曲美他嗪后处理对大鼠心肌缺血再灌注损伤的保护性研究

目的研究曲美他嗪后处理对大鼠急性心肌缺血再灌注后氧自由基损伤及细胞凋亡的影响。方法选择Wistar成年大鼠50只分为假手术组、再灌注损伤模型组(模型组)、曲美他嗪低剂量组(低剂量组)、曲美他嗪高剂量组(高剂量组)、缺血后处理组(后处理组),每组10只。后4组制作缺血再灌注损伤模型后,测定各组大鼠血流动力学变化,超氧化物歧化酶(SOD)、丙二醛水平,光镜、电镜下心肌组织切片观察。结果与假手术组比较,模型组、低剂量组、高剂量组和后处理组左心室舒张末压明显升高,左心室收缩压、左心室内压最大上升和下降速率明显减少(P<0.05,P<0.01)。与模型组比较,高剂量组、后处理组左心室舒张末压明显减低,左心室内压最大上升和下降速率明显增加(P<0.05)。与假手术组比较,模型组、低剂量组、高剂量组、后处理组丙二醛水平明显增高,SOD水平明显降低(P<0.05,P<0.01);与模型组比较,低、高剂量组、后处理组丙二醛水平明显减低,SOD水平明显升高(P<0.05,P<0.01)。结论高剂量曲美他嗪对大鼠心肌缺血再灌注损伤有保护作用。     

环孢菌素A拮抗心肌缺血/再灌注损伤的作用

目的:研究环孢菌素A(CsA)拮抗小型猪心肌缺血/再灌注损伤(MI/RI)的作用及可能的机制。方法:经皮球囊封堵冠状动脉左前降支制备小型猪MI/RI模型。将存活的动物随机分为3组:即对照组(n=4)、CsA组(n=6)及他可英司(FK-506)组(n=6),分别静滴生理盐水100 ml、25 mg/kg CsA及1 mg/kg FK-506。所有动物均经90 min缺血和3 h再灌注。通过病理检查评估心肌梗死(MI)面积。用免疫组化染色法检测心肌细胞凋亡。用透射电子显微镜观察各组心肌细胞线粒体的形态。结果:CsA组MI的面积比对照组[(7.5±0.6)cm2vs.(10.5±2.6)cm2]和FK-506组[(7.5±0.6)cm2vs.(9.6±2.7)cm2]明显减少(P0.01);CsA组心肌细胞的凋亡率(%)比对照组[(11.9±1.88)%vs.(22.3±1.66)%]和FK-506组[(11.9±1.88)%vs.(19.2±1.82)%]明显下降(P0.01)。透射电子显微镜检查显示,CsA组能维持线粒体的形态,线粒体坍塌的百分率为(20%±7%),比对照组(53%±12%)和FK-506组(47%±9%)明显减少(P0.01)。结论:CsA可能对MI/RI具有拮抗作用,其机制可能是通过抑制线粒体膜通透性转换孔(mPTP),保持线粒体形态完整而实现,此种效应不依赖于钙调磷酸酶抑制途径。     

环孢菌素A拮抗心肌缺血／再灌注损伤的作用

目的：研究环孢菌素A（CsA）拮抗小型猪心肌缺血／再灌注损伤（MI／RI）的作用及可能的机制。方法：经皮球囊封堵冠状动脉左前降支制备小型猪MI／RI模型。将存活的动物随机分为3组：即对照组（n=4）、CsA组（n=6）及他可英司（FK-506）组（n=6）,分别静滴生理盐水100ml、25mg／kgCsA及1mg／kgFK-506。所有动物均经90rain缺血和3h再灌注。通过病理检查评估心肌梗死（MI）面积。用免疫组化染色法检测心肌细胞凋亡。用透射电子显微镜观察各组心肌细胞线粒体的形态。结果：CsA组MI的面积比对照组[（7．5±0．6）cm。粥．（10．5±2．6）cm。]和FK-506组[（7．5±0．6）cm。掷．（9．6±2．7）cm。]明显减少（P〈0．01）;CsA组心肌细胞的凋亡率（％）比对照组[（11．9±1．88）％郴．（22．3±1．66）％]和FK-506组[（11．9±1．88）％郴．（19．2±1．82）％]明显下降（JP〈0．01）。透射电子显微镜检查显示,CsA组能维持线粒体的形态,线粒体坍塌的百分率为（20％±7％）,比对照组（53％±12％）和FK-506组（47％±9％）明显减少（P〈0．01）。结论：CsA可能对MI／RI具有拮抗作用,其机制可能是通过抑制线粒体膜通透性转换孔（mPTP）,保持线粒体形态完整而实现,此种效应不依赖于钙调磷酸酶抑制途径。     

