部分：①N组：缺氧复氧后细胞LDH释放量、MDA含量较NC组显著增多,细胞活力、SOD活性显著减弱。缺氧前利多卡因预处理细胞后,其细胞MDA含量较NHR组显著减少,SOD活性显著增强(P均<0.01)；②H组：缺氧复氧后,与HC组比较,其LDH释放量、MDA含量显著增多,而细胞活力、SOD活性显著降低；而利多卡因预处理细胞后,与HHR组比较,其LDH释放量、MDA含量显著减少,细胞活力、SOD活性显著增高(P均<0.01)。③与N组比较：高糖孵育心肌细胞后其LDH释放量、MDA含量较NC组显著增多,而细胞活力、SOD活性则显著降低(P均<0.01)。④高糖与实验处理(分组)两因素的交互效应均具有显著统计学意义(P均<0.01)。
结论
(1)本研究浓度范围内的利多卡因能减轻心肌细胞的缺氧复氧损伤,并呈浓度依赖性；其机制可能与利多卡因的抗自由基作用、离子通道阻滞功能以及抗凋亡作用有关。
(2)缺氧前给予浓度为10μmol/L的利多卡因能够增强H9c2心肌细胞耐受缺氧复氧性氧化损伤的能力,同时抑制缺氧复氧过程中的细胞凋亡,可能与抑制细胞caspase-3活性、钙超载有关。
(3)高糖本身能导致心肌细胞的氧化应激损伤,随后的缺氧复氧则加剧了氧化损伤；一定浓度(10μmol/L)的利多卡因可能是通过抑制活性氧自由基的膜脂质过氧化作用来减轻心肌细胞的缺氧复氧性损伤。缺氧条件下,高糖能以糖酵解的能量代谢途径在一定程度上减轻细胞的缺氧复氧损伤。
Objective
H9c2 rat myoblast cells were treated with hypoxia and reoxygenation as a simulation of myocardial ischemia-reperfusion injury model in vitro, which is respectively devided into three parts:Part One:effects of diversed lidocaine pretreatment on H9c2 cardiomyocytes against hypoxia/reoxygenation(H/R)-induced injury. Part Two:effect of lidocaine pretreatment on caspase-3 activity and calcium overload following injuries of H9c2 cardiomyocytes induced by hypoxia/ reoxygenation. Part Three:protective effects of lidocaine pretreatment on injury of H9c2 cardiomyocytes cultrued in high-glucose culture medium. Therefore, the objectives of this study are as follow:
①To investigate the protective effects of different-concerntration lidocaine pretreatment on H9c2 cardiomyocytes against hypoxia/reoxygenation(H/R)-induced oxidative injury.
②To investigate the protective effects of lidocaine pretreatment on H9c2 cardiomyocytes against H/R-induced apoptosis and its potential mechanisms.
③To investigate the protective effects of lidocaine pretreatment on hypoxia/ reoxygenation-induced injury of H9c2 cardiomyocytes cultured in high-glucose culture medium and roles of high-glucose culture in them.
Methods
Part One:Cultured H9c2 cardiomyocytes were divided randomly into seven groups:(1)normal control group(Group NC); (2) H/R control group(Group HR):A 3-hour hypoxic period was followed by 2 hour of reoxygenation; (3) lidocaine pretreatment group(Group LP):according to the diversed concentration of lidocaine(1,2.5,5,10 and 20μmol/L), Group LP were redevided into five groups, including Group LP1, LP2, LP3, LP4 and LP5, respectivly. Cells were pretreated with lidocaine before H/R treatment. Colorimetric assay was used to detect cell viability and lactate dehydrogenase (LDH) release to evaluate cell injury. Superoxide dismutase (SOD) activity and malonaldehyde (MDA) content were measured by colorimetric assay to evaluate cell antioxidant ability.
Part Two:Cultured H9c2 cardiomyocytes were divided into three groups:(1) NC group; (2) H/R group:A 3-hour hypoxic period was followed by 2 hours of reoxygenation; (3) LPC group:cells were pretreated with lidocaine(10μmol/L) before H/R treatment. Colorimetric assay was used to detect cell viability and lactate dehydrogenase (LDH) release to evaluate cell injury. Apoptotic rate of H9c2 cardiomyocytes were determined by flow cytometer. Caspase-3 activity was detected by immunocytochemistry and intracellular calcium was tested by fluorospectrophotometry.
Part Three:Cultured H9c2 cardiomyocytes were divided randomly into six groups:normal glucose-cultured control group(Group NC), normal glucose-cultured HR group(Group NHR), normal glucose-cultured lidocaine pretreatment group(Group NLP) and high glucose-cultured control group(Group HC), high glucose-cultured HR group(Group HHR), high glucose-cultured lidocaine pretreatment group(Group HLP). Colorimetric assay was used to detect cell viability and lactate dehydrogenase (LDH) release to evaluate cell injury. Superoxide dismutase (SOD) activity and malonaldehyde (MDA) content were measured by colorimetric assay.
Results
Part One:The results showed that after treatment with H/R, cell viability was significantly weakened compared with NC group. LDH release was significantly higher than that in NC group. Lidocaine pretreatment markedly improved cell viability and reduced LDH release. Cells treated with H/R had a lower SOD activity and a higher MDA content compared with Group NC. Cells with lidocaine pretreatment had a higher SOD activity and a lower MDA content compared with HR group. Besides, the change of cell viability was positively correlated significantly to that of LDH release and MDA content, negatively to that of SOD activity.And cell viability was positively correlated significantly to the increasing concentration of lidocaine; LDH release and MDA content were negatively to it.
Part Two:The results showed that after treatment with hypoxia and reoxgenation, cell viability was significantly reduced to 0.188±0.012 compared with NC group (0.325±0.009). LDH release and apoptotic rates were 80.18±4.70U/L and 20.12±2.19% respectively, significantly higher than that of NC group (17.13±2.40U/L and 0.57±0.30%). Lidocaine pretreatment markedly improved cell viability to 0.234±0.015 and reduced LDH activity and apoptotic rates to 38.51±2.45U/L and 8.03±1.50%. Caspase-3 activity and intracellular calcium were increased to 0.163±0.011 and 440.59±22.30nmol/L respectively, significantly higher than NC group after hypoxia/reoxgenation-induced injury. Cells with lidocaine pretreatment had a lower caspase-3 activity of 0.135±0.007 and intracellular calcium of 329.22±13.74nmol/L compared with H/R group.
