金祝秋

　

论文题目：药理性预适应对自由基损伤心脏的保护作用及其细胞和分子机制

作者简介：金祝秋，男，1963年生，1995年师从湖南医科大学陈修教授，于1998年获博士学位。

摘要

近年来,实验发现短暂的心肌缺血后再灌注能显著减轻长时间的心肌缺血再灌注损伤,即心肌缺血预适应(Myocardial ischemic preconditioning, IP)。这种现象已在不同动物（狗，大鼠，家兔等）和临床研究中得到证实。心肌缺血预适应的保护作用表现在缩小心肌梗范围，抗缺血再灌注心律失常，改善心肌代谢及其收缩和舒张功能，减轻心肌顿抑及减轻超微结构的破坏等。现知其机理与心肌释放化学保护物质有关。短暂心肌缺血再灌注可增加心脏内源性物质，如：腺苷，去甲肾上腺素，缓激肽，降钙素基因相关肽等的生成或释放。这些物质分别与相应的G蛋白偶联受体结合，通过细胞内信号传导，产生心脏保护作用。心肌缺血预适应的保护作用可分为两个时相：即急性期（第一窗口）效应和延迟期（第二窗口）效应。急性期效应出现时间快，但维持时间短，延迟期是在短暂心肌缺血后的24小时达到高峰，维持时间长。一般认为与热休克蛋白或抗氧化酶产生有关，但具体机理尚未阐明。基于心肌缺血预适应是释放内源性物质产生的心肌保护作用，因此，近年人们试用外源性化学物质模拟心肌缺血预适应产生心肌保护作用，即心肌药理预适应（Myocardial pharmacological preconditioning）。

血管紧张素转化酶（Angiotensin converting enzyme, ACE）抑制剂是临床上常用的一类抗高血压和治疗心力衰竭的药物。ACE和激肽酶Ⅱ实质上为同一种酶。ACE抑制剂通过抑制激肽酶Ⅱ,可减少缓激肽（Bradykinin,BK）的分解，增加其浓度。BK是机体内的一种重要的内源性物质，有心肌保护作用。应用选择性B2受体阻断剂HOE140能取消大鼠，家兔和狗心肌缺血预适应的心脏保护作用，表明BK是参与心肌缺血预适应的化学物质之一。最近有报道ACE抑制剂预处理离体心脏后，能增强长时间保存后心脏功能的恢复或增强心肌缺血预适应的保护作用。但对ACE抑制剂预适应的抗自由基损伤作用，特别是延迟相的保护心脏作用尚未见报道。

自由基是心肌缺血/再灌注损伤的重要介质。在心肌缺血/再灌注过程中，可产生大量活性氧自由基。它们使生物膜脂质过氧化，是导致心肌损伤的重要原因。本课题重点研究了ACE抑制剂雷米普利（Ramipril, RAM）预处理对自由基损伤心脏和心肌细胞的保护作用；并从细胞和分子水平进行了机理的探讨；为药物启动机体内源性的保护机制，充实药理性预适应的概念与实用性，扩大ACE抑制剂的临床用途提供新的思路。

全文共分为五个部分：

第一部分 一种新的自由基损伤离体心脏模型的建立

目的：1，1-二苯基-2-三硝基苯肼（1,1-Diphenyl-2-Picryl-Hydrazyl, DPPH）是一种脂溶性的稳定自由基，多年来用于检测植物成分的自由基清除作用。我室以前的研究工作表明：DPPH对离体家兔心脏与血管内皮细胞有损伤作用。为建立一种新的自由基损伤心脏模型，我们较系统地研究了DPPH对离体豚鼠心脏的损伤作用，并与次黄嘌呤（HX）-黄嘌呤氧化酶（XO）产生的超氧阴离子（·O2）的心脏损伤进行了比较。

方法：1. 对离体豚鼠心脏功能的影响：实验用雄性豚鼠，Langendorff心脏Krebs-Henseleit (K-H)液灌流；左心室水囊导管记录心脏收缩和舒张功能指标（LVDP, ±dP/dtmax,LVEDP）；量取冠脉流量（CF），并测定流出液的LDH。心脏平衡20分钟后，分别加入100和250 nM DPPH, 记录不同时间内心功能的变化，并测定LDH和心脏脂质过氧化物（TBARS）含量，与HX-XO产生的超氧阴离子（·O2）的心脏损伤进行了比较。另设三组预先给超氧花物歧化酶（SOD，200U/L），二甲基亚砜（DMSO，0.1mM）和左旋半胱氨酸(Cys, 1μM)10分钟，再加入DPPH灌流，观察它们对DPPH自由基损伤心脏的保护作用。