阿利吉仑通过调控线粒体介导的凋亡通路改善心肌缺血再灌注损伤

目的通过体外构建心肌细胞缺氧复氧模型(H/R)模拟体内心肌细胞缺血再灌注,验证阿利吉仑(Aliskiren)对于改善心肌细胞缺血再灌注的药物效果,同时探究细胞凋亡在其中的机制。方法将细胞实验分为四组:正常氧供应组即对照组(Control)、缺氧复氧组(H/R)、阿利吉仑+缺氧复氧组(阿利吉仑+H/R)、NF-κB P65特异抑制剂+缺氧复氧组(bay11-7082+H/R)。使用CCK-8检测不同浓度阿利吉仑处理的心肌细胞存活率,ELISA检测各实验组炎症因子肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)水平。Hoechst33258染色、Annexin V/PI双染流式细胞仪检测各组心肌细胞凋亡比例,JC-1试剂盒测量线粒体膜电位及心肌细胞ATP含量。同时采用Caspase-3试剂盒检测各组心肌细胞凋亡蛋白酶的活性。结果阿利吉仑小于20 mmol/L时,与心肌细胞活性存在正相关关系,而在20 mmol/L和80 mmol/L之间,两者之间存在负相关关系,文章中阿利吉仑的最佳处理浓度是20 mmol/L,此时的心肌细胞活性最高(76.40%±1.64%)。相比H/R组,阿利吉仑能降低TNF-α和IL-6水平[(129.33±5.86) ng/L比(319.00±4.58) ng/L,P0.05;(29.67±1.53) ng/L比(64.67±2.08) ng/L,P0.05],同时显著降低心肌细胞的凋亡率[(7.23%±1.14%)比(32.25%±3.15%),P0.05],并具有降低能量代谢障碍心肌细胞所占比例[(6.9%±1.6%)比(13.5%±1.7%),P0.05]、稳定线粒体膜电位的功能[(3.90±0.60)比(1.80±0.16),P0.05]。另外,抑制凋亡蛋白酶Caspase-3的活性[(2.26±0.35)比(3.26±0.62),P0.05],且阿利吉仑+H/R组与bay11-7082+H/R组的各项实验结果无统计学差异。结论阿利吉仑可以通过抑制炎症反应、调控线粒体受体介导的凋亡,改善缺血心肌细胞缺血再灌注损伤,且阿利吉仑的调控凋亡作用可能与NF-κB表达抑制有关。     

