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.
10.1016/j.joim.2025.05.003
- Author:
Wan-Ling ZHONG
1
;
Jian-Qiong YANG
2
;
Hai LIU
3
;
Ya-Li WU
4
;
Hui-Juan SHEN
1
;
Peng-Yue LI
1
;
Shou-Ying DU
5
,
6
Author Information
1. School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China.
2. The Clinical Medicine Research Center, The First Affiliated Hospital of Gannan Medical University, Ganzhou 341000, Jiangxi Province, China.
3. School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China; National Engineering Research Center for Modernization of Traditional Chinese Medicine (Hakka Medical Resources Branch), Gannan Medical University, Ganzhou 341000, Jiangxi Province, China.
4. Department of Pharmacy, The First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou 450000, Henan Province, China.
5. School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China. Electronic address: dumenzidi123@
6. com.
- Publication Type:Journal Article
- Keywords:
Antithrombotic effect;
Caco-2 cell monolayer;
Fibrinolytic protein;
Intestinal absorption mechanism;
Pheretima vulgaris Chen
- MeSH:
Animals;
Zebrafish;
Humans;
Caco-2 Cells;
Fibrinolytic Agents/pharmacology*;
Thrombosis/drug therapy*;
Intestinal Absorption
- From:
Journal of Integrative Medicine
2025;23(4):415-428
- CountryChina
- Language:English
-
Abstract:
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 than 1.2 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.
