FeSO4／抗坏血酸对大鼠离体心脏缺血再灌注损伤作用的研究

有关心肌缺血再灌流损伤的发生机制多倾间于自由基学说。已有资料证实,外源性自由基发生体系FeSO_4/抗坏血酸可损伤心肌线粒体,但该体系在心肌缺血再灌流损伤中所起的作用却很少报道。本实验     

线粒体移植改善缺血再灌注损伤的作用及机制

当心脏手术、器官移植和血管病变时，血流停止以及之后血液再灌注过程都会对组织器官造成损伤，出现相应的功能障碍，即缺血再灌注损伤（ischemia-reperfusion injury,I/RI）。其中，线粒体功能障碍被认为是器官I/RI的重要原因之一，受损线粒体会导致细胞能量供应减少、活性氧（reactive oxygen species,ROS）生成增加、钙超载等结果，从而激活细胞死亡程序。线粒体是一种高度动态的细胞器，通过不断融合、分裂维持自身稳态，并且，线粒体已被研究证明可在细胞间转移。目前，线粒体提取技术较为成熟，可以从组织中提取出完整的、有活性的线粒体。在以上背景下，出现了线粒体移植（mitochondrial transplantation,MT）技术，通过移植活性线粒体至受损组织内，帮助细胞恢复功能。然而，MT的临床应用还面临着许多挑战，如MT的规范移植程序、作用机制和安全性等仍需要深入研究。本文就近10年MT改善I/RI的作用及相关机制进行了综述。     

离体缺血再灌注鼠心肌钙离子的变化

用液体闪烁计数法测定离体再灌注鼠心肌肌质网(SR)和线粒体(Mit)内 ⁴⁵Ca²⁺放射性强度(cpm),比较能量制剂ATP-MgCl₂,活性氧自由基清除剂SOD和钙阻滞剂Verapamil对离体缺血再灌注鼠心肌细胞SR和Mit钙离子浓度的影响.结果表明,SR内ATp-MgCl₂,SOD和Verapamil组 ⁴⁵Ca²⁺的cpm均高于对照组(P<0.0l或P<0.05),而Mit内均低于对照组(P<0.01).此三种药均能提高离体大鼠心肌细胞内SR ⁴⁵Ca²⁺和降低Mit ⁴⁵Ca²⁺积聚,从而保护了心肌细胞,防止缺血再灌注损伤.     

线粒体在运动保护心肌免受缺血再灌注损伤中的作用

线粒体是参与心肌缺血再灌注(myocardial ischemia and reperfusion,MI/R)损伤的关键细胞器,线粒体活性氧(reactive oxygen species,ROS)爆发、Ca²⁺失调、线粒体通透性转换孔(mitochondrial permeability transition pore,mPTP)开放、线粒体肿胀、促凋亡蛋白释放等都会导致线粒体功能障碍,心肌功能受损。运动是预防MI/R损伤的有效干预手段,其保护作用可能通过线粒体来实现。运动保护MI/R损伤的线粒体机制由多种因素决定,如线粒体能量学、K_ATP通道、mPTP、线粒体跨膜电位(ΔΨ_m)、线粒体蛋白、线粒体脂质、线粒体质量控制、远程调控因子等。本文综述了MI/R产生的线粒体机制,运动对MI/R的保护作用以及线粒体在其中的作用,以期为MI/R损伤的线粒体治疗策略提供参考。     

黄鼠与大鼠离体心脏预缺血处理对缺血再灌损伤的保护作用

本文用黄鼠和大鼠离体心脏比较了两种预缺血处理方案对随后缺血再灌损伤的作用。用Ｌａｎｇｅｎｄｏｒｆｆ方法灌流秋季未入眠的达乌尔黄鼠和Ｗｉｓｔａｒ大鼠离体心脏,以冠脉流出液中肌酸激酶（ＣＫ）释放活性、心律失常发生率、心功能恢复等为指标,检验不同预缺血处理时间对随后缺血再灌损伤的影响。标本平衡１０ｍｉｎ后对照组的两种动物经受缺灌１５ｍｉｎ再灌１５ｍｉｎ预缺血处理,接着进行两次缺灌１０ｍｉｎ再灌１０ｍｉｎ     