Part Three:①In normal glucose-cultured group:after treatment with hypoxia and reoxygenation, cell viability and SOD activity were significantly reduced compared with NC group. Reversely, LDH release and MDA content were significantly higher than that in Group NC. Lidocaine pretreatment markedly improved cell viability and reduced LDH release. Cells treated with lidocaine had a higher SOD activity and a lower MDA content compared with NHR group.②In high glucose-cultured group:after treatment with hypoxia and reoxygenation, cell viability and SOD activity were significantly reduced compared with HC group. Reversely, LDH release and MDA content were significantly higher than that in HC group. Lidocaine pretreatment markedly improved cell viability and reduced LDH release. Cells pretreated with lidocaine had a higher SOD activity and a lower MDA content compared with HHR group;③compared with normal glucose-cultured group, after co-cultured with high glucose, cell viability and SOD activity were lower and LDH release and MDA content were significantly higher in Group HC than that in Group NC;④The interactive effect of two factors between high glucose and experimental treatment (group) was significantly statistically significant.
Conclusions
Based on the results observed, it is concluded that:
①Lidocaine pretreament may protect cardiomyocytes from H/R-induced oxidative injury via inhibiting cell lipoperoxidation in a concerntration-dependent manner. And the better anti-oxidative-injury of it gets, the higher concentrations of lidocaine become.
②Lidocaine pretreatment may protect cardiomyocytes from being injured by H/R. The underlying mechanisms may include repressing caspase-3 activity and reducing intracellular calcium overload.
③Lidocaine(10μmol/L) may protect cardiomyocytes from being oxidatively injury induced by hypoxia and reoxygenation in the both kinds of conditions:normal glucose and high glucose, respectively; High glucose may result in stress injury in the normal condition, but markedly strengthen the anti-H/R ability of H9c2 cells and also have some cardioprective effects in the hypoxic condition.
在缺血基础上恢复血流后组织损伤加重,甚至发生不可逆性损伤的现象称为缺血再灌注损伤(ischemia-reperfusion injury)。现已证实,心、脑、肝、肾、肺、胃肠道、肢体及皮肤等多种组织器官都存在缺血再灌注损伤的现象。心肌缺血再灌注损伤(myocardial ischemia/repeifusion injury, MIRI)为再灌注后缺血的心肌出现舒缩功能降低、心律失常、心肌能量代谢障碍、超微结构的变化和血管无复流等现象。缺血再灌注损伤的发生机制尚未彻底阐明,既往研究证实氧自由基(oxygen free radical, OFR)的作用、白细胞激活、内皮细胞(endothelial cell,EC)功能障碍、细胞内钙超载以及细胞凋亡是引起缺血一再灌注损伤的重要原因。
糖尿病(diabetes mellitus, DM)是常见病、多发病,现已明确DM是冠状动脉粥样硬化性心脏病发病的一个重要因素。流行病学资料显示：DM人群中心绞痛、急性心肌梗死(actue myocardial inarction, AMI)、充血性心力衰竭以及严重心律失常等疾病发病率远高于非DM患者,而且当DM患者并发心肌梗死后行药物或手术(包括冠状动脉搭桥和导管介入)治疗,效果均不及非DM患者。人们将这种不良后果归咎于缺血再灌注(ischemia/repeifusion, I/R),这可能与糖尿病患者冠状动脉粥样病变严重而弥漫、内皮功能障碍、储备能力降低所造成的心肌灌注不良有关,另外DM本身的促凝和炎症状态,易形成冠状动脉内微血栓堵塞；DM心肌缺氧应激,NO活性降低,存在冠脉微循环痉挛。而日本的一项多中心研究却显示：急诊经皮冠状动脉介入治疗(percutaneous coronary intervention, PCI)住院期间患者病死率与是否患有DM无关,而与血糖增高有关,且发现无DM但伴有高血糖者的死亡率最高,该研究推测这可能与DM性冠心病梗死相关血管(infarct-related artery, IRA)再通后I/R损伤增加有关。
基础研究显示DM发生时,心肌组织的结构、功能以及代谢均发生异常,主要表现为：①心肌能量代谢异常,且高糖环境能够直接引起正常心肌细胞功能改变；②细胞内钙调控异常；③微血管病变和微循环障碍,缓激肽释放酶—缓激肽系统在高血糖状态下该系统被抑制；④高血糖时氧化应激增加,信号转导途径改变,激活细胞凋亡。国内外研究证实,DM急性期,NO代偿性合成增加,I/R对DM心肌有保护作用；而DM晚期,由于慢性期NO减少,心肌肥厚,收缩和舒张功能受损,心脏血管周围或间质纤维化, I/R后损伤后将加重心肌损害。
