中文摘要：

目的探讨可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体及其调节因子突触融合蛋白结合蛋白b（SNARE/Munc18b）复合体介导的脓毒症血小板α颗粒释放及一氧化碳释放分子Ⅱ（CORM-2）的干预机制。方法采集健康成人肘静脉血，差速离心法分离出富含血小板血浆（PRP），并随机分为对照组、脂多糖（LPS）组（10 mg/L）、LPS＋iCORM-2组（10 mg/L LPS ＋ 50 μmol/L无活性CORM-2），LPS＋L-CORM-2组（10 mg/L LPS ＋ 10 μmol/L CORM-2）、LPS ＋ H-CORM-2组（10 mg/L LPS ＋ 50 μmol/L CORM-2）。各组于37？℃、湿度95%、5% CO2培养箱孵育30 min取上清液，用酶联免疫吸附试验（ELISA）检测血小板α颗粒释放物质血小板因子4（PF4）、血小板源性生长因子BB（PDGF-BB）和基质金属蛋白酶2（MMP-2）含量；流式细胞仪检测血小板P-选择素的表达；透射电镜和激光共聚焦显微镜下观察血小板α颗粒的分布；蛋白质免疫印迹试验（Western Blot）检测血小板关键膜融合分子Munc18b及相关SNARE蛋白囊泡相关膜蛋白-8（VAMP-8）、突出相关蛋白-23（SNAP-23）、突出融合蛋白-11（STX-11）的表达；免疫沉淀法检测SNARE/Munc18b复合体的形成。结果与对照组比较，LPS组血小板α颗粒释放PF4、PDGF-BB、MMP-2、P-选择素的量明显升高；而低、高浓度CORM-2能有效抑制LPS刺激后血小板α颗粒的释放〔PF4（μg/L）：7.69±0.58、6.03±0.71比10.13±0.82，PDGF-BB（μg/L）：112.71±1.79、102.91±5.86比128.78±1.39，MMP-2（ng/L） ：32.94±2.73、27.58±3.36比53.26±1.21，P-选择素：（17.14±0.57）%、（15.35±0.68）%比（23.78±0.62）%，均P〈0.01〕，且呈剂量依赖趋势。透射电镜和激光共聚焦显微镜下观察，CORM-2能减少LPS刺激后血小板α颗粒向血小板膜周围分布，并可抑制与包膜融合。与对照组比较，LPS刺激后血小板Munc18b和相关SNARE蛋白表达，以及SNARE/Munc18b复合体形成均显著?

英文摘要：

ObjectiveTo investigate the suppressive effect of carbon monoxide-releasing molecule Ⅱ （CORM-2） on LPS induced platelet α-granule exocytosis in sepsis via soluble N-ethylmaleimide-sensitive factor attached protein receptor/mammalian uncoordinated 18b （SNARE/Munc18b） complex formation.MethodsBlood was collected from healthy volunteers＇ cubital vein, then platelets were isolated by differential centrifugation. Platelets were randomly divided into 5 groups. The control group did not undergo any treatment, the LPS group received 10 mg/L LPS simulation, the CORM-2 group and iCORM-2 group underwent LPS simulation and immediate administration of CORM-2 （10 μmol/L and 50 μmol/L） or iCORM-2 （50 μmol/L）, respectively. Samples were incubated in a CO2-incubator at 37？℃, 95% humidity, and 5% CO2. Platelet α-granule contents were detected by using standard enzyme linked immunosorbent assay （ELISA）, including platelet factor 4 （PF4）, platelet derived growth factor-BB （PDGF-BB）, and matrix metalloproteinase-2 （MMP-2）. The expression of P-selectin was detected by flow cytometer. Transmission electron microscope and immunofluorescence microscope was used to assess platelet α-granules distribution. Expressions of Munc18b and SNARE proteins including vesicle-associated membrane protein-8 （VAMP-8）, synaptosomal-associated protein-23 （SNAP-23） and syntaxin-11 （STX-11） were detected by Western Bolt. The SNARE/Munc18b complex formation was detected by immunoprecipitation.ResultsCompared with the control group, levels of PF4, PDGF-BB, MMP-2 and P-selectin in LPSinduced platelets were found to markedly elevated, while CORM-2 （10 μmol/L and 50 μmol/L） could decrease platelet α-granule contents exocytosis： [PF4 （μg/L）： 7.69±0.58, 6.03±0.71 vs. 10.13±0.82; PDGF-BB （μg/L）： 112.71±1.79, 102.91±5.86 vs. 128.78±1.39; MMP-2 （ng/L）： 32.94±2.73, 27.58±3.36 vs. 53.26±1.21; P-selectin： （17.14±0.57）%, （15.35±0.68）% vs. （23.78±0.62）%; all P