榆干离褶伞溶栓酶抗栓过程中对血小板活化、血管内膜炎性损伤的影响及机制研究

Recent studies revealed that some edible fungi including Pleurotus ostreatus, Flammulina velutipes and Armillaria mellea have the activity of thrombolysis. However, these researches are limited to the separation and purification of active components, and the mechanism of antagonism is rare. In our previous work, we have separated and purified one kind of Fibrinolytic enzyme from Lyopyllumulmarium, Recent studies revealed that some edible fungi including Pleurotus ostreatus, Flammulina velutipes and Armillaria mellea have the activity of thrombolysis. However, these researches are limited to the separation and purification of active components, and the mechanism of antagonism is rare. In our previous work, we have separated and purified one kind of Fibrinolytic enzyme from Lyopyllumulmarium, identified as Lyopyllum ulmarium fibrinolytic enzyme (LuFE), which with a molecular weight of 50 kDa. And thrombolytic effect of LuFE has been confirmed in animal models. The injury of vascular endothelial cells and the activation of platelets are not only the key factors of thrombosis, but also the pathological basis of vascular inflammation. This project is intended to study the role of LuFE on vascular endothelial cells injury; and adhesion, aggregation and secretion function of platelet in its antithrombosistic process. We will explore the regulation effect of LuFE on related pathways, including PI3K/Akt pathway and TLR4/ MyD88 pathway, which play important roles in inflammatory reactions and platelet activation. This project will provide the experimental basis not only for study of antithrombosis of LuFE, but also for development of antithrombotic drugs or health food.

研究表明，平菇、金针菇、榛蘑等食用菌具有抗血栓活性。但这些研究仅限于有效成分的分离纯化，而对抗栓作用及机制的研究较少见。前期研究中，我们从榆干离褶伞分离纯化了一种溶栓酶，即榆干离褶伞溶栓酶(Lyopyllum ulmarium fibrinolytic enzyme, LuFE)，且通过动物实验证实了其抗栓作用。血管内皮细胞的损伤及血小板的活化既是血栓形成的关键也是血管发生炎症反应的病理基础。因此，本项目将通过体内、外实验，研究LuFE对血管内皮细胞的炎性损伤，血小板的粘附、聚集及分泌功能的影响。鉴于PI3K/Akt和TLR4/MyD88信号通路在炎症反应及血小板活化过程中的重要作用，本研究将进一步探讨LuFE对上述信号通路的调控作用，以明确该溶栓酶抗炎、抗血小板作用的分子机制和作用靶点。本项研究将为食用菌抗栓作用机制上的多靶点、多途径特点提供实验依据，也为新型的抗栓药物的开发提供依据。

血栓性疾病严重危害人们的健康。血栓形成或栓塞是致死和致残的重要原因。血管内皮细胞的损伤及血小板的异常活化是血栓形成的关键也是血管发生炎症反应的病理基础。血栓形成过程中血管内皮细胞、血小板和白细胞之间的相互作用促进炎症反应。本项目中以榆干离褶伞菌丝体中分离纯化的溶栓酶(Lyopyllum ulmarium fibrinolytic enzyme, LUFE)为研究对象，通过体内外实验探讨了LUFE对血小板的活化及血管内膜炎性损伤的保护作用及其机制。研究结果表明，在血栓形成过程中UFE抑制血小板的活化，降低炎症因子、趋化因子及细胞黏附分子水平，减弱血管内皮细胞与单核细胞、血小板与白细胞之间的黏附作用，从而减轻血管内膜的炎症反应，抑制动脉血栓的形成。LUFE通过调控TLR4/MyD88/NF-κB、MAPK以及Nrf2/HO-1信号通路对致炎因子或氧化应激所致的血管内皮细胞损伤和单核细胞炎症模型有保护作用。在体外，LUFE通过调控PI3K/Akt、GPVI信号通路中的LAT和PLCγ蛋白的活化水平而影响细胞内Ca2+，进而抑制血小板的活化。此外，LUFE抑制尼古丁所致的大鼠血管内膜、肝、肺等组织的损伤，其机制可能与Nrf2/HO-1信号通路的调控有关。本研究探讨了血栓-炎症过程中LUFE对血管内皮细胞-血小板-单核细胞的干预作用，并从抗炎、抗氧化角度阐明了其作用机制。本项目的研究对血栓性疾病的预防及治疗有一定的意义，也为食用菌的开发应用提供了实验依据。