Periodontitis is initiated by the accumulation of microbial plaque that activates the gingival inflammation due to overexpression of matrix metalloproteinases (MMPs) and lead to the tissue destruction. Searching for natural agents for management of periodontitis is a recent focus of investigation. Preliminary results demonstrated that Kaempferia pandurata Roxb., a perennial tropical traditional herb, inhibited MMP-9 expression in human oral epithelial cells. Therefore, K. pandurata is thought to be applied for periodontal therapy. In this study, panduratin A was isolated from the rhizome of K. pandurata and investigated its efficacy on inhibition of oral biofilm and MMP expression.
Preventing and reducing effects of panduratin A were tested against multi-species oral biofilms consisting of Streptococcus mutans, Streptococcus sanguinis, and Actinomyces viscosus. Panduratin A showed MIC of 1 µg/ml for multi-species strains. Panduratin A at 2 × MIC for 8 h possed anti-bactericidal activity against multi-species planktonic cells for 8 h. At 8 × MIC, panduratin A could prevent >50% of biofilm formation. The reduction of >50% biofilm mass was reached after exposing panduratin A at 10 µg/ml for 15 min. Panduratin A showed a dose-dependence manner for preventing and reducing effects of biofilm. These results suggest that panduratin A could be applied as a natural anti-biofilm agent to eliminate oral bacterial colonization in early dental plaque formation.
Further study was aimed to elucidate the molecular mechanism by which panduratin A attenuated signaling pathways involved in MMP expression induced by Porphyromonas gingivalis supernatant in human oral epithelial (KB) and gingival fibroblast (HGF-1) cells. In human oral KB cell system, the gelatin zymogram revealed that the main gelatinase secreted by P. gingivalis supernatant-induced KB cells migrated at 92 kDa, representing MMP-9. Exposure of KB cells to P. gingivalis supernatant significantly upregulated the expression of MMP-9 secretion, protein and gene, and also activation of activator protein-1 (AP-1) and nuclear factor-?B (NF-?B) elements, MAPK phosphorylation (extracellular signal-related kinase 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK)), and transcription factors (Elk1, c-Jun, and c-Fos). MMP-9 protein and mRNA levels were significantly decreased after panduratin A treatment. In contrast, panduratin A had no effect on tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 mRNA. Panduratin A was found to strongly suppress the level of phosphorylated JNK and block AP-1 activity in P. gingivalis supernatant-stimulated KB cells. These results suggest JNK and AP-1 are the major signaling for P. gingivalis supernatant-stimulated MMP-9 expression in KB cells, and panduratin A markedly downregulates MMP-9 expression through inhibition of these signaling. In HGF-1 cell system, MMP-2, indicated by a 72-kDa gelatinolytic band on gelatin zymogram profile, was primarily produced, and its secretion was enhanced by P. gingivalis supernatant. Panduratin A significantly attenuated MMP-2 expression at protein and gene levels. Panduratin A interfered p38 and cyclic AMP response element (CRE) binding protein (CREB) signaling pathways involved in MMP-2 expression in P. gingivalis supernatant-stimulated HGF-1 cells.
Instead of its directly inhibition on the MMP-dependent pathway, panduratin A was found to suppress the expression of urokinase-type plasminogen activator (uPA) secretion, protein, and mRNA in both KB and HGF-1 cell systems. These results indicate that panduratin A also indirectly downregulates MMP expression via a plasmin-dependent pathway in the initiation of extracellular matrix degradation.
Furthermore, the effect of panduratin A on attenuating the expression of MMP-9-dependent gingival inflammation was evaluated by conducting in vivo test using rat gingival model. Lipopolysaccharide (LPS) injection induced the elevated MMP-9 and uPA expression. Panduratin A caused a significant decrease on MMP-9 and uPA secretion, protein and mRNA expression in LPS-induced rat gingival inflammation. In line with these results, panduratin A also repaired the inflammatory morphology of gingival tissue, suggesting that panduratin A may improve tissue remodeling in rat gingival inflammation model through inhibition of MMP-9 and uPA expression .
Taken together, these findings suggest that panduratin A possesses anti-biofilm activity by eliminating oral bacterial colonization during early dental plaque formation. Panduratin A controls periodontal inflammation involving MMP-2 and MMP-9 induction in human gingival fibroblasts and oral epithelial cells in vitro. Panduratin A also exerts inhibitory activity against MMP-9 and uPA expression in LPS-induced rat gingival inflammation in vivo. With dual potent activities as anti-biofilm agent and MMP inhibitor, panduratin A could be applied as a promising candidate for periodontal therapy. |