Intestinal Absorption of Fibrinolytic and Proteolytic Lumbrokinase Extracted from Earthworm, Eisenia andrei

Abstract

To investigate the intestinal absorption of a fibrinolytic and proteolytic lumbrokinase extracted from Eisenia andrei, we used rat everted gut sacs and an in situ closed-loop recirculation method. We extracted lumbrokinase from Eisenia andrei, and then raised polyclonal antibody against lumbrokinase. Fibrinolytic activity and proteolytic activity in the serosal side of rat everted gut sacs incubated with lumbrokinase showed dose- and time-dependent patterns. Immunological results obtained by western blotting serosal side solution using rat everted gut sacs method showed that lumbrokinase proteins between 33.6 and 54.7 kDa are absorbed mostly by the intestinal epithelium. Furthermore, MALDI-TOF mass spectrometric analysis of plasma fractions obtained by in situ recirculation method confirmed that lumbrokinase F1 is absorbed into blood. These results support the notion that lumbrokinase can be absorbed from mucosal lumen into blood by oral administration.

Keywords: Eisenia andrei; Fibrinolytic enzymes; Gut sac; Intestinal proteinase absorption; Recirculation intestinal perfusion.

Figures

Fig. 1

Measurement of fibrinolytic (A) and proteolytic activities (B). The lumbrokinase solutions (0.5, 1.0, and 2.0 mg/ml) were added to Krebs solution on the mucosal sides of small intestine segments. To detect mucosal-to-serosal lumbrokinase transport, aliquots of serosal medium were taken at different times (30, 60, and 120 min, respectively) during incubation, and their fibrinolytic and proteolytic activities were measured. Data are means±SEs from 5 rats. #p<0.01, ##p<0.001 vs. the value obtained with lumbrokinase solution (0.5 mg/ml) at 60 min, **p<0.001 vs. the value obtained with lumbrokinase solution (0.5 mg/ml) at 120 min, ++p<0.001 vs. control.

Fig. 2

Immunoblot and zymogram analyses. Western blotting (left panel) using lumbrokinase polyclonal antibody and fibrin zymography (right panel) using fibrin incorporated in gel were carried out (lane 1: 20 µg of lumbrokinase, lane 2: serosal medium incubated without lumbrokinase for 120 min, lanes 3~5: serosal media incubated with 2 mg/ml lumbrokinase for 30, 60, and 120 min, respectively). Molecular weight standard markers were applied to the left of lane1 in each panel. The blots shown are representative of experiments performed in triplicate.

Fig. 3

MALDI-TOF Mass Spectrometry. Lumbrokinase F1 was separated from plasma obtained by in situ recirculation method, and subjected to 15% SDS-PAGE (Fig. 3C, lane 1-lumbrokinase F1 standard, lane 2: blank plasma without lumbrokinase F1, lane 3: plasma with lumbrokinase F1 for 1 h). The band corresponding to the molecular weight of lumbrokinase F1 was excised and eluted, and the concentrated protein was treated with trypsin. Tryptic digests were analyzed by MALDI-TOF mass spectrometry (Fig. 3A: the spectrum of lumbrokinase F1 (lane 1 of Fig. 3C); Fig. 3B: the spectrum of the plasma obtained from rats with lumbrokinase F1 (lane 3 of Fig. 3C).