Protective Effect Of Fibrinolytic Enzyme From Lyophyllum Ulamarium On Nicotine Induced Endothelial Dysfunction In Rats And Its Antiplatelet Effect

Objective:To investigate the protective effect of fibrinolytic enzyme from Lyophyllum Ulamarium(LuFE)on nicotine-induced vascular endothelial cell damage in rats;to reseach the antiplatelet function and mechanism of LuFE in vitro.Method:(1)In vivo:32 SD rat,weight 180-250g,were divided into five groups:normal control group,nicotine control group(2mg/kg),low-dose LuFE group(nicotine 2mg/kg+LuFE 200mg/kg),high-dose LuFE group(nicotine 2mg/kg+LuFE 400mg/kg).After 4 week,we abserve the morphology changes of aorta abdominalis and lung by HE staining.The levels of TNF-?,IL-1?,ET-1,TM,vWF,PGI2,P-selectin and P-TG in plasma were detected by ELISA;the proteins expression and phosphorylation levels of the PI3K,Akt,I?B and NF-?B were detected by the western blot method.(2)In vitro:In vitro activation of rat platelets induced by collagen.The levels of platelet-rich plasma vWF,P-selectin,P-TG,GP-?b?a were detected by ELISA;the protein phosphorylation levels of Syk,PLC?,p-P47,PI3K,Akt and P38 were observed by the western blot method.Results:(1)Abdominal aorta HE staining showed that,control group vascular endothelial cell structure is intact and smooth,the elastic fibers were continuous uniform.After the nicotine injury,the structural continuity of the abdominal aorta endothelial cell was destroyed.Low and high dose group of LuFE after pretreatment,vascular endothelial structure gradually returned to normal.HE staining results showed that,the alveolar tissue of the control group is intact,and there was no edema,thrombosis or inflammatory cell infiltration in the alveol.After nicotine injury,alveolar tissue integrity was destroyed,alveolar septum was thickened,inflammatory cells infiltrated in alveolar cavity,and microthrombus formation.Compared with the model group,the alveolar septum thickened and a small amount of inflammatory cells infiltrated in the low dose group of LuFE.In the LuFE high dose group,the interstitial thickening of the lung was reduced,no infiltration of inflammatory cells or microthrombosis was observed.ELISA results showed that,compared with the normal group,the levels of TNF-?,IL-1?,ET-1,TM,vWF and P-selectin were significant increased(p<0.01),the levels of PGI2 were significant decreased in the model group;compared with the model group,the levels of TNF-?,IL-1?,ET-1,TM,vWF and P-selectin were reduced(p<0.05),the levels of PGI2 were increased in the drug groups.Western blot results showed,compared with the normal group,nicotine could increased the protein phosphorylation of PI3K,Akt and NF-?B,reduce the protein expressions of I?B in the model groups;compared with the model group,LuFE could reduce the protein phosphorylation of PI3K,Akt and NF-?B,increased the protein expressions of I?B in the drug groups.(2)ELISA results showed that,compared with the normal group,the levels of vWF,P-selectin,?-TG and GP-?b?a were significant increased in the model group(p<0.05);compared with the model group,the levels of vWF,P-selectin,?-TG and GP-?b?a were significant reduced in the drug groups(p<0.05).Westem blot results showed that,LuFE can inhibit the phosphorylation levels of Syk PLCy,P47,PI3K,Akt and P38 proteins,and Akt inhibitors combined with LuFE can enhance the inhibition of platelet activation.Conclusions:(1)Fibrinolytic enzyme from Lyophyllum Ulamarium could play a pr-otection role in nicotine-induced vascular endothelial cell damage in rats,and the protective mechanisms may be related to inhibition of PI3K/Akt signaling pathway.(2)Fibrinolytic enzyme from Lyophyllum Ulamarium can inhibit collagen-induced platelet activation in rats,and its mechanism is related to inhibition of Syk/PLCy/PKC and PI3K/Akt/P38 signaling pathways.