2．离体豚鼠心脏自由基信号的电子自旋共振（ESR）波谱分析：将不同浓度DPPH损伤的离体豚鼠心脏及Cys或SOD+DPPH处理的心脏置于液氮中冷冻，进行ESR测定。

3．对心肌细胞膜流动性的影响：雄性Wistar大鼠，取心脏放入冷Tris分离液内，匀浆，制备心肌细胞膜。加入100和250 nmol/L DPPH 37 ℃ 10 min, 再加入荧光探针DPH，测定不同方向荧光强度，计算荧光偏振强度P，微粘度η和各向异性r, 以反映膜流动性。

结果：1. 所试浓度DPPH对离体豚鼠心脏有不同程度的损伤作用。表现在：（1）降低LVDP，±dP/dtmax, 升高LVEDP。（2）减少CF，增加LDH的释放和TBARS生成。以上指标与超氧阴离子（·O2）的心脏损伤相似。在20分钟时，DPPH与HX-XO分别使LVDP下降至48%±4%与56%±4%。（3）SOD和DMSO对DPPH自由基的损伤心脏均无保护作用。而左旋半胱氨酸可以完全对抗DPPH自由基对心脏的损伤作用。

2．ESR显示，DPPH损伤的离体豚鼠心脏有明显的自由基波谱峰出现，提示有自由基生成。左旋半胱氨酸预处理后自由基信号明显减弱。

3．DPPH能明显升高心肌细胞膜荧光偏振强度P，微粘度η和各向异性r, 表明膜流动性下降。

结论：DPPH自由基能明显降低离体心脏的收缩和舒张功能，是其自由基特性损伤心脏的结果。可作为一种新的简便的自由基损伤心肌模型。

(该部分全文发表于“Journal of Pharmacological and Toxicological Methods 1998; 39: 63-70）

　

第二部分 缓激肽介导豚鼠心肌缺血预适应抗自由基损伤

目的：研究豚鼠心肌缺血预适应对DPPH自由基损伤的保护作用，探讨缓激肽与这种保护作用的关系。

方法：雄性豚鼠，Langendorff心脏灌流，记录指标同第一部分。实验分组为：（1）正常对照组：Krebs-Henseleit (K-H)液连续灌注40分钟。（2）DPPH组：100nM DPPH 40分钟。（3）IP组：全心缺血5min, 再恢复K-H液灌注5min，用DPPH损伤。（4）缓激肽（BK）组：100nM BK预适应10min，再用不含缓激肽的DPPH液损伤。（5）HOE140+BK组：100nM HOE140预灌注5 min，然后加入100nM缓激肽共同灌注10 min，再用DPPH损伤。

结果：豚鼠心脏也具有明显的缺血预适应保护作用，显著对抗DPPH自由基损伤，

改善心脏的收缩和舒张功能，减少LVDP，±dP/dtmax的下降, 使LVEDP升高程

度减轻；减少LDH的释放和TBARS生成。选择性B2受体阻断剂HOE140可以取

消其保护作用。缓激肽预处理10分钟同样具有明显的缺血预适应样心脏保护作

用。HOE140可以完全阻断缓激肽的保护作用。

结论：豚鼠心肌缺血预适应具有显著对抗DPPH自由基的损伤作用。缓激肽是豚

鼠心肌缺血预适应的重要介质之一。外源性缓激肽有类似心肌缺血预适应的

保护作用。

(该部分全文发表于“Clinical and Experimental Pharmacology and

Physiology 1998; 25: 932-935)

　

第三部分 雷米普利酸预处理对离体豚鼠心脏的急性期（早期）保护

作用及其机理

目的：研究有直接作用的雷米普利衍生物雷米普利酸（Ramiprilat, RAMT）预处理的急性期心脏保护作用对抗超氧阴离子（·O2）自由基和DPPH自由基的心脏损伤及其机制。

