Lumbrokinase, a Fibrinolytic Enzyme, Prevents Intra-Abdominal Adhesion by Inhibiting the Migrative and Adhesive Activities of Fibroblast via Attenuation of the AP-1/ICAM-1 Signaling Pathway

Abstract

Intra-abdominal adhesion is a complication following abdominal surgery caused by the suppression of fibrinolytic activity and aggravated fibroblast invasion of the injured area, which may lead to chronic illnesses such as chronic pain, intestinal obstruction, and female infertility. This study hypothesized that lumbrokinase, a fibrinolytic enzyme extracted from the earthworm, supports the wound healing process. Therefore, we assessed the effect of lumbrokinase on intra-abdominal adhesion. Lumbrokinase treatment significantly decreased the severity and the area of intra-abdominal adhesion in vivo in a dose-dependent manner compared with the controls (untreated and hyaluronate-treated). Lumbrokinase-associated adverse effects were not observed. Immunohistochemical analysis of adhesion tissues revealed a loosened adhesive band between tissues, coupled with significantly decreased peritoneal thickening in the lumbrokinase-treated group versus the control group. Three-dimensional spheroid, MTT, and scratch wound migration assays using the IMR-90 human fibroblast cell line demonstrated that lumbrokinase significantly attenuated the migration and adhesive activity of fibroblasts without compromising cell proliferation. The luciferase assay and western blot analysis showed that lumbrokinase inhibited the AP-1/ICAM-1 cell adhesion signaling pathway. Therefore, lumbrokinase decreases intra-abdominal adhesion and peritoneal thickening by augmenting fibrinolytic action and inhibiting fibroblast migration and adhesive activity via attenuation of the AP-1/ICAM-1 signaling pathway. Lumbrokinase is thus a promising agent to prevent intra-abdominal adhesion.

Figure 1

Loosening of adhesion in the central area. Hematoxylin and eosin (H&E) and Masson's trichrome (MT) (40x) staining were performed in the tissue samples obtained after two weeks of lumbrokinase application. Masson's trichrome stain showed fibrosis (blue-stained), which represented the adhesion site between the cecum and abdominal wall. The arrows indicate fibrosis of the peritoneal wall side. Scale bar, 100 μm.

Figure 2

Peritoneal thickness in normal and injured areas. (a) After one week of injury, peritoneum tissue from noninjured and injured areas was obtained and stained with hematoxylin and eosin (H&E). The reduction in peritoneal thickness occurred in a dose-dependent manner. Scale bar, 100 μm. A monolayer of mesothelial cells was observed in the normal peritoneum from the noninjured peritoneum (arrow). The peritoneum of the injured area was thickened in the nontreated group, whereas peritoneal thickness was significantly reduced in the lumbrokinase-treated groups. (b) Quantification of the peritoneal thickness. The thickness measurement is the average obtained at four different sites in each rat based on image analysis with the ImageJ software (p value for the indicated groups compared with the control).

Figure 3

Lumbrokinase inhibits the migration and adhesive ability of fibroblast cells. (a) IMR-90 cells were seeded at a density of 1 × 10⁴ cells/well of a 96-well plate. One day after seeding, the cells were treated with the indicated concentrations of lumbrokinase. After treatment for 24, 48, and 72 h, cell viability was measured by the MTT assay and cell viability ratios were calculated as the ratio of the OD540 value of the lumbrokinase treatment groups relative to the OD540 value of the untreated control (0 U/mL) following each time point. The experiment was performed in triplicate and repeated at least three times. (b) IMR-90 cells (5 × 10⁵ cells/mL) were loaded in the microwell array. The cells aggregated after 20 min of seeding, which was indicative of spheroid formation. One day after seeding, the cells were treated with lumbrokinase every three days. The viability of cell spheroids was measured 14 days later using the MTT assay after preparing a single-cell suspension (left panel, ns, not significant). The shape of the spheroids from lumbrokinase-treated cells was distorted, whereas the control spheroids were circular in shape (right panel). (c) The effect of lumbrokinase on fibroblast cell motility was evaluated by the scratch wound migration assay. After lumbrokinase treatment at the indicated concentration for 24 h, migrated cells were counted based on images generated with the ImageJ software (^∗∗p < 0.01 vs. control (0 U/ml); ^#p < 0.01 indicates a significant difference between the 1,000 and 2,000 U/mL lumbrokinase-treated groups). (d) Schematic diagram of the experimental protocol to assess fibroblast adhesive ability under lumbrokinase treatment. (e) Three-dimensional spheroids were made for 14 days after lumbrokinase treatment. Photos were taken under light microscopy. The spheroids were separated into single cells, after which these cells were recultured in a two-dimensional 6-well culture plate. The number of attached cells was counted under a light microscope on day 14.

Figure 4

Lumbrokinase inhibits the AP-1/ICAM-1 signaling pathway. (a) IMR-90 cells (1 × 10⁵) were seeded into a 24-well culture plate. One day after incubation, the cells were treated with lumbrokinase (2,000 U/mL) for two days. The cells were then transfected with reporter plasmids containing the promoter of AP-1, β-catenin (OT), ERK (ARE), and NF-κB. The cells were harvested two days after transfection, and the transcriptional activity was measured using a dual-luciferase reporter assay kit. ^∗∗p < 0.01 and ^∗p < 0.05. (b) 5 × 10⁵ IMR-90 cells were seeded in 6-well plates, then treated with the indicated concentration of lumbrokinase after 24 h. After 48 h of incubation, the cells were harvested for western blot analysis. (c) 5 × 10⁵ IMR-90 cells were incubated with or without lumbrokinase (2,000 U/ml) in the presence and absence of phorbol 12-myristate 13-acetate (PMA), an AP-1 enhancer, for 48 h. Then, the cells were harvested and subjected to western blotting. The graph was plotted based on the band densities measured using the ImageJ software. ^∗∗p < 0.01 and ^∗p < 0.05. (d) 5 × 10⁵ IMR-90 cells were plated and treated with lumbrokinase (2,000 U/mL) and T-5224 (an AP-1 inhibitor) 24 h later. The cells were harvested after 48 h of incubation in their respective, after which western blotting was performed. The graph was plotted based on the band densities measured using the ImageJ software. ^∗∗ p < 0.01 and ^∗p < 0.05.