神经调节蛋白-1对大鼠心肌缺血/再灌注损伤保护作用的研究

目的:探讨神经调节蛋白-1(NRG-1)对大鼠心肌缺血/再灌注(I/R)损伤的保护作用及其潜在机制。方法:雄性,SD大鼠,分三组:假手术组(n=8)、心肌(I/R)组(n=8)和NRG-1+I/R组(n=9)。通过结扎冠状动脉左前降支45 min,再灌注3 h建立在体大鼠心肌I/R模型。用伊文氏蓝/2,3,5-三苯基氯化四氮唑(TTC)染色法测定心肌梗死范围。用脱氧核糖核苷酸末端转移酶介导的d UTP缺口末端标记法(TUNEL)染色法检测心肌细胞凋亡指数;免疫荧光法测定线粒体膜电位水平;用Western blot方法检测线粒体细胞色素c的转位、凋亡相关因子Bcl-2和Bax的表达;caspase 3试剂盒检测caspase 3活性;用透射电镜观察心肌组织线粒体超微结构。结果:与假手术组相比,I/R组心肌细胞凋亡显著增加[(23.2±3.8)vs.(3.0±1.3)%,P0.01],心肌线粒体膜电位降低[(209.1±13.6)vs.(336.8±10.3)m V,P0.05],细胞色素c向胞浆发生转位,caspase 3活性显著升高[(20.1±3.6)vs.(8,3±1.5),P0.01]。与单纯I/R组比较,NRG-1给药显著降低I/R大鼠心肌的梗死面积/危险区面积[(28.6±9.2)vs.(51.7±7.8)%,P0.01],减少心肌细胞凋亡指数[(11.9±3.5)vs.(23.2±3.8)%,P0.01],升高Bcl-2/Bax蛋白表达比值[(1.647±0.172)vs.(0.490±0.080),P0.01],升高线粒体膜电位[(327.2±15.4)vs.(209.1±13.6)m V,P0.05],抑制细胞色素c向胞浆的转位[(0.207±0.055)vs.(0.483±0.075),P0.01],降低心肌caspase 3活性[(9.3±2.6)vs.(20.1±3.6),P0.01],改善线粒体超微结构。结论:NRG-1具有抗心肌I/R损伤的保护作用,其机制部分通过抑制线粒体介导的心肌细胞凋亡途径实现。     

T细胞参与心肌I/R损伤的研究进展

心肌I/R损伤（MI/RI）是治疗心肌梗死过程中的并发症之一,中性粒细胞、巨噬细胞等固有免疫细胞引起的炎性反应是导致MI/RI的重要原因。众多免疫细胞如何在MI/RI中发挥作用是目前研究的热点问题。近年来,越来越多的研究表明T细胞在MI/RI中发挥重要作用。本文将阐述T细胞在MI/RI中的作用及机制的研究进展,并分析目前存在的问题及发展方向。     

褪黑素减轻心肌缺血/再灌注损伤的作用及机制

目的:探讨褪黑素（melatonin,Mel）在大鼠心肌缺血/再灌注（MI/R）损伤中的拮抗作用及其机制。方法:80只体质量200~250 g雄性SD大鼠随机分为4组:假手术（Sham）组、溶剂对照（MI/R+V）组、Mel治疗（MI/R+Mel）组、Mel+EX527（MI/R+Mel+EX）组。常规结扎左冠状动脉前降支行心肌缺血/再灌注手术,缺血30 min,再灌72 h后超声心动图法检测各组大鼠心功能,再灌6 h后ELISA法检测血清酶学指标,TUNEL法检测心肌细胞凋亡率,Evans blue-TTC双染法测定梗死面积,Western blot法检测沉默信息转录调控因子1（SIRT1）、乙酰化叉头转录因子1（Ac-Foxo1）及凋亡相关蛋白表达水平。结果:Mel治疗可显著改善MI/R损伤后心功能,降低血清肌酸激酶（CK）及乳酸脱氢酶（LDH）水平,降低凋亡率及梗死面积,上调SIRT1表达,下调Ac-Foxo1水平,降低凋亡相关蛋白表达。而使用EX527阻断SIRT1信号后逆转Mel的上述作用（均P<0.01）。结论:Mel可发挥抗凋亡作用减轻MI/R损伤并改善心功能,其作用机制可能与其激活SIRT1信号通路并降低Ac-Foxo1水平有关。     

Inhibition of N-ethylmaleimide-sensitive factor protects against myocardial ischemia/reperfusion injury

Exocytosis of endothelial granules promotes thrombosis and inflammation and may contribute to the pathophysiology of early reperfusion injury following myocardial ischemia. TAT-NSF700 is a novel peptide that reduces endothelial exocytosis by inhibiting the ATPase activity and disassembly activity of N-ethylmaleimide-sensitive factor (NSF), a critical component of the exocytic machinery. We hypothesized that TAT-NSF700 would limit myocardial injury in an in vivo murine model of myocardial ischemia/reperfusion injury. Mice were subjected to 30 minutes of ischemia followed by 24 hours of reperfusion. TAT-NSF700 or the scrambled control peptide TAT-NSF700scr was administered intravenously 20 minutes before the onset of ischemia. Myocardial ischemia/reperfusion caused endothelial exocytosis, myocardial infarction, and left ventricular dysfunction. However, TAT-NSF700 decreased von Willebrand factor levels after myocardial ischemia/reperfusion, attenuated myocardial infarct size by 47%, and preserved left ventricular structure and function. These data suggest that drugs targeting endothelial exocytosis may be useful in the treatment of myocardial injury following ischemia/reperfusion.     