方法：雄性豚鼠，Langendorff心脏灌流，记录指标同第一部分。心脏平衡20

分钟，用10或100nM RAMT预处理10min后，再用不含RAMT的100 nM DPPH自

由基或HX-XO混合产生的·O2自由基灌流40分钟。观察心功能变化，并测定LDH

和TBARS含量。为研究其保护机制，在RAMT预处理前分别先加入：HOE140，

Calphostin C (蛋白激酶C抑制剂)，左旋精氨酸甲酯（NO合酶抑制剂）以及

吲哚美辛（环氧化酶抑制剂），观察它们对RAMT预处理心脏保护作用的影响。

结果：RAMT预处理后呈现显著的保护心脏对抗·O2与DPPH自由基的损伤作用，

维持心脏的收缩和舒张功能，LVDP，±dP/dtmax明显高于自由基损伤组，使LVEDP

降低；HR和CF基本维持恒定，减少LDH释放和TBARS生成。HOE140，Calphostin

C以及吲哚美辛能完全阻断RAMT预处理保护心脏作用；左旋精氨酸甲酯对RAMT

预处理急性期保护作用无影响。

结论：RAMT预处理具有明显的急性期（早期）心脏保护作用，对抗·O2及DPPH

自由基损伤。这种作用与激动缓激肽B2受体，激活蛋白激酶C 及前列腺素释

放有关。

(该部分全文已投稿于“Clinical and Experimental Pharmacology and

Physiology，在退修中)

　

第四部分 雷米普利预处理对大鼠心脏的延迟期保护作用及其机理

目的：1. 研究雷米普利(Ramipril, RAM)预处理的延迟期保护作用以及缓激肽（BK），一氧化氮（NO），前列腺素（PGs）和蛋白质合成与这一保护作用的关系。2. 运用分子生物学方法研究RAM预处理对大鼠心室肌热休克蛋白70（Heat shock protein 70, Hsp70）表达的影响及可能的信号传导通路。

方法：1. RAM预处理的延迟期心脏保护作用：成年雄性Wistar大鼠，用RAM（10μg/kg,50μg/kg, i.v.）预处理。在用药后24h,48h或72h，进行Langendorff心脏灌流。左心室水囊导管记录心脏收缩和舒张功能指标（LVDP, dP/dtmax,LVEDP）；量取冠脉流量（CF），并测定流出液的LDH。心脏平衡20分钟后，加入100nM DPPH自由基20分钟，观察不同时间心功能的变化，测定心肌TBARS含量。为探讨其作用机理，另设四组大鼠，分别在RAM前i.v. B2受体阻断剂HOE140，选择性NO合酶抑制剂N-nitro-L-arginine (N-LA), 环氧化酶抑制剂吲哚美辛(IND)以及RNA转录酶抑制剂放线菌素D（ACT），观察它们对RAM预处理保护作用的影响。另设四组，观察上述受体阻断剂或酶抑制剂自身对DPPH自由基损伤的影响。

2．大鼠心肌Hsp70表达的Western blot 分析：提取大鼠心室肌的蛋白质，制备上样样品；进行SDS-PAGE电泳，每孔注入15μL蛋白样品液（75μg）；加压电泳。将电泳后凝胶进行转膜（0.2 A, 6h），1% 脱脂奶粉封闭，用Hsp 70的单克隆抗体杂交，PBS洗膜，辣根过氧化酶标记羊抗小鼠IgG杂交，PBS洗膜，再用SABC杂交，PBS洗膜；最后用DAN显色，照相保存。对NC膜进行光密度扫描相对定量。