桑叶总黄酮预处理对大鼠缺血再灌注损伤心肌抗氧化作用的研究

为研究桑叶总黄酮预处理对缺血再灌注损伤心肌的抗氧化作用,采用结扎左冠状动脉前降支30min,再灌注2h的方法制备大鼠心肌缺血再灌注损伤模型。将50只大鼠随机分为假手术组、缺血再灌注损伤模型组和桑叶总黄酮高、中、低剂量预处理组,每组10只。实验结束后,取动脉血和心脏。测定各组血清生化指标肌酸激酶(CK)和乳酸脱氢酶(LDH)的含量;测定心肌生化指标超氧化物歧化酶(SOD)的活性和丙二醛(MDA)的含量。结果显示,与模型组相比,桑叶总黄酮预处理组使血清中的CK、LDH含量明显降低,同时使心肌组织中的SOD活性提高,MDA含量降低。结果表明,桑叶总黄酮预处理对缺血再灌注损伤心肌有明显的保护作用,其机制可能与提高心肌SOD活性、清除自由基、增强抗氧化能力有关。     

镁对缺血后再灌注心肌线粒体的影响

大鼠等容心脏制备经低血流缺血６０ｍｉｎ后复灌３０ｍｉｎ。组（２）灌注液中镁浓度始终为１．２ｍｍｏｌ／Ｌ,而组（３）及组（１）则于缺血期分别换用１５ｍｍｏｌ／Ｌ或无镁灌注淮。结果表明,高镁对心肌线粒体Ｃａ＾２＋－ＡＴＰ酶活性,合成ＡＴＰ能力具明显保护作用,并减轻了钙超载现象,部分地防止缺血后再灌注损伤。     

缺血预处理对缺血/再灌注离体心脏的保护作用

目的：探讨连续多次短暂缺血预处理对缺血/再灌注损伤心肌的保护作用及机制。方法：采用大鼠离体心脏Lan-gendorff灌流模型,观察缺血预处理对心肌缺血/再灌注后不同时间点冠脉流出液中AST、CPK、UDH及冠脉流量,心肌组织中SOD、LPO以及再灌注性心律失常的影响。结果：缺血预处理可以减少缺血/再灌注损伤的心肌冠脉流出液中AST、CPK、LDH的含量,提高心肌SOD活性,降低LPO水平,并且抑制再灌注性心律失常的发生,提高再灌注期间的冠脉流量。结论：缺血预处理对心肌缺血/再灌注损伤具有一定保护作用。     

异丙酚和氯胺酮对大鼠离体缺血再灌注损伤心肌脂质过氧化的影响

目的：比较异丙酚和氯胺酮对大鼠离体缺血再灌注损伤心肌脂质过氧化的影响。方法：成年Wistar大鼠18只,雌雄不拘。体重240-300g,随机分为3组（T1=6）：心肌缺血再灌注损伤组（I／R组）,异丙酚组（P组）,氯胺酮组（K组）。采用Langendorff灌装置建立离体心脏缺血再灌注模型,将心脏连接至Langendorff逆灌装置,3组均以K-H液平衡灌注10min后,再分别以K．H液、含30μmol/L。异丙酚的K-H液、含10μmol-L-1氯胺酮的K-H液灌注10min,然后全心停灌25min,再分别以停灌前相同的灌注液恢复灌注30min。留取冠脉流出液测定总LDH活性;灌注末取左室心肌组织置于2．5％的戊二醛固定,观察心肌的超微结构;心尖部心肌组织留待检测8-异前列腺素和SOD活性。结果：与I／R组比较,P组8-异前列腺素含量降低,SOD活性升高,LDH活性降低（P〈0．05）;K组8-异前列腺素含量,SOD及LDH活性均无统计学意义（P〉0．05）;与P组比较,K组8-异前列腺素含量升高,SOD及LDH活性降低（P〈0．05）;P组心肌超微结构损伤较m组和K组也明显改善。结论：异丙酚可显著减轻心肌缺血再灌注损伤大鼠的脂质过氧化和心肌缺血再灌注损伤,而氯胺酮没有抗心肌缺血再灌注损伤心肌脂质过氧化的作用。     