近来,心肌缺血再灌注损伤和心肌保护一直是基础研究和临床研究的重点。1986年Murry等首次提出缺血预处理(Ischemia preconditioning, IPC)可以减轻缺血再灌注所致的心肌损伤,随后的一系列研究则发现许多麻醉药物(包括全身麻醉药、阿片类镇痛药以及局部麻醉药)预处理能产生与经典缺血预处理同样的心肌保护作用,这为预处理心肌保护的临床应用开辟了新的途径。利多卡因在急性心梗、恶性心律失常等引起的工R损伤治疗中具有较好的效果。有关预防性应用利多卡因的临床有效性和安全性一直备受争议。既往的荟萃分析(meta-analysis)表明预防性应用利多卡因虽然可减少急性心肌梗死患者心室纤颤的发生,但却增加了患者的死亡率。然而,一项多国家多中心观察研究表明：预防性应用利多卡因并不增加急性心肌梗死患者的死亡率,预防性应用利多卡因也许是无害的。总而言之,关于利多卡因预处理在心肌缺血再灌注损伤防治中的作用目前尚不明了,更不能确定利多卡因预处理心肌保护效应的具体作用机制。
目的
本研究通过在体外细胞水平以缺氧复氧处理H9c2大鼠成肌细胞建立心肌缺血再灌注损伤模型,初步探讨：①不同梯度浓度利多卡因预处理对缺氧复氧引起的心肌细胞损伤是否具有保护作用；②利多卡因预处理对缺氧复氧引起的H9c2心肌细胞凋亡的影响及其可能机制。③利多卡因预处理对高糖孵育下H9c2心肌细胞缺氧复氧损伤是否具有保护作用及与高糖孵育的关系。
’方法
第一部分：研究不同浓度利多卡因预处理对H9c2大鼠成肌细胞缺氧复氧损伤的影响。实验随机分为7组：正常对照组(NC组)、缺氧复氧损伤对照组(HR组)；利多卡因预处理组(LP组),根据利多卡因的不同浓度对分为LP1(1μmol/L)、LP2(2.5μmol/L)、LP3(5μmol/L)、LP4(10μmol/L)、LP5(20μmol/L)组,每组6孔。心肌细胞损伤程度以心肌细胞活力和乳酸脱氢酶(LDH)活性来表示,同时检测心肌细胞超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。
第二部分：研究利多卡因预处理对H9c2大鼠成肌细胞缺氧复氧损伤后caspase-3活性、钙超载的影响。实验随机分为3组：①正常对照组(NC组)；②缺氧复氧组(H/R组)；③利多卡因预处理组(LPC组)。心肌细胞损伤程度以心肌细胞活力和乳酸脱氢酶(LDH)活性来表示。用流式细胞术检测心肌细胞凋亡率。通过免疫细胞化学技术检测心肌细胞胞浆caspase-3的表达。荧光分光光度法检测细胞内钙离子浓度。
第三部分：研究利多卡因预处理对高糖孵育下H9c2心肌细胞缺氧复氧性损伤是否具有保护作用。实验随机分为二组,即正常糖浓度组(N组)和高糖浓度组(H组),每组又分为正常对照组(NC组和HC组)、缺氧复氧损伤组(NHR组和HHR组)和利多卡因预处理组(NLP组和HLP组)。心肌细胞损伤程度以H9c2细胞活力和培养基中乳酸脱氢酶(LDH)释放量来表示,同时检测H9c2细胞超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。
结果
第一部分：与NC组比较,HR组细胞活力显著减弱,LDH释放量显著增加,并且MDA含量增多,SOD活性降低；与HR组比较,L2-5组细胞活力显著增强,LDH释放量显著降低；并且MDA含量下降,SOD活性增强；且Pearson积差相关分析显示,细胞活力强弱的变化与LDH释放量(r=-0.870,P=0.000)、MDA含量(r=-0.924,P=0.000)呈负相关,与SOD活性呈正相关(r=0.894,P=0.000)。细胞活力的增强与利多卡因浓度的增加呈正相关(r=0.976,P=0.001),与LDH释放量(r=-0.912,P=0.011)和MDA含量(r=-0.969,P=0.001)呈负相关。
第二部分：与NC组比较,缺氧复氧处理细胞后可使其LDH释放量显著增高(P<0.01),细胞活力明显下降(P<0.01),流式细胞仪检测到大量心肌细胞凋亡,凋亡率为20.12±2.19%。利多卡因预处理细胞可显著提高心肌细胞活力(P<0.01),降低LDH释放量和细胞凋亡率(P<0.01)。免疫细胞化学技术检测结果表明,缺氧复氧损伤后心肌细胞中caspase-3活性增强；而利多卡因预处理则可降低caspase-3活性。荧光风光光度法检测显示,缺氧复氧损伤后细胞内钙离子浓度显著升高；而利多卡因预处理则可降低细胞内钙离子浓度。
第三部分：①N组：缺氧复氧后细胞LDH释放量、MDA含量较NC组显著增多,细胞活力、SOD活性显著减弱。缺氧前利多卡因预处理细胞后,其细胞MDA含量较NHR组显著减少,SOD活性显著增强(P均<0.01)；②H组：缺氧复氧后,与HC组比较,其LDH释放量、MDA含量显著增多,而细胞活力、SOD活性显著降低；而利多卡因预处理细胞后,与HHR组比较,其LDH释放量、MDA含量显著减少,细胞活力、SOD活性显著增高(P均<0.01)。③与N组比较：高糖孵育心肌细胞后其LDH释放量、MDA含量较NC组显著增多,而细胞活力、SOD活性则显著降低(P均<0.01)。④高糖与实验处理(分组)两因素的交互效应均具有显著统计学意义(P均<0.01)。
结论
(1)本研究浓度范围内的利多卡因能减轻心肌细胞的缺氧复氧损伤,并呈浓度依赖性；其机制可能与利多卡因的抗自由基作用、离子通道阻滞功能以及抗凋亡作用有关。
(2)缺氧前给予浓度为10μmol/L的利多卡因能够增强H9c2心肌细胞耐受缺氧复氧性氧化损伤的能力,同时抑制缺氧复氧过程中的细胞凋亡,可能与抑制细胞caspase-3活性、钙超载有关。
(3)高糖本身能导致心肌细胞的氧化应激损伤,随后的缺氧复氧则加剧了氧化损伤；一定浓度(10μmol/L)的利多卡因可能是通过抑制活性氧自由基的膜脂质过氧化作用来减轻心肌细胞的缺氧复氧性损伤。缺氧条件下,高糖能以糖酵解的能量代谢途径在一定程度上减轻细胞的缺氧复氧损伤。
Objective
H9c2 rat myoblast cells were treated with hypoxia and reoxygenation as a simulation of myocardial ischemia-reperfusion injury model in vitro, which is respectively devided into three parts:Part One:effects of diversed lidocaine pretreatment on H9c2 cardiomyocytes against hypoxia/reoxygenation(H/R)-induced injury. Part Two:effect of lidocaine pretreatment on caspase-3 activity and calcium overload following injuries of H9c2 cardiomyocytes induced by hypoxia/ reoxygenation. Part Three:protective effects of lidocaine pretreatment on injury of H9c2 cardiomyocytes cultrued in high-glucose culture medium. Therefore, the objectives of this study are as follow:
①To investigate the protective effects of different-concerntration lidocaine pretreatment on H9c2 cardiomyocytes against hypoxia/reoxygenation(H/R)-induced oxidative injury.
②To investigate the protective effects of lidocaine pretreatment on H9c2 cardiomyocytes against H/R-induced apoptosis and its potential mechanisms.
