丹酚酸B對兔缺血再灌注心臟內(nèi)皮細(xì)胞功能和血小板活化的影響

1、丹酚酸B對兔缺血再灌注心臟內(nèi)皮細(xì)胞功能和血小板活化的影響 09-08-14 11:29:00 編輯：studa20 作者：楊富國, 張安勇, 陳作元, 廉哲勛, 劉革新, 董果雄【摘要】 目的：觀察丹酚酸B（salvianolic acid B，SAB）對兔缺血再灌注心臟內(nèi)皮細(xì)胞功能和血小板活化的影響。方法：將24只新西蘭大耳白兔隨機(jī)分為假手術(shù)組、缺血再灌注損傷組（模型組）和SAB治療組。結(jié)扎兔心臟左前降支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。SAB治療組于左前降支結(jié)扎后靜滴SAB溶液，其余各組靜滴等量生理鹽水。分別于結(jié)扎前、缺血0.5 h、再灌注1 h及4 h靜脈采血，

2、用硝酸還原酶法測定血漿一氧化氮（nitric oxide, NO）濃度，放射免疫法測定血漿內(nèi)皮素（endothelin, ET）含量和血小板表面顆粒膜蛋白140（alphagranule membrane protein140, GMP140）的數(shù)目。結(jié)果：假手術(shù)組手術(shù)前后血漿NO濃度、ET含量和血小板表面GMP140差別無統(tǒng)計(jì)學(xué)意義（P0.05）；模型組心肌缺血0.5 h后，血漿NO濃度較術(shù)前及假手術(shù)組相應(yīng)時(shí)間顯著降低（P0.05，并隨再灌注時(shí)間延長呈進(jìn)行性下降，而血漿ET含量和血小板表面GMP140數(shù)目較術(shù)前及假手術(shù)組相應(yīng)時(shí)間顯著升高（P0.05，并隨再灌注時(shí)間延長進(jìn)一步上升；治療組應(yīng)用S

3、AB后，再灌注1 h及4 h時(shí)血漿NO濃度較模型組相應(yīng)時(shí)間明顯升高(P0.01)，而血漿ET含量和血小板表面GMP140數(shù)目較模型組相應(yīng)時(shí)間明顯降低（P0.01）。結(jié)論：心臟缺血再灌注過程中存在血管內(nèi)皮細(xì)胞功能損傷及血小板活化，SAB具有保護(hù)內(nèi)皮細(xì)胞和抑制血小板活化的作用。 【關(guān)鍵詞】 丹酚酸B; 心肌缺血再灌注損傷; 一氧化氮; 內(nèi)皮素; 血小板顆粒膜蛋白140; 兔Methods: A total of 24 New Zealand white rabbits were randomly divided into shamoperated group, ischemiareperfusio

4、n group (untreated group) and SAB group. The hearts of rabbits in untreated group and SAB 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 ischemiareperfusion injury model in v

5、ivo. For shamoperated group, the animals were not subjected to occlusion of LADCA. In SAB treatment group the rabbits were intravenously administered SAB immediately after LADCA occlusion, and the other two groups were given normal saline in the same way instead of SAB. The jugular vein bloods of an

6、imals were collected before LADCA ligation, half an hour after ligation and after 1, 4hour 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 alp

7、hagranule membrane protein140 (GMP140) on platelet surface to identify the activation of the platelet.Results: No significant difference was observed before and after sham LADCA occlusion in shamoperated group in the contents of NO and ET in plasma (P0.05), neither was the count of GMP140 on platele

8、t surface (P0.05). The content of NO in plasma detected 0.5 h after LADCA occlusion was significantly decreased in untreated group compared with the shamoperated group at the corresponding time, and they were also much lower than that before LADCA occlusion in the shamoperated group (P0.05). The pla

9、sma 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 GMP140 on platelet surface were remarkably increased after myocardial ischemia as compared with those before LADCA ligation and those de

10、tected in shamoperated group (P0.05). The content of ET and the count of GMP140 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 GMP140 were both significantly decreased i

11、n SAB group as compared with untreated group after 1 and 4hour myocardial reperfusion, respectively (P0.01).Conclusion: The results show that endothelial dysfunction and platelet activation occur during ischemiareperfusion in rabbit hearts in vivo and SAB protects cardiovascular endothelium cells ag