骨骼肌和心肌缺血后处理对兔缺血/再灌注心肌的保护作用

目的:观察骨骼肌缺血后处理(RPostC)、心肌的缺血后处理(MPostC)对缺血/再灌注心肌保护作用是否存在差异以及两者联合后作用是否叠加。方法:健康新西兰大白兔3O只,随机分为5组(n=6):缺血对照组(Con)、假手术组(sham)、心肌缺血后处理组(MPostC)和肢体缺血后处理组(RPostC)及心肌缺血后处理联合肢体缺血后处理组(MPostC+RPostC)。采用开胸结扎冠状动脉左室支45 min,再灌注120min方法制作缺血/再灌注模型,采用短暂结扎双侧髂外动脉固定部位5 min造成骨骼肌短暂缺血。以Evans蓝标记心肌缺血区范围,以TTC法检测梗死心肌范围,并分别于缺血前、后及再灌注1、2 h测定血浆磷酸肌酸激酶(CPK)活性和乳酸脱氢酶(LDH)含量。结果:和Con组相比,MPostC和RPostC组心肌梗死范围均明显降低(P<0.05);MPostC联合RPostC组心肌梗死范围与MPostC或RPostC组相比,均进一步降低(均P<0.05)。但MPostC组及RPostC组之间心肌坏死范围未见统计学差异。再灌注120 min末血浆CPK活性及LDH含量也显示相似趋势。结论:骨骼肌缺血后处理及心肌后处理对缺血/再灌注心肌均具有明显保护作用;且两者作用可以叠加;但骨骼肌和心肌后处理之间保护作用未显示统计学差异。     

雌激素对去卵巢大鼠离体心脏缺血／再灌注损伤的保护作用

目的：探讨雌激素对去卵巢大鼠离体心脏缺血／再灌注损伤的保护作用。方法：成年SD雌鼠,随机分为假手术组（Sham）,双侧卵巢切除组（Ovx）和双侧卵巢切除后补充17β-雌二醇组（Ovx＋E2）。各组离体心脏再随机分为不同时间的缺血再灌注亚组。测量的指标包括冠脉流出液中LDH及CK含量、心室肌细胞存活率及产率、基础状态和异丙肾上腺素（ISO）刺激状态下收缩幅度。结果：30min缺血及其各复灌纽均显著增加冠脉流出液中LDH、CK的释放量。Ovx组LDH、CK漏出在30min缺血及再灌注条件下,显著高于正常灌注组,而Ovx＋E2组可减轻心肌损伤,减少LDH、CK的释放。10min和20min缺血对心肌细胞存活率、产率及冠脉流出液中LDH、CK含量影响均不明显。Sham、Ovx、Ovx＋E2各组心肌细胞基础收缩幅度在正常和10minⅠ＋30minR灌注条件下无显著差异。Ovx显著增加其他各组心肌细胞基础收缩和ISO刺激收缩幅度,Ovx＋E2可使其降至Sham水平。结论：雌激素对去卵巢大鼠心肌缺血／再灌注损伤具有保护作用。     

