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.
Animals ; Zebrafish ; Humans ; Caco-2 Cells ; Fibrinolytic Agents/pharmacology* ; Thrombosis/drug therapy* ; Intestinal Absorption

Allyl isothiocyanate (AITC) is a natural product commonly used in food preservation and pharmaceutical applications. Toxoplasmosis, caused by the protozoan pathogen Toxoplasma gondii, is prevalent globally while the impact of AITC on toxoplasmosis is unclear. We explored the effect of AITC on acute toxoplasmosis. We infected C57BL/6 mice with T. gondii type I RH strain following AITC administration. On the 4th day after infection, which corresponds to the initial stage of infection, we collected serum for the determination of inflammatory cytokine levels. The mice serum of the AITC-administered group contained significantly lower levels of granulocyte colony-stimulating factor, interferon-gamma, interleukin (IL)-23 subunit p19, IL-4, IL-6, and monocyte chemoattractant protein-1. The lifespan of the mice in the AITC-administered group was significantly reduced. In vitro experiments showed that AITC promoted the proliferation of intracellular T. gondii accompanied by the inhibition of IL-4, IL-1β, and IL-6 production in RAW264.7 macrophages. Our results showed that AITC facilitated T. gondii infection in the early stage by inhibiting the production of several inflammatory cytokines.

Allyl isothiocyanate (AITC) is a natural product commonly used in food preservation and pharmaceutical applications. Toxoplasmosis, caused by the protozoan pathogen Toxoplasma gondii, is prevalent globally while the impact of AITC on toxoplasmosis is unclear. We explored the effect of AITC on acute toxoplasmosis. We infected C57BL/6 mice with T. gondii type I RH strain following AITC administration. On the 4th day after infection, which corresponds to the initial stage of infection, we collected serum for the determination of inflammatory cytokine levels. The mice serum of the AITC-administered group contained significantly lower levels of granulocyte colony-stimulating factor, interferon-gamma, interleukin (IL)-23 subunit p19, IL-4, IL-6, and monocyte chemoattractant protein-1. The lifespan of the mice in the AITC-administered group was significantly reduced. In vitro experiments showed that AITC promoted the proliferation of intracellular T. gondii accompanied by the inhibition of IL-4, IL-1β, and IL-6 production in RAW264.7 macrophages. Our results showed that AITC facilitated T. gondii infection in the early stage by inhibiting the production of several inflammatory cytokines.

Allyl isothiocyanate (AITC) is a natural product commonly used in food preservation and pharmaceutical applications. Toxoplasmosis, caused by the protozoan pathogen Toxoplasma gondii, is prevalent globally while the impact of AITC on toxoplasmosis is unclear. We explored the effect of AITC on acute toxoplasmosis. We infected C57BL/6 mice with T. gondii type I RH strain following AITC administration. On the 4th day after infection, which corresponds to the initial stage of infection, we collected serum for the determination of inflammatory cytokine levels. The mice serum of the AITC-administered group contained significantly lower levels of granulocyte colony-stimulating factor, interferon-gamma, interleukin (IL)-23 subunit p19, IL-4, IL-6, and monocyte chemoattractant protein-1. The lifespan of the mice in the AITC-administered group was significantly reduced. In vitro experiments showed that AITC promoted the proliferation of intracellular T. gondii accompanied by the inhibition of IL-4, IL-1β, and IL-6 production in RAW264.7 macrophages. Our results showed that AITC facilitated T. gondii infection in the early stage by inhibiting the production of several inflammatory cytokines.

Allyl isothiocyanate (AITC) is a natural product commonly used in food preservation and pharmaceutical applications. Toxoplasmosis, caused by the protozoan pathogen Toxoplasma gondii, is prevalent globally while the impact of AITC on toxoplasmosis is unclear. We explored the effect of AITC on acute toxoplasmosis. We infected C57BL/6 mice with T. gondii type I RH strain following AITC administration. On the 4th day after infection, which corresponds to the initial stage of infection, we collected serum for the determination of inflammatory cytokine levels. The mice serum of the AITC-administered group contained significantly lower levels of granulocyte colony-stimulating factor, interferon-gamma, interleukin (IL)-23 subunit p19, IL-4, IL-6, and monocyte chemoattractant protein-1. The lifespan of the mice in the AITC-administered group was significantly reduced. In vitro experiments showed that AITC promoted the proliferation of intracellular T. gondii accompanied by the inhibition of IL-4, IL-1β, and IL-6 production in RAW264.7 macrophages. Our results showed that AITC facilitated T. gondii infection in the early stage by inhibiting the production of several inflammatory cytokines.

Allyl isothiocyanate (AITC) is a natural product commonly used in food preservation and pharmaceutical applications. Toxoplasmosis, caused by the protozoan pathogen Toxoplasma gondii, is prevalent globally while the impact of AITC on toxoplasmosis is unclear. We explored the effect of AITC on acute toxoplasmosis. We infected C57BL/6 mice with T. gondii type I RH strain following AITC administration. On the 4th day after infection, which corresponds to the initial stage of infection, we collected serum for the determination of inflammatory cytokine levels. The mice serum of the AITC-administered group contained significantly lower levels of granulocyte colony-stimulating factor, interferon-gamma, interleukin (IL)-23 subunit p19, IL-4, IL-6, and monocyte chemoattractant protein-1. The lifespan of the mice in the AITC-administered group was significantly reduced. In vitro experiments showed that AITC promoted the proliferation of intracellular T. gondii accompanied by the inhibition of IL-4, IL-1β, and IL-6 production in RAW264.7 macrophages. Our results showed that AITC facilitated T. gondii infection in the early stage by inhibiting the production of several inflammatory cytokines.