③To investigate the protective effects of lidocaine pretreatment on hypoxia/ reoxygenation-induced injury of H9c2 cardiomyocytes cultured in high-glucose culture medium and roles of high-glucose culture in them.
Methods
Part One:Cultured H9c2 cardiomyocytes were divided randomly into seven groups:(1)normal control group(Group NC); (2) H/R control group(Group HR):A 3-hour hypoxic period was followed by 2 hour of reoxygenation; (3) lidocaine pretreatment group(Group LP):according to the diversed concentration of lidocaine(1,2.5,5,10 and 20μmol/L), Group LP were redevided into five groups, including Group LP1, LP2, LP3, LP4 and LP5, respectivly. Cells were pretreated with lidocaine before H/R treatment. Colorimetric assay was used to detect cell viability and lactate dehydrogenase (LDH) release to evaluate cell injury. Superoxide dismutase (SOD) activity and malonaldehyde (MDA) content were measured by colorimetric assay to evaluate cell antioxidant ability.
Part Two:Cultured H9c2 cardiomyocytes were divided into three groups:(1) NC group; (2) H/R group:A 3-hour hypoxic period was followed by 2 hours of reoxygenation; (3) LPC group:cells were pretreated with lidocaine(10μmol/L) before H/R treatment. Colorimetric assay was used to detect cell viability and lactate dehydrogenase (LDH) release to evaluate cell injury. Apoptotic rate of H9c2 cardiomyocytes were determined by flow cytometer. Caspase-3 activity was detected by immunocytochemistry and intracellular calcium was tested by fluorospectrophotometry.
Part Three:Cultured H9c2 cardiomyocytes were divided randomly into six groups:normal glucose-cultured control group(Group NC), normal glucose-cultured HR group(Group NHR), normal glucose-cultured lidocaine pretreatment group(Group NLP) and high glucose-cultured control group(Group HC), high glucose-cultured HR group(Group HHR), high glucose-cultured lidocaine pretreatment group(Group HLP). Colorimetric assay was used to detect cell viability and lactate dehydrogenase (LDH) release to evaluate cell injury. Superoxide dismutase (SOD) activity and malonaldehyde (MDA) content were measured by colorimetric assay.
Results
Part One:The results showed that after treatment with H/R, cell viability was significantly weakened compared with NC group. LDH release was significantly higher than that in NC group. Lidocaine pretreatment markedly improved cell viability and reduced LDH release. Cells treated with H/R had a lower SOD activity and a higher MDA content compared with Group NC. Cells with lidocaine pretreatment had a higher SOD activity and a lower MDA content compared with HR group. Besides, the change of cell viability was positively correlated significantly to that of LDH release and MDA content, negatively to that of SOD activity.And cell viability was positively correlated significantly to the increasing concentration of lidocaine; LDH release and MDA content were negatively to it.
Part Two:The results showed that after treatment with hypoxia and reoxgenation, cell viability was significantly reduced to 0.188±0.012 compared with NC group (0.325±0.009). LDH release and apoptotic rates were 80.18±4.70U/L and 20.12±2.19% respectively, significantly higher than that of NC group (17.13±2.40U/L and 0.57±0.30%). Lidocaine pretreatment markedly improved cell viability to 0.234±0.015 and reduced LDH activity and apoptotic rates to 38.51±2.45U/L and 8.03±1.50%. Caspase-3 activity and intracellular calcium were increased to 0.163±0.011 and 440.59±22.30nmol/L respectively, significantly higher than NC group after hypoxia/reoxgenation-induced injury. Cells with lidocaine pretreatment had a lower caspase-3 activity of 0.135±0.007 and intracellular calcium of 329.22±13.74nmol/L compared with H/R group.
Part Three:①In normal glucose-cultured group:after treatment with hypoxia and reoxygenation, cell viability and SOD activity were significantly reduced compared with NC group. Reversely, LDH release and MDA content were significantly higher than that in Group NC. Lidocaine pretreatment markedly improved cell viability and reduced LDH release. Cells treated with lidocaine had a higher SOD activity and a lower MDA content compared with NHR group.②In high glucose-cultured group:after treatment with hypoxia and reoxygenation, cell viability and SOD activity were significantly reduced compared with HC group. Reversely, LDH release and MDA content were significantly higher than that in HC group. Lidocaine pretreatment markedly improved cell viability and reduced LDH release. Cells pretreated with lidocaine had a higher SOD activity and a lower MDA content compared with HHR group;③compared with normal glucose-cultured group, after co-cultured with high glucose, cell viability and SOD activity were lower and LDH release and MDA content were significantly higher in Group HC than that in Group NC;④The interactive effect of two factors between high glucose and experimental treatment (group) was significantly statistically significant.
Conclusions
Based on the results observed, it is concluded that:
①Lidocaine pretreament may protect cardiomyocytes from H/R-induced oxidative injury via inhibiting cell lipoperoxidation in a concerntration-dependent manner. And the better anti-oxidative-injury of it gets, the higher concentrations of lidocaine become.
②Lidocaine pretreatment may protect cardiomyocytes from being injured by H/R. The underlying mechanisms may include repressing caspase-3 activity and reducing intracellular calcium overload.
③Lidocaine(10μmol/L) may protect cardiomyocytes from being oxidatively injury induced by hypoxia and reoxygenation in the both kinds of conditions:normal glucose and high glucose, respectively; High glucose may result in stress injury in the normal condition, but markedly strengthen the anti-H/R ability of H9c2 cells and also have some cardioprective effects in the hypoxic condition.
引文
[1]Ruiz Gines JA, Lopez Ongil S, Gonzalcz Ruhio M, et al. Reactive oxygen species induce proliferation of bovine aortic endothelial cell [J]. J Card-iovasc Pharmacol,2000,35 (1):109-113.
[2]Mitchell MB, Winter CB, Banerjee A, et al. The relationship between ischemia-reperfusion injury, myocardial stunning and cardiac preconditioning. Surg Gynecol Obstet,1993,177:97-114.
[3]Dhalla NS, Temsah RM, Netticadan T. Role oxidative stress in cardiovascular diseases. J Hypertens,2000,18:655-73.
[4]徐新春.钙超载与心肌缺血再灌注损伤[J].心血管病学进展,2000,21(4)：227-230.