心肌缺血预适应循环血中微囊泡对大鼠心肌I/R损伤的作用

目的：研究心肌缺血预适应（IPC）大鼠循环血中微囊泡（MVs）对大鼠在体心肌缺血/再灌注（I/R）损伤的作用及相关机制。方法：反复短暂结扎/松开大鼠冠状动脉左前降支建立大鼠IPC模型,自腹主动脉取血,超速离心法分离循环血中的IPC-MVs,并对其进行流式鉴定。建立在体大鼠心肌I/R模型,股静脉注射IPC-MVs 7 mg/kg。HE染色观察心肌形态学变化,TTC染色检测心肌梗死范围,TUNEL染色检测心肌细胞凋亡率。比色法测定血清乳酸脱氢酶（LDH）活力,分光光度法测定心肌组织caspase 3活力,Western blot法检测心肌组织Bcl-2、Bax蛋白表达水平。结果：流式细胞术检测IPC-MVs浓度为4380±745个/μl。与I/R组比较,IPC-MVs能够减轻I/R大鼠心肌组织损伤,缩小心肌梗死范围（P<0.01）,减少心肌细胞凋亡数量（P<0.01）,明显降低血清LDH活力（P<0.01）,降低心肌组织caspase 3活力（P<0.01）,升高Bcl-2蛋白表达（P<0.01）,降低Bax蛋白表达（P<0.01）,升高Bcl-2/Bax比值（P<0.01）。结论：IPC-MVs显著减轻大鼠在体心肌I/R损伤,通过上调心肌组织中Bcl-2的蛋白表达,下调Bax的蛋白表达,升高Bcl-2/Bax比值,降低caspase 3活力而发挥心肌保护作用。     

Diverse effects of L-arginine on cardiac function of rats subjected to myocardial ischemia and reperfusion in vivo

In vivo administration of L-arginine at different time points during the course of myocardialischemia and reperfusion(MI/R)has been shown to differentially regulate postischemic apoptosis.Cardiacfunction is one of the most important indexes used to judge the degree of myocardial injury.The presentstudy attempted to determine whether in vivo administration of L-arginine at different stages of MI/R has adiverse influence on cardiac function of ischemic reperfused hearts and,if so,to investigate the mechanismsinvolved.Male adult rats were subjected to 30 min myocardial ischemia followed by 5 h reperfusion.Anintravenous L-arginine bolus was given either 10 min before and 50 min after reperfusion(early treatment)or3 h and 4 h after reperfusion(late treatment).Early treatment with L-arginine markedly increased the leftventricular systolic pressure(LVSP)and dP/dt_(max),and decreased myocardial nitrotyrosine content.In strictcontrast,late treatment with L-arginine resulted in a significant decrease in LVSP and dP/dt_(max)from 4 h to 5h after reperfusion,and increase in toxic peroxynitrite formation as measured by nitrotyrosine.These resultssuggest that the administration of L-arginine at different time points during the course of MI/R leads todiverse effects on cardiac dysfunction.Early supplementation decreased the nitrative stress and improvedleft ventricular function.However,late treatment with L-arginine increased the formation of peroxynitriteand aggravated cardiac functional injury.     

11,12.环氧二十碳三烯酸预处理与后处理对缺血/再灌注大鼠心肌的保护作用

采用Langendorff离体灌流装置,通过停灌40 min/复灌30 min复制大鼠心肌缺血/再灌注(ischemia/reperfusion,IR)损伤模型,观察11,12-环氧二十碳三烯酸(11,12-epoxyeicosatrienoic acid,11,12-EET)预处理和后处理对心肌线粒体功能以及心功能的影响,探讨11,12-EET顸处理和后处理对IR大鼠心肌的作用及其机制.将30只Sprague-Dawley大鼠随机分为对照组、IR组、EET预处理组(Pre-EET)、EET后处理组(Post-EET),每组6只.除对照组外,其它各组全心缺血40 min,再灌注30 min.监测左心室内压差(ALVP)和左心室内压升降的最大变化率(±dp/dtmax)等心功能指标,测定灌流液中乳酸脱氢酶(1actate dehydrogenase,LDH)的活性.灌流结束后,测定心肌线粒体琥珀酸脱氢酶(succinate dehydrogenase,SDH)、Ca"ATPase、Na - K -ATPase活性以及心肌超氧化物歧化酶(superoxide dismutase,SOD)活性、丙二醛(malondialdehyde,MDA)含量.结果显示:(1)与IR组相比,Pre-EET组及Post.EET组Na -K -ATPase和SDH活性均增强,Ca2 -ATPase活性均减弱,有显著性差异(P<0.05);而Pre-EET与Post-EET组间没有显著性差异.(2)与IR组相比,Pre-EET组及Post-EET组心功能明显改善,LDH漏出显著减少,心肌SOD活性明显增强,MDA含量明显降低,有显著性差异(P<0.05);而Pre-EET与Post-EET组间没有显著性差异.结果表明,11,12-EET预处理及后处理均可通过上调心肌线粒体Na -K -ATPase、SDH活性以及下调Ca2 -ATPase活性改善线粒体功能和心肌能量代谢,拮抗心肌IR损伤;11,12-EET预处理及后处理还可通过提高心肌SOD活性、降低MDA含量改善IR心肌的氧化应激.     