大鼠心肌缺血再灌注心功能变化与JAK-STAT信号通路相关性研究

目的观察JAK-STAT信号通路在离体大鼠心肌缺血再灌注(I/R)损伤过程中激活的时程及意义。方法采用Langendorff离体灌流模型,将42只雄性Wistar大鼠随机分为7组:对照组:用改良的KH液持续灌流180min;I/R组:按再灌注时间分为R0、R5、R15、R30、R60、R120 6组,用改良的KH液灌流平衡30 min后,全心停灌30 min,分别再灌注0、5、15、30、601、20 min。连续监测左室舒张压(LVDP)、左室压力最大升降速率(+dp/dtmax)以评价心功能,免疫印记法检测再灌注不同时程磷酸化的信号转导与转录激活子-1(STAT1)、STAT3蛋白表达的变化。结果与对照组相比,再灌注120 min时,LVDP从(96.0±7.4)mm Hg降至(46.2±3.8)mm Hg,+dp/dtmax从(2002±215)mm Hg降至(642±124)mm Hg(P<0.01)。再灌注各时程STAT1、STAT3均处于激活状态,与对照组相比差异有统计学意义(P<0.01),但二者表达存在时间差异,p-STAT1在缺血30 min增高不明显,再灌注过程中处于显著激活状态(P<0.01);p-STAT3在缺血30 min即明显升高(P<0.01),而再灌注期未见进一步升高(P>0.05),p-STAT1/p-STAT3与LVDP及+dp/dtmax呈明显的相关性(r=-0.894,r=-0.886,P<0.001)。结论JAK-STAT信号通路参与了心肌I/R损伤,磷酸化的STAT1与STAT3的比值可能决定了再灌注损伤的严重程度。     

Pharmacological possibilities for protection against myocardial reperfusion injury

Reperfusion through thrombolysis or percutananeous coronary angioplasty is standard treatment in impending acute myocardial infarction. Although restoration of blood flow to the jeopardised myocardial area is a perquisite for myocardial salvage, reperfusion itself may lead to accelerated and additional myocardial injury beyond that generated by ischemia alone. This is referred to as the "reperfusion injury". Since the reperfusion injury is initiated by the treatment of myocardial infarction, it is of importance to limit the extent of the injury. Several studies aimed at preventing reperfusion injury by means of pharmacological agents have therefore been conducted. The design of such studies is crucial for the results. Factors of importance are the timing of drug administration, animal species used, the degree of collateral flow and the duration of ischemia. A variety of pharmacological compounds have been investigated in different experimental models of myocardial ischemia and reperfusion. These include oxygen free radical scavengers, antioxidants, calcium channel blockers, inhibitors of neutrophils, nitric oxide, adenosine-related agents, inhibitors of the renin-angiotensin system, endothelin receptor antagonists, Na(+)/H(+) exchange inhibitors, and anti-apoptotic agents. All these groups of pharmacological agents have been demonstrated to protect from reperfusion injury determined as limitation of infarct size, improved myocardial and endothelial function, and reduced incidence of arrhythmias. The mechanism behind the protective effect may differ between different groups of compounds, but some compounds may exert cardioprotection via common pathways. Such a pathway may be via maintained bioavailability of nitric oxide.     

Melatonin receptor‐mediated protection against myocardial ischemia/reperfusion injury: role of SIRT1