This study aimed to investigate the effects of nanoparticles PLGA-NPs and mesoporous silicon nanoparticles(MSNs) of different stiffness before and after combination with menthol or curcumol on the mechanical properties of bEnd.3 cells. The particle size distributions of PLGA-NPs and MSNs were measured by Malvern particle size analyzer, and the stiffness of the two nanoparticles was quantified by atomic force microscopy(AFM). The bEnd.3 cells were cultured in vitro, and the cell surface morphology, roughness, and Young's modulus were examined to characterize the roughness and stiffness of the cell surface. The changes in the mechanical properties of the cells were observed by AFM, and the structure and expression of cytoskeletal F-actin were observed by a laser-scanning confocal microscope. The results showed that both nanoparticles had good dispersion. The particle size of PLGA-NPs was(98.77±2.04) nm, the PDI was(0.140±0.030), and Young's modulus value was(104.717±8.475) MPa. The particle size of MSNs was(97.47±3.92) nm, the PDI was(0.380±0.016), and Young's modulus value was(306.019±8.822) MPa. The stiffness of PLGA-NPs was significantly lower than that of MSNs. After bEnd.3 cells were treated by PLGA-NPs and MSNs separately, the cells showed fine pores on the cell surface, increased roughness, decreased Young's modulus, blurred and broken F-actin bands, and reduced mean gray value. Compared with PLGA-NPs alone, PLGA-NPs combined with menthol or curcumol could allow deepened and densely distributed surface pores of bEnd.3 cells, increase roughness, reduce Young's modulus, aggravate F-actin band breakage, and diminish mean gray value. Compared with MSNs alone, MSNs combined with menthol could allow deepened and densely distributed surface pores of bEnd.3 cells, increase roughness, reduce Young's modulus, aggravate F-actin band breakage, and diminish mean gray value, while no significant difference was observed in combination with curcumol. Therefore, it is inferred that the aromatic components can increase the intracellular uptake and transport of nanoparticles by altering the biomechanical properties of bEnd.3 cells.
Animals ; Mice ; Menthol/pharmacology* ; Actins/metabolism* ; Endothelial Cells/metabolism* ; Nanoparticles/chemistry*

By preparing 15 batches of substance benchmarks of Taohong Siwu Decoction, the methodology of the characteristic spectrums of substance benchmarks was established. The paste-forming rate range, the contents and the transfer rate range of the index components, hydroxy safflower yellow A, ferulic acid and paeoniflorin, the characteristic peaks and the similarity range of the characteristic spectrums of Taohong Siwu Decoction were determined to define key quality attributes of substance benchmarks of Taohong Siwu Decoction.In the 15 batches of substance benchmarks of Taohong Siwu Decoction, the similarity of characteristic spectrums was higher than 0.9. Furthermore, based on summarization of the characteristic peak information, there were 13 characteristic peaks in the whole decoction. Baishao had three characteristic peaks, Honghua had seven characteristic peaks, and Chuanxiong and Danggui had three characteristic peaks. The paste-forming rate of the 15 batches of substance benchmarks was controlled at 33.11%-40.62%. The content of hydroxy safflower yellow A was 0.129%-0.203%, with the average transfer rate of 16.596%±0.669%.The content of ferulic acid was 0.043%-0.055%, with the average transfer rate of 20.489%±1.772%.The content of paeoniflorin was 0.676%-0.943%, with the average transfer rate of 29.112%±3.273%.The quality value transfer of substance benchmarks of classical prescription Taohong Siwu Decoction was analyzed by the combination of characteristic spectrums, paste-forming rate and the content of index components. The established substance benchmark quality evaluation method was stable and feasible, and could provide a basis for quality control and subsequent development of relevant preparations of Taohong Siwu Decoction.
Benchmarking ; Drugs, Chinese Herbal ; Quality Control

Hematoma/etiology* ; Humans ; Percutaneous Coronary Intervention/adverse effects* ; Retroperitoneal Space

Objective:A strong antithrombotic protein component， named PvQ， was purified and enriched from total protein of Pheretima vulgaris， a traditional Chinese medicine. Moreover， we evaluated its fibrinolytic and anticoagulant activity， and expected to provide reference for the research on antithrombotic substances of Pheretima. Method:A rapid in vitro activity-oriented separation combined with the AKTA-Pure protein purification system conducted on P. vulgaris. Meanwhile， the fibrinolytic and anticoagulant activities of PvQ were measured by fibrin plate method and fibrinogen-thrombin time （Fibg-TT） method. And the in vitro thrombolysis assay was used for evaluating the lysis ability of PvQ to thrombus. Then the stability of PvQ was also analyzed for its anticoagulant activity at different pH and temperature. Result:The PvQ was successfully enriched and its activity was determined to have significant fibrinolytic and anticoagulant activities. And the result of in vitro thrombolysis assay revealed that PvQ could hydrolyze more than 80% of thrombus after 5 h of incubation at 37 ℃. In addition， the changes of temperature and pH had significant effects on antithrombotic activity， and this study showed that PvQ was rapidly inactivated at ≥60 ℃ or in acidic conditions （pH<7）. While， the activity of PvQ was unaffected or less affected at ≤50 ℃ and under alkaline conditions. Conclusion:A feasible preparation method of PvQ is established， and it can affect fibrin and fibrinogen at the same time， thus exerting a dual fibrinolytic effect and possessing significant fibrinolytic and anticoagulant activities. It provides a scientific interpretation for the treatment of thrombotic diseases by PvQ and a reference for the development of antithrombotic protein products of Pheretima.