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

Figure 3

Lumbrokinase inhibits the migration and adhesive ability of fibroblast cells. (a) IMR-90 cells were seeded at a density of 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 ( 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 ( vs. control (0 U/ml); ^# 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.

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