丹酚酸B对兔缺血再灌注心脏内皮细胞功能和血小板活化的影响
作者：杨富国, 张安勇, 陈作元, 廉哲勋, 刘革新, 董果雄
【摘要】 目的：观察丹酚酸B（salvianolic acid B，SA?B）对兔缺血再灌注心脏内皮细胞功能和血小板活化的影响。
方法：将24只新西兰大耳白兔随机分为假手术组、缺血再灌注损伤组（模型组）和SA?B组。结扎兔心脏左前降支0.5 h造成心肌缺血后再灌注4 h制成心肌缺血再灌注模型。SA?B治疗组于左前降支结扎后静滴SA?B溶液，其余各组静滴等量生理盐水。分别于结扎前、缺血0.5 h、再灌注1 h及4 h静脉采血，用硝酸还原酶法测定血浆一氧化氮（nitric oxide, NO）浓度，放射免疫法测定血浆内皮素（endothelin, ET）含量和血小板表面α颗粒膜蛋白140（alpha?granule membrane protein?140, GMP?140）的数目。
结果：假手术组手术前后血浆NO浓度、ET含量和血小板表面GMP?140差别无统计学意义（P＞0.05）；模型组心肌缺血0.5 h后，血浆NO浓度较术前及假手术组相应时间显著降低（P＜0.05﹚，并随再灌注时间延长呈进行性下降，而血浆ET含量和血小板表面GMP?140数目较术前及假手术组相应时间显著升高（P＜0.05﹚，并随再灌注时间延长进一步上升；治疗组应用SA?B后，再灌注1 h及4 h时血浆NO浓度较模型组相应时间明显升高(P＜0.01)，而血浆ET含量和血小板表面GMP?140数目较模型组相应时间明显降低（P＜0.01）。
结论：心脏缺血再灌注过程中存在血管内皮细胞功能损伤及血小板活化，SA?B具有保护内皮细胞和抑制血小板活化的作用。
【关键词】 丹酚酸B; 心肌缺血再灌注损伤; 一氧化氮; 内皮素; 血小板α颗粒膜蛋白140; 兔
Methods: A total of 24 New Zealand white rabbits were randomly divided into sham?operated group, ischemia?reperfusion group (untreated group) and SA?B group. The hearts of rabbits in untreated group and SA?B group underwent half an hour of left anterior descending coronary artery (LADCA) occlusion via ligation technology, which was followed by 4 hours of reperfusion to prepared ischemia?reperfusion injury model in vivo. For sham?operated group, the animals were not subjected to occlusion of LADCA. In SA?B treatment group the rabbits were intravenously administered SA?B immediately after LADCA occlusion, and the other two groups were given normal saline in the same way instead of SA?B. The jugular vein bloods of animals were collected before LADCA ligation, half an hour after ligation and after 1?, 4?hour reperfusion, respectively. The content of plasma nitric oxide (NO) was determined by nitrate reductase process. Radioimmunoassay was applied to detect the endothelin (ET) content in plasma and the count of alpha?granule membrane protein?140 (GMP?140) on platelet surface to identify the activation of the platelet.
Results: No significant difference was observed before and after sham LADCA occlusion in sham?operated group in the contents of NO and ET in plasma (P＞0.05), neither was the count of GMP?140 on platelet surface (P＞0.05). The content of NO in plasma detected 0.5 h after LADCA occlusion was significantly decreased in untreated group compared with the sham?operated group at the corresponding time, and they were also much lower than that before LADCA occlusion in the sham?operated group (P＜0.05). The plasma content of NO in untreated group showed a progressive decrease in response to the myocardial reperfusion. However, the content of ET in plasma and the count of GMP?140 on platelet surface were remarkably increased after myocardial ischemia as compared with those before LADCA ligation and those detected in sham?operated group (P＜0.05). The content of ET and the count of GMP?140 in the untreated group were further increased corresponding to the aggressive reperfusion. The content of NO was significantly increased while the content of ET and the count of GMP?140 were both significantly decreased in SA?B group as compared with untreated group after 1? and 4?hour myocardial reperfusion, respectively (P＜0.01).