结果：RAM预处理后的24h, 48h均有明显的保护心脏作用，LVDP，±dP/dtmax

明显高于自由基损伤组，HR和CF基本维持恒定，减少LDH释放和TBARS生成。

其中，24h保护心脏作用最强。在72h, 部分心功能指标得到改善：LVDP降低

为41%±8%；TBARS为235nmol/g （P<0.05, 与DPPH对照组比较）。HOE140， N-LA

和ACT均能取消RAM预处理的延迟期心脏保护作用；IND对这一作用无影响。

RAM预处理后的24h, 48h，72h均能明显增加有明显增加大鼠心室肌Hsp70

的表达；HOE14，N-LA和ACT均能降低Hsp70的表达。

结论与讨论：ACE抑制剂RAM预处理能够引起延迟期心脏保护作用，对抗自由

基损伤，这一作用可持续到48小时。利用生物检定法发现，i.v.50μg/kg RAM 24

小时后，抑制ACE作用减弱，48小时抑制ACE作用消失，ACE活性恢复。说明

RAM 预处理的延迟期心脏保护作用与ACE的抑制无直接关系。在预处理前，分

别应用B2受体阻断剂HOE140，选择性NO合酶抑制剂N-nitro-L-arginine (N-

LA), RNA转录酶抑制剂放线菌素D（ACT），这种保护作用完全消失。说明RAM

延迟期心脏保护作用是通过缓激肽B2受体介导，有NO及内源性蛋白的合成参

与其作用。RAM预处理后的大鼠心室肌Hsp70的表达明显增加；表明Hsp70可

能与RAM延迟期心脏保护作用有关。

(该部分全文发表于“British Journal of Pharmacology 1998; 125: 556-562)

　

第五部分 雷米普利酸预处理对大鼠乳鼠原代心肌细胞的保护作用及

其机理

目的：研究雷米普利酸（RAMT）预处理对体外培养的心肌细胞自由基损伤的保

护作用及其机理。

方法：1. 急性期保护作用：取出生1-2天的Wistar大鼠心脏，常规方法培养大鼠原代心肌细胞。在培养第四天后，进行心肌细胞实验。分组为：（1）溶剂对照组：培养液中，加入0.025%丙二醇（DPPH溶剂）3小时。（2）DPPH自由基损伤组：培养液中，加入0.1μM DPPH 3小时。(3) RAMT预处理组：培养液中，先加入100nM RAMT 10 min, 再换用不含RAMT的0.1μM DPPH 培养3小时。为研究RAMT的急性保护作用机理，另设四组，分别在给RAMT前加入HOE140，Calphostin C, N-LA 或IND 10分钟。然后加入RAMT共孵化10分钟，再换用不含RAMT的0.1μM DPPH 培养3小时。分别测定1h，2h，3h上清液和细胞的乳酸脱氢酶（LDH）活性和脂质过氧化物（TBARS）含量。采用0.1%胰蛋白酶消化结合吹打法使心肌细胞脱壁，台酚蓝染色，检查心肌细胞死亡率。同时，用放射免疫法测定心肌细胞液6-酮-PGF1α含量。

2．延迟期保护作用：心肌细胞培养和观测指标同前。实验分组为：（1）正常对照组。（2）HX-XO损伤组：加入HX (0.06 mM)和XO (2U/L)于心肌细胞培养液中，观察细胞形态学和生化指标的变化。（3）RAMT预处理组：在培养液中加入100 nM RAMT 10 min, 再换用不含RAMT的DMEM培养液培养，24h后，加入HX-XO于心肌细胞培养液中，观察细胞形态学和生化指标的变化。为研究RAMT的这一保护作用机理，另设二组，分别在给RAMT前加入Calphostin C或N-LA 10分钟。观察其对RAMT的影响。用Western blot方法检测给药后心肌细胞Hsp70的表达。

结果：1. DPPH自由基对心肌细胞有明显的损伤作用，表现为：心肌细胞多退缩成圆性，其死亡率明显增加，为68.3%±3.0%; LDH和TBARS含量也明显高于溶剂对照组。RAMT预处理能明显减轻DPPH对心肌细胞的损伤。细胞死亡率为35.8%±5.6% (P<0.01, 与DPPH组比较)；LDH，TBARS含量也明显降低；心肌细胞仍大多为不规则多角形。HOE140，Calphostin C或IND均能够阻断RAMT的保护作用。DPPH能明显增加心肌细胞PGI2的释放，为71±21 pg/ml; RAMT预处理后，PGI2释放减少为5.8±4.4 pg/ml (P<0.01, 与DPPH组比较)。

2．RAMT预处理后24h有明显的延迟期心肌细胞保护作用，对抗超氧阴离子自由基的损伤。使LDH释放量和细胞死亡率明显减少。N-LA和Calphostin C均能够阻断RAMT的保护作用。Western Blot显示，RAMT可促进Hsp70的表达。

结论与讨论：RAMT预处理对大鼠乳鼠原代心肌细胞有急性期和延迟期的保护作

用，对抗自由基损伤。其急性期的保护作用与激动B2受体，激活PKC，促进前

列环素生成有关。RAMT预处理的延迟期保护作用是通过PKC和NO介导的，与

Hsp70的表达有关。

　