[5]Fenvary D, Lopaschuk GD. Controversies on the sensitivity of the diabetic heart to ischemic injury:the sensitivity of the diabetic injury is decreased[J]. Cardivasc Res,1997,34(1):113-120.
[6]Claeys MJ,Bosmans J, Ceuninck M, et al. Effect of intracoronary adenosion infusion injury during coronary intervention on myocardial referfusion injury in patients with acute myocardial infarction[J]. Am J Cardiol,2004,94(1):9-13
[7]Antoniucci D, Valenti R, Migliorini A, et al. Impact of insulin-requiring diabetes mellitus on effectiveness of reperfusion and outcome of patients undergoing primary percutaneous coronary intervention for acute myocardial infraction[J]. Am J Cardiol,2004,93(9):1170-1172.
[8]Prasad A, Stone GM, Stucket TD. Impact of diabetes mellitus on Myocardial perfusion after angniplast in patients with Acute Myocardial Infraction [J].Circulation,2005,15(45):508-514.
[9]Msami K, kazuo K, sanuo K, et al. Effects of Glucose Abnormalitises on In-Hospital Outcome after cornnary intervention for Acute Myocardial Infraction[J]. J Circulation,2005,69(4):375-379.
[10]沈絮华,贾三庆,李宏伟.血糖对直接经皮冠状动脉介入治疗后患者心肌灌注的影响[J].中华心血管病杂志,2006,34(2)：138-142.
[11]张丽,金玉华.糖代谢异常对心脏结构和功能的影响[J].中华老年心脑血管病杂志,2008,10(2)：229-231.
[12]Jweied EE, McKinney RD, Walker LA, et al. Depressed cardiac myofilament function in human diabetesmellitus[J]. Am J PhysiolHeart Circ Physiol, 2005,289(12):H2478-H2483.
[13]Tsch C, Walther T, Escher F, et al. Transgenic activation of the kallikrein-kinin system inhibits intramyocardial inflammation,endothelial dysfunction,and oxidative stress inexperimental diabetic cardiomyopathy[J]. FASEB J,2005,19 (12):2057-2059.
[14]Fujio Y, Nguyen T, Wencker D, et al. Akt promotes survival of cardiomyocytes in vitro and protects against ischemiare-perfusion injury in mouse heart[J]. 2000,101(6):660-667.
[15]马世玉,吴基良,向继洲,等.倒卵叶五加总皂甙对大鼠心肌缺血再灌注损伤后Bcl-2、Bax蛋白表达的影响[J].华中科技大学学报,2003,32(2)：145-156.
[16]Ravingerova T, Neckar J, Kolar F. Ischemic tolerance of rat hearts in acute and chronic phases of experimental diabetes[J]. Mol Cell Biochem,2003,249(1-2):167-174.
[17]Xu G, Takashi E, Kudc M, et al. Contradictory effects of short-and long-term hyperglycemias on ischemic injury of myocardium via intracellular signaling pathway[J]. Exp Mol Pathol,2004,76(1):57-65.
[18]Chen H, Wu XJ, Lu XY, et al. Phosphorylated heat shock protein 27 is involved in enhanced heart tolerance to ischemia in short-term type 1 diabetic rats[J]. Acta Pharmacol Sin,2005,26(8):806-812.
[19]Jansson PA. Endothelial dysfunction in insulin insistance and type 2 diabetes.[J]. Intern Med,2007,262(2):173-183.
[20]Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia:a delay of lethal cell injury in ischemic myocardium. Circulation.1986,74:1124-36.
[21]MacMahon S, Collins R, Peto R, et al. Effects of prophylactic lidocaine in suspected acute myocardial infarction:an overview of results from the randomized, controlled trials. JAMA,1988;260:1910-1916.
[22]Alexander JH, Granger CB, Sadowski Z, et al. Prophylactic lidocaine use in acute myocardial infarction:incidence and outcomes from two international trials. The GUSTO-I and GUSTO-IIb Investigators. Am Heart J,1999; 137: 799-805.
[23]Schmid RA, Yamashita M, Ando K, et al. Lidocaine reduces reperfusion injury and neutrophil migration in canine lung allografts. Ann Thorac Surg,1996,61: 949-55.
[24]Borg T, Modig J. Potential anti-thrombotic effects of local anaesthetics due to their inhibition of platelet aggregation. Acta Anaesthesiol Scand,1985,29: 739-42.
[25]Das KC, Misra HP. Lidocaine:a hydroxyl radical scavenger and singlet oxygen quencher. Mol Cell Biochem,1992,115:179-85.
[26]Pike MM, Kitakaze M, Chacko VP, et al. Lidocaine prevents the increase in myocardial free calcium during ischemia and reperfusion:an antiarrhythmic mechanism? Circulation,1987,6:IV-I6.
[1]Lei, B, J.E. Cottrell and I.S. Kass, Neuroprotective effect of low-dose lidocaine in a rat model of transient focal cerebral ischemia[J]. Anesthesiology, 2001,95(2):445-451.
[2]Fried E, Amorim P, Chambers G, et al. The importance of sodium for anoxic transmission damage in rat hippocampal slices:Mechanisms of protection by lidocaine[J]. J Physiol (Lond) 1995,489:557-65
[3]杜敏逸,纪方,吴新民.在体外乳鼠心肌细胞缺氧的损伤及利多卡因的保护作用[J].中华麻醉学杂志,1999,19(3)：161-163
[4]Luo ZQ, Feng DD, Zhou FW, et al. Protective effect of low concentration endothelin-1 on the reactive oxygen-induced inhibition of pulmonary surfactant lipid synthesis[J]. Acta Physiol Sin,2002,64(2):89-93
[5]庄心良,曾因明,陈伯銮.现代麻醉学[M].3版.北京：人民卫生出版社,2003：628-630
[6]张一,杨伊林,焦志华,等.低浓度利多卡因对大鼠海马CA1区缺氧神经元持续钠电流的影响[J].中华麻醉学杂志,2005,25(8)：576-578
[7]Hinokiyama K, Hatori N, Ochi M, et al. Myocardial protective effect of lidocaine during experimental off-pump coronary artery bypass grafting [J]. Ann Thorac Cardiovasc Surg.2003,9(1):36-42.