Free fatty acid metabolism during myocardial ischemia and reperfusion

Long chain free fatty acids (FFA) are the preferred metabolic substrates of myocardium under aerobic conditions. However, under ischemic conditions long chain FFA have been shown to be harmful both clinically and experimentally. Serum levels of free fatty acids frequently are elevated in patients with myocardial ischemia. The proposed mechanisms of the detrimental effects of free fatty acids include: (1) accumulation of toxic intermediates of fatty acid metabolism, such as long chain acyl-CoA thioesters and long chain acylcarnitines, (2) inhibition of glucose utilization, particularly glycolysis, during ischemia and/or reperfusion, and (3) uncoupling of oxidative metabolism from electron transfer. The relative importance of these mechanisms remains controversial. The primary site of FFA-induced injury appears to be the sarcolemmal and intracellular membranes and their associated enzymes. Inhibitors of free fatty acid metabolism have been shown experimentally to decrease the size of myocardial infarction and lessen postischemic cardiac dysfunction in animal models of regional and global ischemia. The mechanism by which FFA inhibitors improve cardiac function in the postischemic heart is controversial. Whether the effects are dependent on decreased levels of long chain intermediates and/or enhancement of glucose utilization is under investigation. Manipulation of myocardial fatty acid metabolism may prove beneficial in the treatment of myocardial ischemia, particularly during situations of controlled ischemia and reperfusion, such as percutaneous transluminal coronary angioplasty and coronary artery bypass grafting. (Mol Cell Biochem 166: 85-94, 1997)     

缺血后处理对缺血／再灌注大鼠心肌基质金属蛋白酶-2表达的影响

目的：探讨缺血后处理对缺血／再灌注大鼠心肌基质金属蛋白酶-2（MMP-2）表达的影响及其与心肌间质和心功能变化的关系。方法：2,4只sD大鼠随机分为3组（n=8）：假手术组（SC组）、缺血／再灌注组（I／R组）和缺血后处理组（n,rc组）。记录各组左室血流动力学变化,观察心肌胶原含量,测定血浆中丙二醛（MJ）A）和超氧化物歧化酶（SOD）浓度改变。以№ternblot测定MMP-2蛋白的活性,以RT-PCR法测定MMP-2rrffLNA表达的变化。结果：IFIE组心肌MMP-2蛋白活性及MMP-2mRNA表达明显降低,而心肌胶原含量、左室舒缩功能明显高于L／R组。同时,血浆SOD活力增强而MDA含量降低。结论：IFIE对I／R心肌的保护作用之一可能是通过减少自由基产生,抑制MMP-2的活性和表达,减轻了心肌间质的损伤而实现的。     

Protection against in vivo focal myocardial ischemia/reperfusion injury-induced arrhythmias and apoptosis by Hesperidin

Among the heart diseases, ischemia and reperfusion (I/R) induced arrhythmias contribute to episodes of sudden death. Cardiac arrhythmias during ischemia reperfusion are believed to be related to oxidative stress. Therefore, the aim of this study was to examine whether treatment with Hesperidin alleviates arrhythmias and infarct size in experimentally-induced myocardial I/R injury using an in vivo rat model. In this study haemodynamics parameters, markers of inflammation, biomarkers of oxidative stress and tissue nitrite level and infarct size of the heart were estimated in various groups. I/R showed a significant decrease in tissue nitrite and antioxidant level and significant increase in arrhythmias, inflammation and myocardial cell apoptosis. Treatment with Hesperidin showed a significant increase in tissue nitrite, antioxidant level and reduction in inflammation, arrhythmias and apoptosis. In conclusion, the protecting effect of Hesperidin in I/R induced arrhythmias is due to reduction in inflammation and oxidative stress.     