小结

1．1，1-二苯基-2-三硝基苯肼（1,1-Diphenyl-2-Picryl-Hydrazyl, DPPH）自由基能损伤离体豚鼠心脏，该损伤作用与其自由基特性有关，是一种新的，简易的自由基损伤离体豚鼠心脏模型。

2．豚鼠心肌缺血预适应具有显著对抗DPPH自由基的损伤作用。缓激肽是豚鼠

心肌缺血预适应的重要介质之一。外源性缓激肽有类似心肌缺血预适应的保护

作用。

3．ACE抑制剂雷米普利酸预处理具有明显的急性期（早期）心脏保护作用，对

抗自由基损伤。这种作用与激动缓激肽B2受体，激活蛋白激酶C 及前列腺素

释放有关。

4．ACE抑制剂雷米普利预处理能够引起延迟期心脏保护作用，对抗自由基损伤，

这一作用可持续到48小时。这种保护作用是通过缓激肽B2受体介导，有NO及

内源性蛋白的合成参与其作用。Hsp70的表达可能与RAM延迟期心脏保护作用

有关。

5．雷米普利酸预处理对大鼠乳鼠原代心肌细胞也有急性期和延迟期的保护作

用，对抗自由基损伤。其急性期的保护作用与激动B2受体，激活PKC，促进前

列环素生成有关。RAMT预处理的延迟期保护作用是通过PKC和NO介导的。

综上所述，ACE 抑制剂雷米普利酸预处理有急性期和延迟期保护心脏和心肌细胞的作用，其机理存在着相同和不同之处，它们有一个共同的触发机制：即激动缓激肽B2受体，激活蛋白激酶C。但是，急性期保护有PGI2的参与，与NO无关；而延迟期需要NO介导和蛋白质合成的参与。前列腺素合成是RAMT急性期心脏保护所必须的；然而，放免法直接测定表明，DPPH自由基能明显刺激心肌细胞PGI2的合成与释放，RAMT预处理后，PGI2的合成与释放明显减少，这一矛盾现象及其意义有待进一步研究。

　

Ph.D. DISSERTATION

Pharmacological Preconditioning Induces Cardioprotection against Free Radical Injury and Its Cellular and Molecular Mechanisms

　

Abstract

Myocardial Ischemic preconditioning (IP) induced by single or repetitive short periods of ischemia followed by intermittent reperfusion, renders the heart more resistant to a subsequent longer ischemic period. This cardioprotection limits infarct size, reduces the risk of ischemia-reperfusion arrhythmias and improves recovery of ventricular function. This cardioprotective effect has been observed in different species, including rats, rabbits, dogs, pigs and human beings. Several endogenous substances, such as adenosine, noradrenalinem bradykinin (BK) and calcitonin gene-related peptide (CGRP), have been reported to be involved in IP. These substances activate G-protein coupled receptors and protect the heart. IP can be classified into two phases: early protection and delayed protection. The early cardioprotection appears soon and lasts shortly. The maximum effect of the delayed preconditioning appears 24 hours later and sustains for 2-3 days. Protein synthesis (heat shock protein, SOD or CAT,etc) is involved in the delayed preconditioning.

Pharmacological preconditioning (PP) is a cardioprotection induced by drugs or chemicals which trigger endogenous protective substances release. The prerequisite for PP is that the protection should depend on endogenous mediators triggered by the drug and exist after elimination of the drug. Angiotension-converting enzyme (ACE) inhibitors are used extensively for treatment of hypertension and chronic heart failure, and its cardioprotection against ischemia-reperfusion injury has been shown in various animal models. The mechanism of its cardioprotective effect has been attributes to the blockade of degradation of BK and the subsequent activation of BK receptor. Previous studies have shown that both endogenous and exogenous BK exerts protection against myocardial ischemia-reperfusion injury. Recently, it was reported that enalaprilat pretreatment enhanced functional recovery after long term cardiac preservation possibly via activations of BK receptor and protein kinase C (PKC). Captopril can potentiate cardioprotective effect of myocardial IP against ischemia-reperfusion injury.

However, litter is known about the cardioprtoection induced by pretreatment with ACE inhibitors against free radical injury and particularly, the delayed cardioprotection. Furthmore, protection of cardiac myocytes with ACE inhibitors has not been fully investigated. Therefore, the primary aim of the present study was to evaluate whether ACE inhibitor (Ramipril) pretreatment affords early and especially delayed protection against free radical insult to heart and cardiac myocytes. The possible mechanisms of action were furtherly studied.