Melatonin confers cardioprotective effect against myocardial ischemia/reperfusion (MI/R) injury by reducing oxidative stress. Activation of silent information regulator 1 (SIRT1) signaling also reduces MI/R injury. We hypothesize that melatonin may protect against MI/R injury by activating SIRT1 signaling. This study investigated the protective effect of melatonin treatment on MI/R heart and elucidated its potential mechanisms. Rats were exposed to melatonin treatment in the presence or the absence of the melatonin receptor antagonist luzindole or SIRT1 inhibitor EX527 and then subjected to MI/R operation. Melatonin conferred a cardioprotective effect by improving postischemic cardiac function, decreasing infarct size, reducing apoptotic index, diminishing serum creatine kinase and lactate dehydrogenase release, upregulating SIRT1, Bcl‐2 expression and downregulating Bax, caspase‐3 and cleaved caspase‐3 expression. Melatonin treatment also resulted in reduced myocardium superoxide generation, gp91^phox expression, malondialdehyde level, and increased myocardium superoxide dismutase (SOD) level, which indicate that the MI/R‐induced oxidative stress was significantly attenuated. However, these protective effects were blocked by EX527 or luzindole, indicating that SIRT1 signaling and melatonin receptor may be specifically involved in these effects. In summary, our results demonstrate that melatonin treatment attenuates MI/R injury by reducing oxidative stress damage via activation of SIRT1 signaling in a receptor‐dependent manner.     

心肌缺血再灌注损伤研究进展

急性心肌梗死是主要的致死致残原因,再灌注治疗是其标准的治疗方案,然而再灌注治疗伴随着再灌注损伤,再灌注损伤的机理目前还未完全清楚,分子、细胞、组织上的改变均与参与再灌注损伤,本文就心肌缺血再灌注损伤的研究进展作一综述。     

褪黑素抗心肌缺血/再灌注损伤的室旁核机制

目的 探讨外周输注褪黑素通过下丘脑室旁核（PVN）调节交感神经活性对抗心肌I/R损伤的机制。方法 腹腔注射褪黑素或者无菌生理盐水,24 h后建立C57BL/6J小鼠心肌I/R损伤模型。超声心动图测定心功能,用Evans blue/TTC双染色法测定再灌注24 h后心肌梗死面积。免疫荧光检测下丘脑室旁核（PVN）IL-10和IL-1β及心肌酪氨酸羟化酶（TH）水平;Western blot检测下丘脑室旁核NF-κB水平;ELISA检测血浆去甲肾上腺素(NE)水平。结果 外周给予褪黑素可抑制PVN区NF-κB水平（P<0.01）,进而降低PVN区IL-1β水平（P<0.01）,升高IL-10水平（P<0.01）,降低NE及TH活性,缩小心肌梗死面积（P<0.01）,改善C57BL/6J小鼠心功能。结论 外周注射褪黑素,通过调控PVN区炎性细胞因子水平,抑制交感神经活性,改善心肌I/R损伤所致心功能降低。     

Superoxide dismutase conjugated to polyethylene glycol provides sustained protection against myocardial ischemia/reperfusion injury in canine heart

Disagreement regarding the cardioprotective role of superoxide dismutase may relate to the use of different durations for induction of ischemic injury and reperfusion. The present study employed superoxide dismutase conjugated to polyethylene glycol (PEG-SOD), which has a half-life greater than 30 hours. Two protocols differing in the mode of administration and the duration of the reperfusion interval were used. Dogs were subjected to occlusion of the circumflex coronary artery for 90 minutes, then reperfused for 6 hours (Protocol A) or 4 days (Protocol B). The dogs received either polyethylene glycol conjugated to albumin (PEG-ALB) or PEG-SOD (1,000 U/kg). In Protocol A, treatment was administered starting 15 minutes before coronary occlusion and continued for 2 hours, terminating 15 minutes after reperfusion. Infarct size was determined 6 hours later. In Protocol B, the conjugated proteins were given 15 minutes before reperfusion and ended simultaneously with reperfusion. Infarct size was measured after 4 days. Infarct size (percentage of area at risk) in control (n = 9) and treated (n = 9) dogs in Protocol A differed between groups: 46.7 +/- 3.5% versus 28.3 +/- 2.9%, respectively (p less than or equal to 0.005); risk regions did not differ: 42.8 +/- 1.5% versus 43.8 +/- 2.1%, respectively. Myocardial salvage also was observed in Protocol B. Infarct size in control (n = 13) and treated (n = 13) groups was 44.2 +/- 2.6% versus 29.2 +/- 1.6%, respectively (p less than or equal to 0.005), with risk regions being 44.4 +/- 1.4% versus 46.0 +/- 1.6% (p = NS). Hemodynamic variables did not differ during the period of coronary artery occlusion. The respective collateral blood flows to the inner two thirds of the ischemic myocardium determined 60 minutes after occlusion were 0.05 +/- 0.01 ml/min/g and 0.06 +/- 0.04 ml/min/g (p = 0.806) for the PEG-ALB and PEG-SOD treated groups, respectively. Infarct size was related inversely to collateral blood flow in the PEG-ALB treated group. This relation shifted downward (analysis of covariance, p = 0.017). Plasma SOD activity in Protocols A sustained for 6 hours. Significant enzymatic activity was present after 4 days in Protocol B. Previous negative studies with native SOD may be related to the short half-life of its free-radical scavenging capacity, which compromises the chances of observing a protective effect after 4 days of reperfusion. The present results support our previous observations, as well as those of other investigators, demonstrating that superoxide dismutase can reduce that component of myocardial injury associated with reperfusion.     

