Anticoagulation Effect Of Enteromorpha's Polysacchride And The Preliminary Research Of The Mechanism

Object:To research the anticoagulation effect of Enteromorpha's Polysacchride(EP)and the preliminary research of the mechanismMethod:1.Take the enteromorpha which is produced by fujian province as the research object.The EP is extracted by hot water diffusion.Then remove protein by neutral protease,remove salts by materialto dialysis bag,remove pigment by DA201 resin.Combinated with water extract-alcohol precipitation to extract the CEP.Analysis CEP about polysaccharide content by phenol sulfuric acid method,sulfate radical content by barium sulfate turbidimetry.2.The adult female SD rats was used.The animals were randomly divided into six groups with each consisting of 12 rats.The experiment include six groups: Normal group(ND),Model group(HD),enteromorpha's polysacchride dose groups(HEP150,HEP300,HEP600),positive control group(Heparin).Normal group was given normal diet;model group,enteromorpha's polysacchride dose groups and positive control group were high fat feed.Six groups were given enough drinking water.Enteromorpha's polysacchride dose groups,respectively with the concentration of 150 mg/kg,300mg/kg and 600 mg/kg polysaccharide aqueous solution to fill the stomach every day;Normal group,model group and positive control group gastric by distilled water every day.For 35 days in a row.From the 33 rd day,the positive control group was given 0.5ml/100 g tail vein injection of heparin sodium injection,and the other 5 groups were injected with saline for the tail vein.The tail vein was injected for 3 days.At the last delivery,all groups stop feeding for 12 hours.3.At the end of the experiment,weighed agina,then take blood by removalling eyeball.The second drop was used to determinate the clotting time.Collection of residual blood to determinate total cholesterol,triglycerides,high density lipoprotein cholesterol,low density lipoprotein cholesterol,prothrombin time,activated partial thromboplastin time,thrombin time,Fibrinogen,coagulation factor 5,coagulation factor 7,coagulation factor 9,coagulation factor 10,coagulation factor 11,coagulation factor 12,antithrombin and protein C.Dissecting the rat? and separating the liver.Reverse tanscription PCR(RT-PCR)deteminated the AT?,NF-?B,TNF-?,p50,p52,p53 m RNA expression level in liver tissue.Result:1.EP can inhibit the coagulation casued by high fat feedCompared with normal group,model group's CT,PT and TT significantly decreased(P<0.05);And model group's FIB significantly increased(P<0.05).Compared with model group,HEP600 and Heparin group's CT significantly increased(P<0.05);Heparin group's CT significantly increased(P<0.05);HEP150,HEP300,HEP600 and Heparin group's FIB significantly decreased(P<0.05).2.Preliminary research to the anticoagulation mechanism of EPCompared with normal group,model group's AT and ? Protein C significantly decreased(P<0.05).Compared with model group,HEP300,HEP600 and Heparin group's AT and ? Protein C significantly increased(P<0.05).And the dose-dependent relationship was presented,that in,as the dose increased,the AT? and Protein C increased accordingly.The experimental results of coagulation factors show that: Compared with normal group,model group's F5?F9?F10?F11?F12 significantly increased(P<0.05);model group's F7 also increased,but has no statistical differences(P>0.05).Compared with model group,HEP300,HEP600 and Heparin group's F5?F7?F9?F10?F11?F12 significantly decreased(P<0.05).3.Research on related genes of anticoagulationCompared with normal group,model group can make the AT amount of m RNA ?expression decrease(P<0.05).Compared with model group,HEP600 group can make the AT increased(P<0.05).? Compared with normal group,model group can increased the P50,p52,p53,p65 and TNF-? amount of m RNA expression(P<0.05).Compared with model group,HEP600 group can decrease the p65 amount of m RNA expression(P<0.05);HEP150,HEP300,HEP600 and Heparin group can decrease the p52 amount of m RNA expression(P<0.05);HEP150,HEP300,HEP600 and Heparin group have no diffenrence on p50 and p53 amount of m RNA expression(P>0.05);HEP300,HEP600 and Heparin group can decrease the TNF-? amount of m RNA expression(P<0.05).Conclusion:1.The high-fat diet can form the hypercoagulant state of rat blood.The average clotting time in the model group was only 30 % compared with the normal group.2.EP have good anticoagulant effect and can effectively reduce the level of fibrinogen.At the same time,EP can increase the AT and protein C.And then,it canregulate a ?series of clotting factors,which coincide with heparin anticoagulation.3.In the mechanism of EP anticoagulant,NF-?B-related genes play an inportant role.Its possible mechanism is: The anticoagulation of AT was increased under the ?action of EP.The expression of TNF-? was inhibited because of increasing AT?.Furthermore,downstream gene was also inhibited.Therefore,the damage of vascular endothelial cells was protected,and vascular endothelial cells continued to release coagulation factors and clot promoting factors was decreased.