Role of insulin in the anti-apoptotic effect of glucose-insulin-potassium in rabbits with acute myocardial ischemia and reperfusion

OBJECTIVE: To study the effects of glucose-insulin-potassium (GIK) cocktail on cardiac myocyte apoptosis and cardiac functional recovery following myocardial ischemia/reperfusion (MI/R), and to further determine the role of insulin in the GIK-induced cardioprotective effect in vivo . METHODS: Forty eight male rabbits were subjected to 40 min MI followed by R for 3 h and were randomly received one of the following treatments: saline, GIK (glucose: 150 g/L, insulin: 60 U/L and KCl: 80 mmol/L), or insulin (n = 16 in each group) at 1 ml x kg(-1) x h(-1), beginning 30 min before MI and continuing throughout the 3 h-reperfusion. Blood glucose, electrolytes, arterial blood pressure and left ventricular pressure (LVP) were monitored throughout the experiment. Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) activity were measured spectrophotometrically. Myocardial infarction and myocardial apoptosis (both DNA laddering and TUNEL analysis) were determined in a blinded manner. RESULTS: MI/R caused significant cardiac dysfunction and myocardial apoptosis (both strong DNA ladder formation and TUNEL-positive staining). Compared with vehicle, GIK-treated rabbits showed protection against MI/R as evidenced by reduced myocardial infarction (19.7% +/- 2.6% vs . 26.8% +/- 3.3% of vehicle, n = 10, P < 0.05), marked decrease in DNA fragmentation and apoptotic index (11.0% +/- 2.1% vs . 20.1% +/- 3.1% of vehicle, n = 6, P < 0.01), significant decrease of plasma CK and LDH and improved recovery of cardiac systolic/diastolic function at the end of R. Treatment with insulin alone decreased blood glucose significantly but still exerted cardioprotective effects comparable with that of GIK. CONCLUSIONS: GIK exerts cardioprotective effects against postischemic myocardial injury and improves cardiac functional recovery in vivo . Insulin, mainly through the anti-apoptotic effect, plays a key role in the GIK-elicited myocardial protection in MI/R.     

下肢骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响

目的:探讨在体情况下,骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响。方法:新西兰大白兔36只,随机分成3组(每组随机选取6只进行梗死范围的测定,另外6只进行凋亡测定):①假手术组(Sham组);②缺血/再灌注组(I/R组);③远端后处理组(RPostC组)。在缺血前、后及再灌注60 min、120 min分别抽血测定肌酸激酶(CK),乳酸脱氢酶(LDH)的活性。采用伊文思兰(evans blue)和三苯基氯化四氮唑(TTC)染色方法确定心肌缺血区范围以及心肌坏死区范围。用Tunel法检测兔心肌缺血区细胞凋亡情况,免疫组织化学方法检测心肌缺血区蛋白caspase-3、Bcl-2及Bax的表达。结果:RPostC组心肌坏死程度、再灌注末CK活性较I/R组明显减低。RPostC组缺血区心肌Tunel阳性指数显著低于I/R组(21.79%±1.07%vs35.81%±1.10%,P<0.05)。而RPostC组缺血区心肌细胞caspase-3阳性指数显著低于I/R组(25.03%±1.16%vs39%±2.43%,P<0.05)。与Sham组比较,I/R组及RPostC组Bax蛋白表达指数、Bcl-2蛋白表达指数均升高;但RPostC组的Bax/Bcl-2比值降低,而I/R组的Bax/Bcl-2比值升高。与I/R组相比较,RPostC组Bax蛋白表达指数及Bax/Bcl-2比值显著降低,Bcl-2表达指数显著升高,差异均有统计学意义。结论:远端后处理能够明显的减少缺血/再灌注心肌细胞的坏死和凋亡,其减轻心肌细胞凋亡的机制可能与抑制促凋亡基因caspase-3的活化及Bcl-2表达的上调有关。