[8]Kaczmarek DJ, Herzog C, Larmann J, et al. Lidocaine protects from myocardial damage due to ischemia and reperfusion in mice by its antiapoptotic effects[J]. Anesthesiology,2009,110(5):1041-1049.
[9]Ross JD, Ripper R, Law WR, et al. Adding bupivacaine to high-potassium cardioplegia improves function and reduces cellular damage of rat isolated hearts after prolonged, cold storage[J]. Anesthesiology,2006,105(4):746-52
[1]Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ Res,1996,79:949-956
[2]Zhao ZQ, Nakamura M, Wang NP, et al. Progressively developed myocardial apoptotic cell death during late phase of reperfusion[J]. Apoptosis,2001; 6:279-290
[3]Ling H, Lou Y. Total flavones from Elsholtzia blanda reduce infarct size during acute myocardial ischemia by inhibiting myocardial apoptosis in rats[J]. Ethnopharmacol,2005,101:169-175
[4]Hahnenkamp K, Theilmeier G, Van Aken HK, Hoenemann CW:The effects of local anesthetics on perioperative coagulation, inflammation, and microcirculation [J].Anesth Analg 2002,94:1441-7
[5]Kaczmarek DJ, Herzog C, Larmann J, et al. Lidocaine protects from myocardial damage due to ischemia and reperfusion in mice by its antiapoptotic effects[J]. Anesthesiology,2009,110:1041-1049
[6]Pei TS, Xu CQ, Li HZ, et al. Effect of quercetin on rat cardiomyocyte apop tosis induced by adriamyc in in vitro[J]. Chin Pharmacol Bull,2008, 24:534-538.
[7]Fried E, Amorim P, Chambers G, et al. The importance of sodium for anoxic transmission damage in rat hippocampal slices:Mechanisms of protection by lidocaine[J]. J Physiol(Lond),1995,489:557-65.
[8]Maulin K, Yoshida T, Das DK. Oxidative stress developed during the reperfusion of ischemic myocardium induces apoptosis [J].Free Radiac Biol Med,1998,24:869-875
[9]Hamm CW, Katus HA. New biochemical markers for myocardial cell injury[J]. Curr Opin Cardiol,1995,10:355-360
[10]Kevin LG, Camara AK, Riess ML, et al. Ischemic preconditioning alters real-time measure of 02 radicals in intact hearts with ischemia and reperfusion[J]. Am J Physiol Heart Circ Physiol,2003,284:H566-H574
[11]Hollmann MW, Durieux ME:Local anesthetics and the inflammatory response: A new therapeutic indication? Anesthesiology,2000,93:858-75
[12]12Honda HM, Korge P, Weiss JN. Mitochondria and ischemia/repeffusion injury[J]. Ann N Y Acad Sci.2005,047:248-258
[13]Borutaite V, Jekabsone A, Morkuniene R, et al. Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction cytochrome c and apoptosis induced by heart ischemia[J]. Mol Cell Cardiol,2003,35:357-366
[14]Chen SJ, Bradley M, Lee TC. Chemical hypoxia triggers apoptosis of cultured neonatal rat cardiac myocytes modulation by calium-regulated proteases and protein kinases[J]. Mol Cell Bichem,1998; 178:41-49
[1]Ceriello A, Quagliaro L, D' Amico M, et al. Acute hyperglycemia induces nitrotyrosine formation and apoptosis in perfused heart from rat[J]. Diabetes, 2002,51(4):1076-1082
[2]Jovanovic S, Jovanovic N, Jovanovic A. High glucose protects single beating adult cardiomyocytes against hypoxia[J]. Biochem Biophys Res Commun,2006,341(1):57-66.
[3]Kaczmarek DJ, Herzog C, Larmann J, et al. Lidocaine protects from myocardial damage due to ischemia and reperfusion in mice by its antiapoptotic effects[J]. Anesthesiology,2009,110(5):1041-1049.
[4]Luo ZQ, Feng DD, Zhou FW, et al. Protective effect of low concentration endothelin-1 on the reactive oxygen-induced inhibition of pulmonary surfactant lipid synthesis[J]. Acta Physiol Sin,2002,64(2):89-93
[5]庄心良,曾因明,陈伯銮.现代麻醉学[M].3版.北京：人民卫生出版社,2003：628-630
[6]Fried E, Amorim P, Chambers G, et al. The importance of sodium for anoxic transmission damage in rat hippocampal slices:Mechanisms of protection by lidocaine[J]. J Physiol(Lond),1995,489:557-65.
[7]Hinokiyama K, Hatori N, Ochi M, et al. Myocardial protective effect of lidocaine during experimental off-pump coronary artery bypass grafting[J]. Ann Thorac Cardiovasc Surg.2003;9(1):36-42
[8]Ross JD, Ripper R, Law WR, et al. Adding bupivacaine to high-potassium cardioplegia improves function and reduces cellular damage of rat isolated hearts after prolonged, cold storage[J]. Anesthesiology,2006,105(4):746-752.
[9]Yu XY, Song YH, Geng YJ, et al. Glucose induces apoptosis of cardiomyocytes via microRNA-1 and IGF-1[J]. Biochem Biophys Res Commun,2008,376(3):548-52
[10]Allen DA, Yaqoob MM, Harwood SM. Mechanisms of high glucose-induced apoptosis and its relationship to diabetic complications [J]. J Nutr Biochem, 2005,16(12):705-713.
[11]Williams SB, Goldfme AB, Timimi FK, et al. Acute hyperglycemia attenuates endothelium-dependent vasodilation in humans in vivo[J]. Circulation. 1998,97(17):1695-1701.
[12]Verma S, Maitland A, Weisel RD, et al. Hyperglycemia exaggerates ischemia/reperfusion-induced cardiomyocyte injury:reversal with endothelin antagonism[J]. J Thorac Cardiovasc Surg,2002 123(6):1120-1124.
[13]刘涛,徐美英,都大伟等糖尿病大鼠离体心脏对缺血/再灌注的耐受性[J].中华麻醉学杂志,2005,25(2)：126-128.
[2]Mitchell MB, Winter CB, Banerjee A, et al. The relationship between ischemia-reperfusion injury, myocardial stunning and cardiac preconditioning. Surg Gynecol Obstet,1993,177:97-114.
