Objectives: Co-expression of cysteine cathepsins (CTs) and matrix metalloproteinases (MMPs) in dentin-pulp complex has been recently shown (Tersariol et al., 2010). Chlorhexidine (CHX) demonstrated to be an efficient inhibitor of MMPs activity. This study aimed to investigate whether CHX can also inhibit CTs.
Methods: Recombinant enzymes (Cathepsin B – CT-B and Cathepsin L – CT-L) were used in this study. The inhibitory potential of CHX on CT-B and CT-L was spectrofluorometrically evaluated (Hitachi F-2500) by using a specific fluorgenic substrate (Z-FR-MCA, Sigma). The enzymes (5 µM) and substrate (10 µM) were incubated in assay buffer (pH 6.3) containing different concentrations of CHX: 0 (negative control) to 1 wt%. The excitation and emission wavelengths for MCA substrate were, respectively, 380 and 460 nm. The inhibitory profile of CHX was monitored by measuring the substrate hydrolysis under time course scan and the inhibitory constants were calculated by linear regression. Data were analyzed by ANOVA and Holm-Sidak tests.
Results: Cysteine CTs activities were efficiently inhibited by different concentrations of CHX. CT-B had 50% of its activity inhibited with 0.01% CHX, while CT-L had 50% of its activity inhibited with 0.001% CHX.
Conclusion: The inhibitory activity of CHX on cysteine cathepsins was successfully demonstrated by a fluorimetric substrate assay. The present results suggest that the well-demonstrated, protective effect of CHX on preserving the integrity of dentin collagen may be not only due to the inhibition of matrix metalloproteinases, but also to the inhibition of cathepsins.
Funding: FAPESP 07/54618-4 (P.I. Carrilho)