Objective:EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen,an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome.Its composition,anticoagulant ...Objective:EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen,an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome.Its composition,anticoagulant and fibrinolytic activities,and relevant mechanisms have been confirmed through in vitro experiments.However,whether it has antithrombotic effects in vivo and can be absorbed by the gastrointestinal tract is unknown.This study evaluates the antithrombotic effect in zebrafish and investigates the gastrointestinal stability and intestinal absorption mechanism of this protein in vitro.Methods:The antithrombotic effect of EPF3 in vivo was verified using the zebrafish thrombus model induced by arachidonic acid and FeCl3.Then,the protein bands of EPF3 incubated with simulated gastric fluid(SGF),simulated intestinal fluid(SIF),and homogenate of Caco-2 cells(HC2C)were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis to evaluate its gastrointestinal stability.Finally,the transport behavior and absorption mechanism of EPF3 were studied using Caco-2 cell monolayer.Results:EPF3 could significantly enhance the returned blood volume and blood flow velocity in zebrafish with platelet aggregation thrombus induced by arachidonic acid.It could also prolong the formation time of tail artery thrombus and increase the blood flow velocity in zebrafish with vessel injury thrombus induced by FeCl3.EPF3 was stable in SIF and HC2C and unstable in SGF.The permeability of EPF3 in Caco-2 monolayer was time-dependent and concentration-dependent.The efflux ratio was less than1.2 during transport,and the transport behavior was not affected by inhibitors.EPF3 could reversibly reduce the expression of tight junction-related proteins,including zonula occludens-1,occludin,and claudin-1 in Caco-2 cells.Conclusion:EPF3 could play a thrombolytic and antithrombotic role in zebrafish.It could be transported and absorbed into the intestine through cellular bypass pathway by opening the intestinal epithelium tight junction.This study provides a scientific explanation for the antithrombotic effect of earthworm and provides a basis for the feasibility of subsequent development of EPF3 as an antithrombotic enteric-soluble preparation.Please cite this article as:Zhong WL,Yang JQ,Liu H,Wu YL,Shen HJ,Li PY,Du SY.Antithrombotic effect in zebrafish of a fibrinolytic protein EPF3 from Dilong(Pheretima vulgaris Chen)and its transport mechanism in Caco-2 monolayer through cell bypass pathway.J Integr Med.2025;23(4):415–428.展开更多
Thrombin,the ultimate proteinase of the coagulation cascade,is an attractive target for the treatment of a variety of cardiovascular diseases.A bromophenol derivative named (+)-3-(2,3-dibromo-4,5-dihydroxy-phenyl)-4-b...Thrombin,the ultimate proteinase of the coagulation cascade,is an attractive target for the treatment of a variety of cardiovascular diseases.A bromophenol derivative named (+)-3-(2,3-dibromo-4,5-dihydroxy-phenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroiso-benzofuran 1,isolated from the brown alga Leathesia nana exhibited significant thrombin inhibitory activity.In this study,we investigated the inhibition of human thrombin in vitro with this bromophenol derivative,and its antithrombotic efficacy in vivo using the arteriovenous shunt model and the ferric chloride-induced arterial thrombosis model in rats.The results show that the bromophenol derivative is a potential inhibitor of thrombin (IC50=1.03 nmol/L).In antithrombotic experiments in vivo,the bromophenol derivative also shows good effect comparing with the control group.These data indicate that the bromophenol derivative is a potential drug for prophylaxis and the treatment of thrombotic diseases.展开更多
基金supported by grants from the Fundamental Research Funds for the Central Universities(No.2020-JYB-ZDGG-032)National Natural Science Foundation of China(No.82104352)。
文摘Objective:EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen,an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome.Its composition,anticoagulant and fibrinolytic activities,and relevant mechanisms have been confirmed through in vitro experiments.However,whether it has antithrombotic effects in vivo and can be absorbed by the gastrointestinal tract is unknown.This study evaluates the antithrombotic effect in zebrafish and investigates the gastrointestinal stability and intestinal absorption mechanism of this protein in vitro.Methods:The antithrombotic effect of EPF3 in vivo was verified using the zebrafish thrombus model induced by arachidonic acid and FeCl3.Then,the protein bands of EPF3 incubated with simulated gastric fluid(SGF),simulated intestinal fluid(SIF),and homogenate of Caco-2 cells(HC2C)were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis to evaluate its gastrointestinal stability.Finally,the transport behavior and absorption mechanism of EPF3 were studied using Caco-2 cell monolayer.Results:EPF3 could significantly enhance the returned blood volume and blood flow velocity in zebrafish with platelet aggregation thrombus induced by arachidonic acid.It could also prolong the formation time of tail artery thrombus and increase the blood flow velocity in zebrafish with vessel injury thrombus induced by FeCl3.EPF3 was stable in SIF and HC2C and unstable in SGF.The permeability of EPF3 in Caco-2 monolayer was time-dependent and concentration-dependent.The efflux ratio was less than1.2 during transport,and the transport behavior was not affected by inhibitors.EPF3 could reversibly reduce the expression of tight junction-related proteins,including zonula occludens-1,occludin,and claudin-1 in Caco-2 cells.Conclusion:EPF3 could play a thrombolytic and antithrombotic role in zebrafish.It could be transported and absorbed into the intestine through cellular bypass pathway by opening the intestinal epithelium tight junction.This study provides a scientific explanation for the antithrombotic effect of earthworm and provides a basis for the feasibility of subsequent development of EPF3 as an antithrombotic enteric-soluble preparation.Please cite this article as:Zhong WL,Yang JQ,Liu H,Wu YL,Shen HJ,Li PY,Du SY.Antithrombotic effect in zebrafish of a fibrinolytic protein EPF3 from Dilong(Pheretima vulgaris Chen)and its transport mechanism in Caco-2 monolayer through cell bypass pathway.J Integr Med.2025;23(4):415–428.
基金Supported by the National High Technology Research and Development Program of China (863 Program) (No. 2007AA09Z410)the National Major Research Program of China "The Creation for Significant Innovative Drugs" (No. 2009ZX09103-148)
文摘Thrombin,the ultimate proteinase of the coagulation cascade,is an attractive target for the treatment of a variety of cardiovascular diseases.A bromophenol derivative named (+)-3-(2,3-dibromo-4,5-dihydroxy-phenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroiso-benzofuran 1,isolated from the brown alga Leathesia nana exhibited significant thrombin inhibitory activity.In this study,we investigated the inhibition of human thrombin in vitro with this bromophenol derivative,and its antithrombotic efficacy in vivo using the arteriovenous shunt model and the ferric chloride-induced arterial thrombosis model in rats.The results show that the bromophenol derivative is a potential inhibitor of thrombin (IC50=1.03 nmol/L).In antithrombotic experiments in vivo,the bromophenol derivative also shows good effect comparing with the control group.These data indicate that the bromophenol derivative is a potential drug for prophylaxis and the treatment of thrombotic diseases.
