| Thrombotic diseases,one of the causes of high-mortality rate,is a serious threat to human life and health.The main therapeutic approach to treat this kind of disease is thrombolysis therapy,and the thrombolytic agents are widely used in clinics.However,the existing thrombolytic agents have various side effects,such as:short high-life,poor specificity for fibrin,abnormal hemorrhage risk and high cost.Thus,searching for safe and efficient ideal thrombolytic products is the focal points in the field of thrombolysis.Cordyceps cicadae,one of the famous Chinese traditional medicinal mushrooms,is a kind of entomogenous fungi belonging to Clavicipitaceae and Ascomycotina.Modern research shows that it contains several kinds of active components such as amino acids,polysaccharides,cordycepin,and other active ingredients,which can regultate human immunity,improve renal function and so on.C.cicadae is extremely similar to the Cordyceps sinensis in active component and pharmacodynamic action,so it has been used as the substitute of C.sinensis in clinic.However,little information is available from C.cicadae on the antithrombotic and thrombolytic activities.Therefore,we purified the novel fibrinolytic protein from C.cicadae and partially studied physical and chemical properties for the first time in the present study,and evaluated the anti-thrombolytic effect in vitro,and investigated its protective effect on H2O2-induced human umbilical endothelial cells.It would lay a function for further research and development of fibrinolytic protein from C.cicadae.The main achievements in this paper are shown as follows:(1)Preparation of fibrinolytic protein from C.cicadaeThe crude extraction is received from dried C.cicadae using the PBS freezing and thawing technique.And the crude protein was primary separated by adding the ammonium sulfate to the rough extraction of 30%～70%saturation and dialysis into water,and then the crude protein powder was gotten after freeze drying.Next,the fibrinolytic protein was obtained after a combination of Source 15Q ion-exchange chromatography and dextran G-200 gel filtration column chromatography.The purified fibrinolytic protein displayed a single band in SDS-PAGE,and its apparent molecular weight was about 15 kDa.And the fibrinolytic activity was detected by the fibrin plate method.(2)Study on physicochemical properties of fibrinolytic protein from C.cicadaeTh e protein fibrinolytic activity was measured by azocasein assay.The results indicated that the optimal pH and the optimal temperature of the fibrinolytic protein is 8.0 and 45℃separately.More than 90%of the activity wsa found stable after 1h in the pH range of 7.0～10.0,and 75%of the activity wsa stable after 5h in the temperature range of 20℃～45℃.The fibrinolytic activity was enhanced in the presence of metal ions such as K+and Li+ions,while Cu2+、Fe3+and Zn2+ions inhibited the activity.By contrast,Ba2+、Ca2+、Mg2+ and Mn2+have little influence on the fibrinolytic activity of the fibrinolytic protein.Furthermore,the experiment results showed that SDS and PMSF almost completely inhibited the activity,while β-mercaptoethanol,EDTA and DTT inhibited the fibrinolytic activity at some degree,and urea almost has no effect,which suggested that this fibrinolytic protein could be a kind of serine protease.(3)Study on anticoagulant and thrombolytic effects of fibrinolytic protein from C.cicadae in vitroVenous blood was got from rat and the anticoagulant activity was detected by adding the fibrinolytic protein into the blood immediately.The results indicated that the fibrinolytic protein has a favorable effect on anticoagulantion.Then,venous blood was got from rat and fomed clot in room temperature.The thrombolysis activity was detected by adding the different concentration of fibrinolytic protein(100μg/mL、75μg/mL、50μg/mL and 25μg/mL)into the clots.The results showed that fibrinolytic protein from C.cicadae exhibited good thrombolytic effect in vitro,and the dissolution rate of blood clots increased with a dose-response manner in different concentration of fibrinolytic protein group.And more than 80%of blood clot could be dissolved after 5h in the high dose group(100μg/mL).Besides,the dissolution rate of blood clots setting time less than 5h could reach 70%,and it had poor degradation ability of blood clots setting time more than 6h.(4)Fibrinolytic and fibrinogeolytic activity of fibrinolytic protein from C.cicadaeThe fibrinolytic manner of the fibrinolytic protein was detected according to the difference of transparent circle on fibrin plate with or without plasminogen.The result indicated that the fibrinolytic protein from C.cicadae could degrade fibrin directly.The determination of fibrinolysis and showed that the fibrinolytic protein from C.cicadae could hydrolyze the y-y-chain of fibrin followed by α-chain and β-chain,and the fibrinogen degrading pattern of the fibrinolytic protein was Aα-chain>Bβ-chain>γ-chain in time-dependent manner.(5)Protective effect of the fibrinolytic protein from C.cicadae on H2O2-induced oxidative damage of vascular endothelial cellsA model of HUVEC injured by hydroxide(H2O2)was established to investigate the protective effect of the fibrinolytic protein from C.cicadae.After the treatment of different concentration of the fibrinolytic protein samples,the contents of NO,LDH,MDA,SOD and GSH were measured to explore the effects of the samples on the oxidative stress of HUVECs.The result showed that the fibrinolytic protein from C.cicadae could enhance the content of NO and decrease the content of MDA and LDH,and restore activities of SOD and GSH.Which proved that the fibrinolytic protein from C.cicadae may have the protective effect on HUVECs injured by H2O2.To summarize,the fibrinolytic protein from C.cicadae has the fibrinolysis activity in vitro and protective effects on HUVEC.It’s important and potential for the drug of clinical therapy to treat thrombotic diseases. |
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