12、ainst ischemiareperfusion injury and inhibits the activation of platelet during myocardial ischemia and reperfusion.Keywords: salvianolic acid B; myocardical reperfusion injury; nitric oxide; endothelin; platelet alphagranule membrane protein140; rabbits及時(shí)恢復(fù)血流再灌注是治療缺血性心臟病普遍采用的措施，可以挽救嚴(yán)重缺血或?yàn)l臨壞死的心肌，并限制

13、或縮小梗死面積，有效地保護(hù)心臟功能，降低心臟事件的發(fā)生率，改善臨床預(yù)后。然而，再灌注早期冠狀動脈血流的快速恢復(fù)可引起心肌進(jìn)一步損傷即心肌缺血再灌注損傷，血小板聚集、白細(xì)胞浸潤、細(xì)胞內(nèi)鈣離子超負(fù)荷和氧自由基爆發(fā)等在其中發(fā)揮重要作用1。中藥減輕心肌缺血再灌注損傷的研究對于急性心肌梗死的治療具有重要意義。中藥丹參為唇形科植物丹參Salvia miltiorrhiza Bge.的干燥根及根莖，具有活血祛瘀的功效。丹酚酸B（salvianolic acid B, SAB）作為丹參中的主要水溶性成分，具有清除氧自由基、抗氧化、保護(hù)心肌的作用25。本實(shí)驗(yàn)通過觀察內(nèi)皮細(xì)胞和血小板在心臟缺血再灌注時(shí)的變化，觀察

14、SAB對該病理過程的影響，以期為臨床用藥提供依據(jù)。1 材料與方法1.1 藥物及試劑 SAB，淡黃色粉針劑，純度大于98%，批號為111562200403（中國藥品生物制品檢定所）。LKB1275型免疫計(jì)數(shù)儀（芬蘭WALLAC公司）；Miniplus 2型電子蠕動泵（法國CSA公司）。一氧化氮（nitric oxide, NO）檢測試劑盒（南京建成生物工程研究所提供）、內(nèi)皮素（endothelin, ET）放射免疫檢測試劑盒（解放軍總醫(yī)院科技開發(fā)中心放免研究所提供）和血小板表面顆粒膜蛋白140（alphagranule membrane protein140, GMP140）檢測試劑盒（蘇州醫(yī)學(xué)

15、院止血與血栓研究室提供）。1.2 心肌缺血再灌注損傷模型制備 健康雄性新西蘭大耳白兔24只，體質(zhì)量（3.250.56）kg，由青島市藥品檢驗(yàn)所提供，實(shí)驗(yàn)動物合格證號為SCXK（魯）20030010。 參考文獻(xiàn)方法6建立心肌缺血再灌注損傷模型。20%烏拉坦5 ml/kg耳緣靜脈麻醉，氣管插管，開胸，暴露心臟，在距冠狀動脈左前降支（left anterior descending coronary artery, LADCA）根部0.5 cm處穿線，結(jié)扎LADCA，制備急性心肌缺血模型。以心電圖導(dǎo)聯(lián)ST段出現(xiàn)弓背向上抬高、T波高聳，心肌組織顏色由正常變蒼白再變暗為心肌缺血模型成功標(biāo)志。LADCA結(jié)

16、扎0.5 h后剪斷穿線使其再通形成再灌注，局部反應(yīng)性充血表示心肌再灌注開始，再灌注持續(xù)4 h。假手術(shù)組大鼠于LADCA根部0.5 cm處穿一膠管，內(nèi)引絲線，但不予結(jié)扎。1.3 動物分組及給藥 24只新西蘭兔按隨機(jī)數(shù)字表法隨機(jī)分為假手術(shù)組、模型組和SAB組，每組8只。SAB組動物模型制作方法同模型組，并于結(jié)扎后即刻由電子蠕動泵經(jīng)耳緣靜脈1 h內(nèi)勻速滴入20 ml SAB溶液（按3 mg/kg精確稱取SAB，溶于20 ml生理鹽水）。其余各組于相應(yīng)時(shí)間內(nèi)滴入20 ml生理鹽水。1.4 指標(biāo)測定 于結(jié)扎前、結(jié)扎0.5 h、再灌注1 h及4 h由頸靜脈取血3.8 ml。其中1 ml血肝素抗凝后，以3 000 r/min 離心15 min，取上清液置于20 冰箱