榆干离褶伞溶栓酶对大鼠动脉血栓的抑制作用

目的：研究榆干离褶伞溶栓酶（Lyophyllum ulmarium fibrinolytic enzyme，LUFE）对大鼠动脉血栓形成的抑制作用及其机制。方法：将SD大鼠随机分为假手术组、模型组、阳性对照组和LUFE低、高剂量组，除假手术组以外，其余4 组均以三氯化铁建立颈动脉血栓模型。造模前阳性对照组和LUFE低、高剂量组分别以10 mg/kg mb阿司匹林以及100、400 mg/kg mb LUFE灌胃7 d。观察血栓质量和颈动脉血管组织病理变化；酶联免疫吸附测定法检测肿瘤坏死因子α（tumor necrosis factor alpha，TNF-α）、白细胞介素-6（interleukin-6，IL-6）、血小板活化因子（platelet-activating factor，PAF）、可溶性血栓调节蛋白（soluble thrombomodulin，sTM）、E-选择素和组织因子（tissue factor，TF）水平；流式细胞仪检测血小板CD62P表达水平。为观察LUFE对血小板功能的影响，在体外以胶原诱导血小板活化，观察LUFE对大鼠富血小板血浆中P-选择素、β-血小板球蛋白（β-thromboglobulin，β-TG）和血小板膜糖蛋白IIbIIIa（glycoprotein IIbIIIa，GPIIbIIIa）水平的影响；Western blot方法观察LUFE对大鼠血小板磷脂酰肌醇3激酶（phosphatidylinositol 3 kinase，PI3K）、蛋白激酶B（protein kinase B，Akt）和p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase，p38 MAPK）表达水平的影响。结果：LUFE能明显减轻血栓质量和血管受损程度，抑制血栓模型大鼠血清sTM、E-选择素、TF、TNF-α、IL-6和PAF水平的上调，下调血小板CD62P的表达。同时，在体外LUFE能够下调胶原诱导的富血小板血浆中P-选择素、β-TG和GPIIbIIIa水平，下调血小板PI3K、Akt和p38 MAPK蛋白的磷酸化水平。结论：LUFE具有抗血栓作用，其机制可能与LUFE对血管内皮细胞的保护作用以及对血小板活化的抑制作用有关。

Antithrombotic Effect of Fibrinolytic Enzyme from Lyophyllum ulmarium on Arterial Thrombosis in Rats

Objective:To explore the inhibitory effect of the fibrinolytic enzyme from Lyophyllum ulmarium(LUFE)on rat thrombosis.Methods:SD rats were randomly divided into sham operation,model,positive control and low-and high-dose LUFE groups.All groups except the sham operation group were used to establish a ferric chloride(FeCl3)-induced carotid arterial thrombosis model.Before modeling,the rats in the positive control group and the low-and high-dose LUFE groups were administered with 10 mg/kg mb of aspirin and 100 and 400 mg/kg mb of LUFE,respectively,by gavage for 7 days.The changes in thrombus mass and carotid vascular histopathology were observed.The levels of tumor necrosis factor alpha(TNF-α),interleukin-6(IL-6),platelet-activating factor(PAF),soluble thrombomodulin(sTM),E-selectin and tissue factor(TF)were detected by enzyme-linked immunosorbent assay.The expression level of CD62P in platelets was detected by flow cytometry.In order to observe the effect of LUFE on platelet function,collagen-induced platelet activation was performed in vitro to observe the effect of LUFE on P-selectin,β-thromboglobulin(β-TG)and platelet membrane glycoprotein IIbIIIa(GPIIbIIIa)levels in platelet-rich plasma.Western blot was used to evaluate the effect of LUFE on the expression levels of phosphatidylinositol 3 kinase(PI3K),protein kinase B(Akt)and p38 mitogen-activated protein kinase(p38 MAPK)in platelets.Results:LUFE significantly reduced thrombus mass and vascular damage,inhibited the increase in serum sTM,E-selectin,TF,TNF-α,IL-6 and PAF levels in the thrombus model rats,and decreased the expression of platelet CD62P.LUFE could in vitro reduce the levels of P-selectin,β-TG and GPIIbIIIa in collagen induced platelet-rich plasma,and down-regulated the phosphorylation levels of platelet PI3K,Akt and p38 MAPK protein.Conclusion:LUFE has an anti-thrombotic effect,and its mechanism may be related to the protection of vascular endothelial cells and the inhibition of platelet activation.