[3]Dhalla NS, Temsah RM, Netticadan T. Role oxidative stress in cardiovascular diseases. J Hypertens,2000,18:655-73.
[4]徐新春.钙超载与心肌缺血再灌注损伤[J].心血管病学进展,2000,21(4)：227-230.
[5]Fenvary D, Lopaschuk GD. Controversies on the sensitivity of the diabetic heart to ischemic injury:the sensitivity of the diabetic injury is decreased[J]. Cardivasc Res,1997,34(1):113-120.
[6]Claeys MJ,Bosmans J, Ceuninck M, et al. Effect of intracoronary adenosion infusion injury during coronary intervention on myocardial referfusion injury in patients with acute myocardial infarction[J]. Am J Cardiol,2004,94(1):9-13
[7]Antoniucci D, Valenti R, Migliorini A, et al. Impact of insulin-requiring diabetes mellitus on effectiveness of reperfusion and outcome of patients undergoing primary percutaneous coronary intervention for acute myocardial infraction[J]. Am J Cardiol,2004,93(9):1170-1172.
[8]Prasad A, Stone GM, Stucket TD. Impact of diabetes mellitus on Myocardial perfusion after angniplast in patients with Acute Myocardial Infraction [J].Circulation,2005,15(45):508-514.
[9]Msami K, kazuo K, sanuo K, et al. Effects of Glucose Abnormalitises on In-Hospital Outcome after cornnary intervention for Acute Myocardial Infraction[J]. J Circulation,2005,69(4):375-379.
[10]沈絮华,贾三庆,李宏伟.血糖对直接经皮冠状动脉介入治疗后患者心肌灌注的影响[J].中华心血管病杂志,2006,34(2)：138-142.
[11]张丽,金玉华.糖代谢异常对心脏结构和功能的影响[J].中华老年心脑血管病杂志,2008,10(2)：229-231.
[12]Jweied EE, McKinney RD, Walker LA, et al. Depressed cardiac myofilament function in human diabetesmellitus[J]. Am J PhysiolHeart Circ Physiol, 2005,289(12):H2478-H2483.
[13]Tsch C, Walther T, Escher F, et al. Transgenic activation of the kallikrein-kinin system inhibits intramyocardial inflammation,endothelial dysfunction,and oxidative stress inexperimental diabetic cardiomyopathy[J]. FASEB J,2005,19 (12):2057-2059.
[14]Fujio Y, Nguyen T, Wencker D, et al. Akt promotes survival of cardiomyocytes in vitro and protects against ischemiare-perfusion injury in mouse heart[J]. 2000,101(6):660-667.
[15]马世玉,吴基良,向继洲,等.倒卵叶五加总皂甙对大鼠心肌缺血再灌注损伤后Bcl-2、Bax蛋白表达的影响[J].华中科技大学学报,2003,32(2)：145-156.
[16]Ravingerova T, Neckar J, Kolar F. Ischemic tolerance of rat hearts in acute and chronic phases of experimental diabetes[J]. Mol Cell Biochem,2003,249(1-2):167-174.
[17]Xu G, Takashi E, Kudc M, et al. Contradictory effects of short-and long-term hyperglycemias on ischemic injury of myocardium via intracellular signaling pathway[J]. Exp Mol Pathol,2004,76(1):57-65.
[18]Chen H, Wu XJ, Lu XY, et al. Phosphorylated heat shock protein 27 is involved in enhanced heart tolerance to ischemia in short-term type 1 diabetic rats[J]. Acta Pharmacol Sin,2005,26(8):806-812.
[19]Jansson PA. Endothelial dysfunction in insulin insistance and type 2 diabetes.[J]. Intern Med,2007,262(2):173-183.
[20]Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia:a delay of lethal cell injury in ischemic myocardium. Circulation.1986,74:1124-36.
[21]MacMahon S, Collins R, Peto R, et al. Effects of prophylactic lidocaine in suspected acute myocardial infarction:an overview of results from the randomized, controlled trials. JAMA,1988;260:1910-1916.
[22]Alexander JH, Granger CB, Sadowski Z, et al. Prophylactic lidocaine use in acute myocardial infarction:incidence and outcomes from two international trials. The GUSTO-I and GUSTO-IIb Investigators. Am Heart J,1999; 137: 799-805.
[23]Schmid RA, Yamashita M, Ando K, et al. Lidocaine reduces reperfusion injury and neutrophil migration in canine lung allografts. Ann Thorac Surg,1996,61: 949-55.
[24]Borg T, Modig J. Potential anti-thrombotic effects of local anaesthetics due to their inhibition of platelet aggregation. Acta Anaesthesiol Scand,1985,29: 739-42.
[25]Das KC, Misra HP. Lidocaine:a hydroxyl radical scavenger and singlet oxygen quencher. Mol Cell Biochem,1992,115:179-85.
[26]Pike MM, Kitakaze M, Chacko VP, et al. Lidocaine prevents the increase in myocardial free calcium during ischemia and reperfusion:an antiarrhythmic mechanism? Circulation,1987,6:IV-I6.
[1]Lei, B, J.E. Cottrell and I.S. Kass, Neuroprotective effect of low-dose lidocaine in a rat model of transient focal cerebral ischemia[J]. Anesthesiology, 2001,95(2):445-451.
[2]Fried E, Amorim P, Chambers G, et al. The importance of sodium for anoxic transmission damage in rat hippocampal slices:Mechanisms of protection by lidocaine[J]. J Physiol (Lond) 1995,489:557-65
[3]杜敏逸,纪方,吴新民.在体外乳鼠心肌细胞缺氧的损伤及利多卡因的保护作用[J].中华麻醉学杂志,1999,19(3)：161-163
[4]Luo ZQ, Feng DD, Zhou FW, et al. Protective effect of low concentration endothelin-1 on the reactive oxygen-induced inhibition of pulmonary surfactant lipid synthesis[J]. Acta Physiol Sin,2002,64(2):89-93
[5]庄心良,曾因明,陈伯銮.现代麻醉学[M].3版.北京：人民卫生出版社,2003：628-630
[6]张一,杨伊林,焦志华,等.低浓度利多卡因对大鼠海马CA1区缺氧神经元持续钠电流的影响[J].中华麻醉学杂志,2005,25(8)：576-578
[7]Hinokiyama K, Hatori N, Ochi M, et al. Myocardial protective effect of lidocaine during experimental off-pump coronary artery bypass grafting [J]. Ann Thorac Cardiovasc Surg.2003,9(1):36-42.