Conclusion: The results show that endothelial dysfunction and platelet activation occur during ischemia?reperfusion in rabbit hearts in vivo and SA?B protects cardiovascular endothelium cells against ischemia?reperfusion injury and inhibits the activation of platelet during myocardial ischemia and reperfusion.
Keywords: salvianolic acid B; myocardical reperfusion injury; nitric oxide; endothelin; platelet alpha?granule membrane protein?140; rabbits
及时恢复血流再灌注是治疗缺血性心脏病普遍采用的措施，可以挽救严重缺血或濒临坏死的心肌，并限制或缩小梗死面积，有效地保护心脏功能，降低心脏事件的发生率，改善临床预后。然而，再灌注早期冠状动脉血流的快速恢复可引起心肌进一步损伤即心肌缺血再灌注损伤，血小板聚集、白细胞浸润、细胞内钙离子超负荷和氧自由基爆发等在其中发挥重要作用［1］。中药减轻心肌缺血再灌注损伤的研究对于急性心肌梗死的治疗具有重要意义。中药丹参为唇形科植物丹参Salvia miltiorrhiza Bge.的干燥根及根茎，具有活血祛瘀的功效。丹酚酸B（salvianolic acid B, SA?B）作为丹参中的主要水溶性成分，具有清除氧自由基、抗氧化、保护心肌的作用［2?5］。本实验通过观察内皮细胞和血小板在心脏缺血再灌注时的变化，观察SA?B对该病理过程的影响，以期为临床用药提供依据。
1 材料与方法
1.1 药物及试剂 SA?B，淡黄色粉针剂，纯度大于98%，批号为111562?200403（药品生物制品检定所）。LKB?1275型γ免疫计数仪（芬兰WALLAC公司）；Miniplus 2型蠕动泵（法国CSA公司）。一氧化氮（nitric oxide, NO）检测试剂盒（南京建成生物工程研究所提供）、内皮素（endothelin, ET）放射免疫检测试剂盒（解放军总科技开发中心放免研究所提供）和血小板表面α颗粒膜蛋白140（alpha?granule membrane protein?140, GMP?140）检测试剂盒（苏州医学院止血与血栓研究室提供）。
1.2 心肌缺血再灌注损伤模型制备 健康雄性新西兰大耳白兔24只，体质量（3.25±0.56）kg，由青岛市药品检验所提供，实验动物合格证号为SCXK（鲁）20030010。
方法［6］建立心肌缺血再灌注损伤模型。20%乌拉坦5 ml/kg耳缘静脉麻醉，气管插管，开胸，暴露心脏，在距冠状动脉左前降支（left anterior descending coronary artery, LADCA）根部0.5 cm处穿线，结扎LADCA，制备急性心肌缺血模型。以心电图Ⅱ导联ST段出现弓背向上抬高、T波高耸，心肌组织颜色由正常变苍白再变暗为心肌缺血模型成功标志。LADCA结扎0.5 h后剪断穿线使其再通形成再灌注，局部反应性充血表示心肌再灌注开始，再灌注持续4 h。假手术组大鼠于LADCA根部0.5 cm处穿一胶管，内引丝线，但不予结扎。
1.3 动物分组及给药 24只新西兰兔按随机数字表法随机分为假手术组、模型组和SA?B组，每组8只。SA?B组动物模型制作方法同模型组，并于结扎后即刻由电子蠕动泵经耳缘静脉1 h内匀速滴入20 ml SA?B溶液（按3 mg/kg精确称取SA?B，溶于20 ml生理盐水）。其余各组于相应时间内滴入20 ml生理盐水。
