The Protective Effect Of Fibrinolytic Enzyme From Lyophyllum Ulamarium On LPS-induced Pre-DIC In Rats

Objective:to investigate the protective effect of fibrinolytic enzyme from Lyophyllum Ulamarium on LPS induce rat early disseminated intravascular coagulation model.Method:(1)In vitro:The survival rate of HUVEC was detected after fibrinolytic enzyme from Lyophyllum ulmarium treatment by MTT method in the inflammation damage model induced with 10 ?g/L LPS.LDH,ICAM-1,IL-1?,AT-III,P-selectin,Fib and TNF-a activity were assayed in HUVEC culture fluid by ELISA method.(2)In vivo:the rat model of early disseminated intravascular coagulation was established by 10mg/kg LPS through the intraperitoneal injection,then the morphology changes of liver,lung and aorta abdominalis were tested by HE staining.The levels of plasma TM,ET-1,D2D,IL-1?,AT-?,P-selectin,TNF-? and Fib were detected by ELISA;the levels of ALT,AST,ALB,Cr and BUN were assayed by the colorimetric.The protein expressions of TLR4,p-TAK1,I?KB-?,NF-?B,p-NF-KB and COX-2 were observed by the western blot method.Results:(1)MTT result showed that fibrinolytic enzyme from Lyophyllum Ulamarium played a protect role for LPS-induced endothelial cell injury.The survival rate of HUVEC was improved after fibrinolytic enzyme from Lyophyllum ulmarium treatment.The LDH activity of cell culture fluid was significantly decreased than that of model group.The levels of ICAM-1,IL-1? and TNF-a were decreased than that of model group too.(2)HE staining results showed that,In the control group,the central vein was well structured and the hepatic cord was radial and directed to the central vein.There were significant changes after LPS induced liver,lung and aorta abdominalisdamage.Congestion was noted in the central vein,the tendency of hepatic cords were disorder;part of the liver cells appeared cell structure dissolved,liver cells necrosis.After the treatment of fibrinolytic enzyme from Lyophyllum Ularmarium,in the drug gtoups,the congestion of central vein was significantly decreased than model group,The condition of hepatic cords and liver cells were improved.The alveolar structure of the normal group is complete.The control group had good alveolar structure,no edema in the interstitial lung,and no inflammatory cell infiltration in the alveoli.The pathological changes of acute lung injury caused by lipopolysaccharide are characterized,the destruction of alveolar structure and thickening of alveolar septum,inflammatory cell infiltration in alveolar cavity and vascular cavity,and formation of microthrombus.Compared with the model group,the positive control group had relatively complete alveolar structure,large alveolar congestion and obvious inflammatory cell infiltration,without thrombosis.In the low dose group of fibrinolytic enzyme from Lyophyllum Ulamarium,the alveolar septal thickening was severe,but no obvious inflammatory cell infiltration,no thrombus formation.In the high dose group,interstitial thickening of lung was reduced,no inflammatory cells were infiltrated and no thrombus.Abdominal aortic HE staining showed that the control group vascular endothelial cell structure is good,after the lipopolysaccharide damage,aorta abdominalis endothelial cell rupture,after pretreatment with dexamethasone,the positive control group endothelial cell structure is acceptable,but the inflammatory cells adhere to the vascular endothelium.Fibrinolytic enzyme from L.u of low and high dose group after pretreatment,vascular endothelial structure gradually returned to normal.ELISA result showed that,compared with the normal group,the levels of TM,ET-1,D2D,IL-1?,TNF-a,P-selectin and Fib were significantl increased,the levels of ALT,AST,Cr and BUN were increased,while ALB and AT-? levels were reduced in the model group;compared with the model group,the levels of TM,ET-1,D2D,IL-1?,TNF-a and Fib were significantl reduced,the levels of ALT,AST,Cr and BUN were reduced,while ALB and AT-? levels were increased in the drug groups.Western blot results showed,compared with the normal group,LPS could increased the protein expressions of TLR4,p-TAK1,NF-?B,COX-2 and p-NF-KB,reduce I?KB-? in the model groups;compared with the model group,fibrinolytic enzyme from Lyophyllum Ulamarium could reduce the protein expressions of TLR4,p-TAK1,NF-?B,COX-2 and p-NF-?B,increased IicB-a in the drug groups.Conclusions:.(1)Fibrinolytic enzyme from Lyophyllum Ulamarium have the anti-infl ammatory effect.(2)Fibrinolytic enzyme from Lyophyllum Ulamarium could play a prot-ection role on LPS-induced rat early disseminated intravascular coagulation model.(3)Fibrinolytic enzyme from Lyophyllum Ulamarium may regulate the signaling pathway of TLR4/NF-?B/COX-2 to inhibite the activity of early disseminated intravascular coa-gulation.