[8]Kaczmarek DJ, Herzog C, Larmann J, et al. Lidocaine protects from myocardial damage due to ischemia and reperfusion in mice by its antiapoptotic effects[J]. Anesthesiology,2009,110(5):1041-1049.
[9]Ross JD, Ripper R, Law WR, et al. Adding bupivacaine to high-potassium cardioplegia improves function and reduces cellular damage of rat isolated hearts after prolonged, cold storage[J]. Anesthesiology,2006,105(4):746-52
[1]Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ Res,1996,79:949-956
[2]Zhao ZQ, Nakamura M, Wang NP, et al. Progressively developed myocardial apoptotic cell death during late phase of reperfusion[J]. Apoptosis,2001; 6:279-290
[3]Ling H, Lou Y. Total flavones from Elsholtzia blanda reduce infarct size during acute myocardial ischemia by inhibiting myocardial apoptosis in rats[J]. Ethnopharmacol,2005,101:169-175
[4]Hahnenkamp K, Theilmeier G, Van Aken HK, Hoenemann CW:The effects of local anesthetics on perioperative coagulation, inflammation, and microcirculation [J].Anesth Analg 2002,94:1441-7
[5]Kaczmarek DJ, Herzog C, Larmann J, et al. Lidocaine protects from myocardial damage due to ischemia and reperfusion in mice by its antiapoptotic effects[J]. Anesthesiology,2009,110:1041-1049
[6]Pei TS, Xu CQ, Li HZ, et al. Effect of quercetin on rat cardiomyocyte apop tosis induced by adriamyc in in vitro[J]. Chin Pharmacol Bull,2008, 24:534-538.
[7]Fried E, Amorim P, Chambers G, et al. The importance of sodium for anoxic transmission damage in rat hippocampal slices:Mechanisms of protection by lidocaine[J]. J Physiol(Lond),1995,489:557-65.
[8]Maulin K, Yoshida T, Das DK. Oxidative stress developed during the reperfusion of ischemic myocardium induces apoptosis [J].Free Radiac Biol Med,1998,24:869-875
[9]Hamm CW, Katus HA. New biochemical markers for myocardial cell injury[J]. Curr Opin Cardiol,1995,10:355-360
[10]Kevin LG, Camara AK, Riess ML, et al. Ischemic preconditioning alters real-time measure of 02 radicals in intact hearts with ischemia and reperfusion[J]. Am J Physiol Heart Circ Physiol,2003,284:H566-H574
[11]Hollmann MW, Durieux ME:Local anesthetics and the inflammatory response: A new therapeutic indication? Anesthesiology,2000,93:858-75
[12]12Honda HM, Korge P, Weiss JN. Mitochondria and ischemia/repeffusion injury[J]. Ann N Y Acad Sci.2005,047:248-258
[13]Borutaite V, Jekabsone A, Morkuniene R, et al. Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction cytochrome c and apoptosis induced by heart ischemia[J]. Mol Cell Cardiol,2003,35:357-366
[14]Chen SJ, Bradley M, Lee TC. Chemical hypoxia triggers apoptosis of cultured neonatal rat cardiac myocytes modulation by calium-regulated proteases and protein kinases[J]. Mol Cell Bichem,1998; 178:41-49
[1]Ceriello A, Quagliaro L, D' Amico M, et al. Acute hyperglycemia induces nitrotyrosine formation and apoptosis in perfused heart from rat[J]. Diabetes, 2002,51(4):1076-1082
[2]Jovanovic S, Jovanovic N, Jovanovic A. High glucose protects single beating adult cardiomyocytes against hypoxia[J]. Biochem Biophys Res Commun,2006,341(1):57-66.
[3]Kaczmarek DJ, Herzog C, Larmann J, et al. Lidocaine protects from myocardial damage due to ischemia and reperfusion in mice by its antiapoptotic effects[J]. Anesthesiology,2009,110(5):1041-1049.
[4]Luo ZQ, Feng DD, Zhou FW, et al. Protective effect of low concentration endothelin-1 on the reactive oxygen-induced inhibition of pulmonary surfactant lipid synthesis[J]. Acta Physiol Sin,2002,64(2):89-93
[5]庄心良,曾因明,陈伯銮.现代麻醉学[M].3版.北京：人民卫生出版社,2003：628-630
[6]Fried E, Amorim P, Chambers G, et al. The importance of sodium for anoxic transmission damage in rat hippocampal slices:Mechanisms of protection by lidocaine[J]. J Physiol(Lond),1995,489:557-65.
[7]Hinokiyama K, Hatori N, Ochi M, et al. Myocardial protective effect of lidocaine during experimental off-pump coronary artery bypass grafting[J]. Ann Thorac Cardiovasc Surg.2003;9(1):36-42
[8]Ross JD, Ripper R, Law WR, et al. Adding bupivacaine to high-potassium cardioplegia improves function and reduces cellular damage of rat isolated hearts after prolonged, cold storage[J]. Anesthesiology,2006,105(4):746-752.
[9]Yu XY, Song YH, Geng YJ, et al. Glucose induces apoptosis of cardiomyocytes via microRNA-1 and IGF-1[J]. Biochem Biophys Res Commun,2008,376(3):548-52
[10]Allen DA, Yaqoob MM, Harwood SM. Mechanisms of high glucose-induced apoptosis and its relationship to diabetic complications [J]. J Nutr Biochem, 2005,16(12):705-713.
[11]Williams SB, Goldfme AB, Timimi FK, et al. Acute hyperglycemia attenuates endothelium-dependent vasodilation in humans in vivo[J]. Circulation. 1998,97(17):1695-1701.
[12]Verma S, Maitland A, Weisel RD, et al. Hyperglycemia exaggerates ischemia/reperfusion-induced cardiomyocyte injury:reversal with endothelin antagonism[J]. J Thorac Cardiovasc Surg,2002 123(6):1120-1124.
[13]刘涛,徐美英,都大伟等糖尿病大鼠离体心脏对缺血/再灌注的耐受性[J].中华麻醉学杂志,2005,25(2)：126-128.