1.4 指标测定 于结扎前、结扎0.5 h、再灌注1 h及4 h由颈静脉取血3.8 ml。其中1 ml血肝素抗凝后，以3 000 r/min 离心15 min，取上清液置于－20 ℃冰箱保存，硝酸还原酶法测定血浆NO浓度；另外1 ml血注入含10%依地酸二钠30 μl和抑肽酶40 μl的试管中混匀，4 ℃ 3 000 r/min离心10 min，取上清液置－20 ℃冰箱保存，放射免疫法测定血浆ET含量；余1.8 ml血以依地酸二钠抗凝，用放射免疫法测定血小板表面GMP?140的数目，操作严格按照试剂盒的要求进行，结果以分子数/血小板表示［7］。
1.5 统计学方法 应用SPSS 11.0统计学软件进行统计分析，数据采用x±s表示，采用单因素方差分析进行组间比较，采用q检验进行两两比较。检验水准为α=0.05。
2 结果
2.1 各时间点血浆NO浓度及ET含量的变化 假手术组各时间点间比较，血浆NO浓度、ET含量差异无统计学意义（P＞0.05）。模型组LADCA结扎0.5 h后，兔血浆NO浓度较术前及假手术组显著降低（P＜0.05），再灌注后仍进行性下降（P＜0.01），而兔血浆ET含量较术前及假手术组显著升高（P＜0.05），并随再灌注时间延长进一步上升（P＜0.01）。SA?B组LADCA结扎0.5 h后与结扎前比较，兔血浆NO浓度降低（P＜0.05），ET含量升高（P＜0.05）；再灌注1 h和4 h后与缺血0.5 h比较，兔血浆NO浓度、ET含量差异无统计学意义，但和模型组比较，NO浓度显著提高（P＜0.01），ET含量显著降低（P＜0.01）。见表1和表2。
2.2 各时间点血小板表面GMP?140数目的变化 假手术组各时间点血小板表面GMP?140数目差异无统计学意义（P＞0.05）。模型组LADCA结扎0.5 h后，血小板表面GMP?140数目上升，并随时间延长而进一步增加，与结扎前及假手术组相比，差异有统计学意义（P＜0.05, P＜0.01）。SA?B组LADCA结扎0.5 h后，血小板表面GMP?140数目较结扎前显著升高（P＜0.05），再灌注1 h和4 h后维持在结扎0.5 h水平，但较模型组显著下降（P＜0.01）。见表3。
表1 各组血浆NO浓度变化（略）
Table 1 Change of NO content in plasma in three groups
*P＜0.05, **P＜0.01, vs before LADCA ligation; △P＜0.05, △△P＜0.01, vs sham?operated group; ▲▲P＜0.01, vs untreated group.
表2 各组血浆ET含量变化（略）
Table 2 Change of ET content in plasma in three groups
*P＜0.05, **P＜0.01, vs before LADCA ligation; △P＜0.05, △△P＜0.01, vs sham?operated group; ▲▲P＜0.01, vs untreated group.
表3 各组血小板表面GMP?140数目变化（略）
Table 3 Count of GMP?140 on platelet surface in three groups
*P＜0.05, **P＜0.01, vs before LADCA ligation; △P＜0.05, △△P＜0.01, vs sham?operated group; ▲▲P＜0.01, vs untreated group.