微小RNA在心肌缺血再灌注损伤中的作用及机制

微小RNA（microRNA,miRNA）是一类在进化上高度保守的小分子非编码RNA,大约南19～25个核苷酸组成,具有转录后渊控蛋白质编码基因表达的功能,     

IPC和缬沙坦在心肌缺血/再灌注损伤中的作用

目的 研究缺血预适应（ischemic preconditioning,IPC）和缬沙坦(valsartan)在缺血/再灌注(I/R)损伤时与信号转导及转录激活因子3(STAT3)的关系,及其对心脏的保护作用。方法 将32只Wistar 大鼠随机分为4组(每组8只),分别为I/R组、IPC组、缬沙坦I/R（VIR）组、和缬沙坦IPC（VIPC）组,I/R组和IPC组合称为生理盐水组,VIR组和VIPC组合称为缬沙坦组。用ELISA方法检测血清IL-6的水平,并留取梗死区心肌组织用免疫组化染色法,检测STAT3 的磷酸化。结果 IPC 组与I/R组相比,STAT3的磷酸化明显增加(P<0.01),IL-6 的水平降低(P<0.01)。缬沙坦组与生理盐水组相比,STAT3的磷酸化和血清IL-6的水平明显降低(P<0.01)。结论 IPC通过STAT3磷酸化,可激活心肌存活通路,抑制炎症因子的表达,减轻I/R损伤。缬沙坦可部分抑制STAT3 活化,抑制IL-6的表达,并产生心脏保护作用。     

mTOR信号介导的自噬在褪黑素减轻心脏缺血/再灌注损伤中的作用

目的 探讨mTOR信号介导的自噬在褪黑素（melatonin,Mel）减轻心脏缺血/再灌注损伤中的作用。方法 将60只8周龄C57BL/6小鼠随机分为假手术（Sham）组、单纯褪黑素10 mg/(kg·d)处理(Mel)组、缺血/再灌注（ischemia reperfusion,I/R）组和褪黑素10 mg/(kg·d)干预I/R（Mel+I/R）组。采用冠状动脉左前降支结扎术制备心肌I/R模型,HE染色观察心肌组织形态学变化,试剂盒检测各组血清中LDH的含量,TUNEL染色检测各组细胞凋亡情况,蛋白印迹法（Western blot）检测自噬相关蛋白微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3,LC3）I和II、Beclin1和mTOR磷酸化的表达,免疫荧光染色法检测LC3B的表达。结果 与Sham组相比,Mel组各项指标均无明显变化;I/R组心肌纤维断裂明显排列紊乱,血清中LDH含量明显增加（P<0.01）,TUNEL阳性细胞明显增多（P<0.01）,LC3II和Beclin1表达显著升高（P<0.01）,而磷酸化mTOR的表达降低（P<0.01）,免疫荧光结果显示LC3B表达增加（P<0.01）; Mel+I/R组可明显减轻心肌纤维的断裂,降低血清中LDH含量（P<0.01）,减少TUNEL阳性细胞数（P<0.01）,减少LC3II和Beclin1的表达（P<0.01）,降低免疫荧光染色中LC3B的表达（P<0.01）。结论 褪黑素通过调节mTOR信号介导的自噬减轻心脏I/R损伤。