Part One: A simple reproducible model of free radical-injured isolated heart induced by 1,1-diphenyl-2-picryl-hydrazyl (DPPH)

AIM: 1,1-diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical, has been used for detecting free radical scavenging effect and antioxidant activity in chemical analysis. However, it is still unknown if DPPH triggers free radical injury in cardiac tissue. In order to establish a simple free radical-injured isolated heart model, we investigated the injury induced by DPPH on isolated guinea pig heart to ascertain that this injury is due to its free radical character.

METHOD: Langendorff guinea pig heart was prepared. A fluid-filled latex balloon connected to a pressure transducer by a polyethylene tubing was inserted into the left ventricule to record the left ventricular developed pressure (LVDP), ±dP/dtmax, left ventricular end-diastolic pressure (LVEDP) and heart rate (HR). Lactate dehydrogenase (LDH) in coronary effluent and thiobarbituric acid reactive substances (TBARS) formation in cardiac tissue were detected. Effect of DPPH on cardiac performance was compared with superoxide anion (·O2), generated by hypoxnthine (HX)-xanthine oxidase (XO) system. Free radical scavengers, dimethyl sulphoxide (DMSO), superoxide dismutase (SOD) and L-cysteine, were also used to analyze the characteristic of the DPPH free radical-induced cardiac dysfunction. The free radical signals of DPPH-injured heart were detected by electron spin resonance (ESR). Effect of DPPH on cardiac membrane fluidity was also studied.

RESULTS: 100 nM and 250 nM DPPH in Krebs-Henseleit solution significantly decreased the LVDP and ±dP/dtmax, elevated LVEDP, increased LDH release and TBARS formation. L-cysteine improved the DPPH-impared cardiac function, while DMSO and SOD had no beneficial effect in this injury. The cardiac membrane fluidity was decreased by DPPH. Free radical signals, detected by ESR in the DPPH-injured heart, were reduced by L-cysteine treatment.

CONCLUSION: DPPH-induced isolated heart dysfunction is attributed to its free radical damage and can serve as a simple and reproducible heart model of free radical injury.

　

(The original paper was published in Journal of Pharmacological and Toxicological Methods 1998; 39: 63-70)

Part two: Bradykinin Mediates Myocardial Ischemic Preconditioning aginst Free Radical Injury in Isolated Guinea Pig Heart

AIM: To determine whether ischemic preconditioning (IP) can protect the heart against free radical injury in isolated guinea pig heart and the role of bradykinin (BK) in the cardiac protection; to examine whether pretreatment with BK can mimic the cardiac protectionm of IP.

METHODS: DPPH was used for triggering free radical injury in Langendorff perfusion guinea pig heart with the same method used in part one. Effect of IP (5 min global ischemia and 5 min perfusion) against DPPH free radical-induced cardiac dysfunction in isolated guinea pig heart was investigated. To examine the role of BK in IP, the isolated heart was pretreated with HOE140, a selective B2receptor antagonist, prior to IP. In other two group, 100 nM BK or HOE140+BK was perfused to isolated guinea pig heart and subjected to DPPH insult.

RESULTS: Myocardial IP exerted cardioprotection in isolated guinea pig heart. LVDP, ±dP/dtmax, HR and CF were significantly improved. TBARS formation in cardiac tissue was reduced. Pretreatment with HOE140 abolished the IP-induced cardioprotection. 100 nM BK perfusion for 10 min protected the heart against free radical injury. HOE140 completed reversed this protection.

CONCLUSION: Myocardial ischemic preconditioning exerted cardiac protection against free radical injury. Endogenously generated BK may mediate IP in the isolated guinea pig heart through activation of BK B2receptor.

　

(The original paper was published in Clin Exp Pharmacol Physiol 1998; 25: 932-935)

Part Three: Ramiprilat Induces Early Cardioprotection against Free Radical Injury in Isolated Guinea Pig Heart: Involvement of Bradykinin, PKC and Prostaglandins

AIM: To investigate the early cardioprotection induced by ramiprilat (RAMT) pretreatment and its possible mechanism of action.

METHOD: Langendorff guinea pig heart was prepared and perfused with Krebs-Henseleit solution. The monitoring procedure and parameters of function were the same as those in part one. After pretreatment with 10 or 100 nM RAMT for 10 min, the heart was injured with 100 nM DPPH or superoxide anion (·O2), produced by HX-XO, for 40 min. To study th eprotective mechanism of RAMT pretreatment, HOE 140, Calphostin C (a PKC inhibitor), L-NAME (a NO synthase inhibitor) or indomethacin (a cycloxygenase inhibitor) were added individually before RAMT pretreatment.