3 讨论
心肌缺血再灌注损伤是多细胞参与和多种递质共同发挥作用的复杂病理生理过程，常发生于冠心病、心肌梗死及其后的溶栓、介入、冠状动脉旁路移植手术等过程中［8］。
NO和ET为血管内皮细胞分泌的两种效应相反的血管活性物质。ET是一种来源于内皮细胞具有广泛生物学活性的多肽类物质，是目前已知的作用最强和作用时间最持久的缩血管物质，也是一种内源性损伤因子［9, 10］。NO作为细胞间信息交换载体，是一种最有效的内皮源性血管扩张剂，其生物学效应与ET相反，对ET具有负反馈调节。生理情况下，二者呈动态平衡，对维持心血管系统正常的舒缩状态、调节冠状动脉基础张力有重要作用［11］。本实验表明，缺血再灌注时血浆NO含量降低、ET水平升高，可能原因为缺血再灌注过程中，缺氧后恢复血氧供应使细胞内钙超载，NO合成减少，而ET生成增多；缺血再灌注时产生的大量氧自由基使NO灭活加速，间接反馈性促进ET的产生。
GMP?140是血小板活化依赖性颗粒表面膜蛋白，主要存在于血小板α颗粒膜上，部分存在于内皮细胞中，正常时不在血小板表面显露，而只表达在活化的血小板表面，是具有特异性的血小板活化分子标记物。活化血小板的黏附、聚集、填塞和血小板性微小血栓可引起心肌缺血再灌注后无复流现象［12?14］。本实验结果表明，缺血使血小板表面GMP?140增加，再灌注恢复血液供应并不能使缺血期增高的血小板表面GMP?140立即恢复正常，提示血小板活化可能参与缺血再灌注损伤。GMP?140在缺血再灌注过程中增高的原因可能为：恢复血氧供应时产生的大量氧自由基破坏了血小板膜及颗粒膜的稳定性；内皮细胞的功能紊乱，白细胞的趋化、聚集以及炎症反应释放出的一些炎症介质均对血小板的活化起到了一定的促进作用。
SA?B是丹参的主要水溶性成分之一，具有很强的清除自由基的作用［15, 16］。本实验显示，应用SA?B后，血浆NO浓度增加、ET含量降低，血小板表面GMP?140下降，提示SA?B对缺血再灌注损伤有保护作用，部分是通过保护内皮细胞和抑制血小板活化实现的。可能的作用机制有：SA?B能对抗自由基生成，直接促进自由基清除，或通过增强超氧化物歧化酶及过氧化物酶活性间接加强自由基清除；还能拮抗钙离子，防止内皮细胞及血小板内钙超载，稳定内皮细胞及血小板膜，从而保护内皮细胞及抑制血小板活化。
【】
1 Hoffman JW Jr, Gilbert TB, Poston RS, et al. Myocardial reperfusion injury: etiology, mechanisms, and therapies. J Extra Corpor Technol. 2004; 36(4): 391?411.
2 Xu J, Fan WH. Effect of salvianolate on migration of human vascular endothelial cells. Zhong Xi Yi Jie He Xue Bao. 2003; 1(3): 211?214. Chinese with abstract in English.
徐杰, 范维琥. 丹参多酚酸盐对人血管内皮细胞迁移的影响. 中西医结合学报. 2003; 1(3): 211?214.
3 Liu W, Zhang Y, Zuo J. Protective effects and mechanism of salvianolic acid B (SAB) on PC12 glucose deprivation injury. Shanghai Zhong Yi Yao Za Zhi. 2006; 40(10): 66?68. Chinese with abstract in English.
刘雯, 张艳, 左伋. 丹酚酸B对PC12细胞缺糖损伤的保护作用及机制. 上海中医药杂志. 2006; 40(10): 66?68.
4 Wu J, Xu L, Xu L. Protective effect of salvianolic acid B against anoxia?reoxygenation injury in isolated rabbit hearts. Zhongguo Yao Li Xue Yu Du Li Xue Za Zhi. 2007; 21(3): 212?216. Chinese with abstract in English.
武骏, 徐立, 许立. 丹酚酸B对兔离体心脏缺氧再复氧损伤的保护作用. 药与毒理学杂志. 2007; 21(3): 212?216.
5 Wang XN, Hu YY, Liu CH, et al. Effects of salvianolic acid B on expressions of TGF?β1 and its receptors in liver of rats with dimethylnitrosamine?induced hepatic fibrosis. Zhong Xi Yi Jie He Xue Bao. 2005; 3(4): 286?289. Chinese with abstract in English.
