FIGURE 3.

Inhibitory activity of HEF against gingipain. (A,B) Inhibitory effects of HEF against enzymatic activities of (A) Rgps and (B) Kgp. P. gingivalis wild-type strain culture supernatant was preincubated with HEF at various concentrations, then substrates for Rgps (A) and Kgp (B) were incubated with the culture supernatant that contains both Rgps and Kgp. Residual enzyme activity was measured. The value for enzymatic activity in the vehicle control (absence of HEF) was considered to be 100%. Data are expressed as the mean ± SD from results obtained in three independent experiments. *p ≤ 0.05 vs. vehicle control. Statistical analysis was performed using ANOVA and Tukey’s multiple comparison test. (C,D) Ca9-22 cell detachment assay (C) Ca9-22 cells were incubated with culture supernatants of P. gingivalis wild-type, gingipain triple mutant, or without culture supernatant. (D) Ca9-22 cells were incubated with culture supernatant of P. gingivalis wild-type without or with preincubation with different concentrations of HEF at 20, 40, 80, 160, and 320 μg/mL. Bars: 30 μm.