RESULTS: RAMT pretreatment exerts significant cardioprotection against free radical injury immediately. Cardiac function (LVDP, ±dP/dtmax, LVEDP) was improved. LDH release and TBARS formation were reduced. HOE140 or calphostin C or indomethacin can abolish the protective effect of RAMT. Whereas, L-NAME has no effect on the cardioprotection induced by RAMT.

CONCLUSION: RAMT pretreatment can induce early cardioprotection against either ·O2 or DPPH free radical injury. The protective effect depends on activation of B2 receptor and PKC. Prostaglandins synthesis is also involved.

　

(The original paper was submitted to Clin Exp Pharmacol Physiol, in revision)

Part four: Ramipril Induced Delayed Myocardial Preconditioning against Free Radical Injury Involves Bradykinin B2 Receptor-NO pathway and Protein Synthesis

AIM: 1. To examine whether Ramipril (RAM) can induce delayed myocardial protection against free radical injury ex vivo and to determine the possible role of bradykinin B2 receptor-NO pathway, PGs and protein synthesis in this protection.

2. To investigate whether RAM pretreatment increase the expression of heart shock protein (HSP)70 in rat heart and its possible signal transducing pathway.

METHODS: 1. Rats were pretreaed with RAM (10 or 50 μg/kg, i.v.) and hearts were isolated after 24, 48, 72 h. Langendorff hearts were perfused and subjected to DPPH free radical-induced injury. Cardiac function (LVDP, +dp/dtmax, CF and HR), LDH in coronary effluent and TBARS formation in myocardium were measured. To elucidate the mechanism of action, HOE140, N-LA, Indomethacin and actinomycin D (a RNA transcription inhibitor), were used prior to RAM pretreatment.

2. Wetern blot analysis: the protein sample of rat ventricule myocardium was prepared and submitted to SDS-PAGE. Proteins in gel were transferred to nitrocellulose membrane, which was blocked with 1% defatted milk powder for 1 h. The blots were incubated with monoclonal mouse anti-human Hsp 70 antibody and horseradish peroxidase-conjugated sheep anti-mouse IgG individualy. The blots were visualized with DAB.

RESULTS: In DPPH control group, 20 min after DPPH perfusion, LVDP, +dP/dtmax, CF and HR were declined, whereas TBARS and LDH were increased significantly. The above cardiac function parameters were improved in RAM-pretreated rats after 24h and 48 h. Treatment with HOE140, N-LA or actinomycin D respectively, prior to RAM pretreatment, abolished the beneficial effects of RAM at 24h, while indomethacin had no effect on RAM-induced delayed protection. The Hsp70 expression in cardiac tissue was enhanced after RAM pretreatment. HOE140, N-LA and actinomycin D diminished the Hsp70 expression.

CONCLUSION: RAM pretreatment induces delayed cardioprotection against DPPH free radical injury. This protection sustains at least 48 h. In our bioassay experiment (ACE converts Ang I to Ang II, thus the blood pressure is raised), ACE activity is partially recovered 24h after RAM 50 μg/kg, i.v. and completely recovered at 48 h. This result implicates that RAM-induced delayed cardioprotection is a consequence of its ACE inhibition, which triggers endogenous protective mechanism. BK B2 receptor, NO and protein synthesis are involved in RAM-induced delayed preconditioning. Hsp70 was increased 24 h, 48 h and 72 h after RAM pretreatment.

(The original paper was published in British Journal of Pharmacology 1998; 125: 556-562)

Part Five: Protection Induced by Ramiprilat Pretreatment in Cultured Cardiac Myocytes of Neonatal Rat and Its Mechanism of Action

　

AIM: To examine whether Ramiprilat (RAMT) can induce early or/and delayed protection in cultured cardiac myocytes of neonatal rats and its possible signal transduction.

METHODS: 1. RAMT-induced early protection: In DPPH free radical group, 100 nM DPPH was used for triggering free radical injury in the primary culture neonatal rat cardiac myocytes. In RAMT pretreatment group, the cardiac myocyte was pretreated with 100 nM RAMT for 10 min and Followed by 100 nM DPPH insult for 3 h. To ascertain the signal transduction pathway, HOE140, calphostin C, N-LA or IND were used respectively prior to RAMT. PGI2 formation in cardiac myocyte was detected by using radioimmunoassay.