王晓柠, 胡义扬, 刘成海, 等. 丹酚酸B盐对二甲基亚硝胺诱导的肝纤维化大鼠肝脏转化生长因子β1及其受体蛋白表达的影响. 中西医结合学报. 2005; 3(4): 286?289.
6 Yao Z, Chen YF, Feng JZ, et al. Effect of experimental ischemic preconditioning on reducing myocyte apoptosis in rabbits. Zhongguo Dong Mai Ying Hua Za Zhi. 1999; 7(1): 20?23. Chinese with abstract in English.
姚震, 陈颜芳, 冯建章, 等. 实验性家兔缺血预适应减轻心肌细胞凋亡. 中国动脉硬化杂志. 1999; 7(1): 20?23.
7 Wu GX, Ruan CG. Comparison of four different activation?dependent proteins on the platelet surface in detecting platelet activation. Suzhou Yi Xue Yuan Xue Bao. 1993; 13(4): 263?265. Chinese with abstract in English.
吴国新, 阮长耿. 检测体外血小板活化程度时4种血小板表面活化标志蛋白的比较. 苏州医学院学报. 1993; 13(4): 263?265.
8 Moens AL, Claeys MJ, Timmermans JP, et al. Myocardial ischemia/reperfusion?injury, a clinical view on a complex pathophysiological process. Int J Cardiol. 2005; 100(2): 179?190.
9 Doggrell SA. The endothelin system and its role in acute myocardial infarction. Expert Opin Ther Targets. 2004; 8(3): 191?201.
10 Hou XL, Li BL, Zhao L, et al. Effects of Xuefu Zhuyu Capsule on endothelin?1 release in myocardium vascular endothelium and nitric oxide/nitric oxide synthase system of swines after acute myocardial infarction and reperfusion. Zhong Xi Yi Jie He Xue Bao. 2008; 6(4): 381?386. Chinese with abstract in English.
侯宵雷, 李白翎, 赵雷, 等. 血府逐瘀胶囊对急性心肌梗死再灌注后心肌及内皮素?1、一氧化氮/一氧化氮合酶体系影响的实验研究. 中西医结合学报. 2008; 6(4): 381?386.
11 Kaneda H, Taguchi J, Kuwada Y, et al. Coronary artery spasm and the polymorphisms of the endothelial nitric oxide synthase gene. Circ J. 2006; 70(4): 409?413.
12 Elesber AA, Solomon H, Lennon RJ, et al. Coronary endothelial dysfunction is associated with erectile dysfunction and elevated asymmetric dimethylarginine in patients with early atherosclerosis. Eur Heart J. 2006; 27(7): 824?831.
13 Yang YJ, Zhao JL, You SJ, et al. Different effects of tirofiban and aspirin plus clopidogrel on myocardial no?reflow in a mini?swine model of acute myocardial infarction and reperfusion. Heart. 2006; 92(8): 1131?1137.
14 Alfayoumi F, Srinivasan V, Geller M, et al. The no?reflow phenomenon: epidemiology, pathophysiology, and therapeutic approach. Rev Cardiovasc Med. 2005; 6(2): 72?83.
15 Zhou Y, Gu J, Xu LM. Effect and mechanism of salvianolic acid B in attenuating elevated portal pressure in a rat model of portal hypertension induced by endothelin?1. Zhong Xi Yi Jie He Xue Bao. 2007; 5(1): 61?64. Chinese with abstract in English.
周扬, 顾杰, 徐列明. 丹参酚酸B盐抑制内皮素?1所致大鼠门静脉压力升高的药效及其作用机制. 中西医结合学报. 2007; 5(1): 61?64.
16 Liu GS, Xu GF. Research advance in antioxidation of salvianolic acid. Shi Pin Yu Yao Pin. 2007; 9(1): 69?71. Chinese with abstract in English.