2. RAMT-induced delayed protection: The neonatal rat cardiac myocyte was pretreated with 100 nM RAMT for 10 min and exposed to superoxide anion free radical (·O2) 24 h later. To clarify the signal transduction, calphostin C or N-LA were used respectively prior to RAMT. Heat shock protein 70 expression was detected by using Western blot method.

RESULTS: 1. RAMT-induced early protection: RAMT pretreatment attenuated the damage of cardiac myocyte. The percentage of dead cells was decreased to 35.8±8.6% (P<0.05, compared with 68.3±3.0% in DPPH group. LDH and TBARS formation were also reduced. DPPH free radical significantly enhanced PGI2 release to 71±21 pg/ml (P<0.01 VS Control). After RAMT pretreatment, the PGI2 release was decreased to 5.8±4.4 pg/ml (P<0.05, vs DPPH group). HOE140,IND and calphostin C can abolished the beneficial effects of RAMT.

2. RAMT-induced delayed protection: RAMT pretreatment significantly protected the cardiac myocyte against free radical injury 24 later. The percentage of dead cells was decreased to 35.4±8.2% (P<0.05, compared with 72.2±3.5% in HX-XO group). LDH leakage was also attenuated to 28.0±3.5 IU/L. Hsp70 expression was increased in RAMT group.

CONCLUSION: RAMT exerts either early or delayed protection of primary cardiac myocytes. The early protection depends on activation of B2 receptor and PKC. PGI2 is involved in this protection. Whereas, NO and PKC mediate the delayed protection of cardiac myocytes.

　

Summary

In the present study, our results show that: (1) DPPH-injured isolated heart can serve as a simple and reproducible heart model of free radical injury. (2) myocardial ischemic preconditioning is observed in isolated guinea pig heart and bradykinin mediates the cardioprotection. (3) RAMT pretreatment induces early cardioprotection against free radical injury in isolated heart. This protection involves activations of B2 receptor, PKC and prostaglandins synthesis. (4) RAM pretreatment induces delayed cardioprotection against free radical damage via B2 receptor-NO- protein synthesis. Hsp70 is increaed after RAM pretreatment. (5) RAMT induces early and delayed protection of cardiac myocytes against free radical injury. The early protection depends on activation of B2 receptor and PKC. PGI2 is involved in this protection. Whereas, NO and PKC mediate the delayed protection of cardiac myocytes.

In conclusion, pretreatment with RAM, an ACE inhibitor, exerts early and delayed cardioprotection against free radical injury. Activations of BK B2 receptor and PKC are the common triggering mechanism of early and delayed protection. However, PGs mediates the early cardioprotection. NO and protein synthesis mediate the delayed cardioprotection. Hsp 70 expression is increased in rat heart and neonatal rat cardiac myocytes after RAM or RAMT pretreatment 24 h later. A provocative finding in present study is that DPPH free radical apparently increased PGI2 release in cardiac myocytes. RAMT pretreatment attenuated the release of PGI2. The apparent discrepant effects of DPPH and RAMT on PGI2, a well-known cytoprotective factor, and the possible interaction between PGI2 and free radicals is a problem to be investigated.

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

　

本文常用略缩语 (Abbreviation)

ACE: Angiotensin Converting Enzyme ACT: Actinomycin D,

BK: BradykininCAL CAL: Calphostin C,

Cys: L-Cysteine DMSO: Dimethyl sulphoxide

DPPH: 1,1-Diphenyl-2-Picryl-Hydrazyl ESR: Electron spin resonance

Hsp70: Heat shock protein 70 HX: Hypoxanthine

IND: Indomethacin IP: Ischemic preconditioning

K-H: Krebs-Henseleit LDH: Lactate dehydrogenase

LVDP: Left ventricular developed pressure

LVEDP: Left ventricular end-diastolic pressure

N-LA: Nω-nitro-L-arginine NO: Nitric oxide

PGs: Prostaglandins PKC: Protein kinase C

RAM: Ramipril RAMT: Ramiprilat

SOD: Superoxide dismutase

TBARS: Thiobarbituric acid reactive substances

XO: Xanthine oxidase

　回主页home.gif (1910