Anti-Porphyromonas gingivalis and anti-inflammatory activities of A-type cranberry proanthocyanidins

Abstract

A-type cranberry proanthocyanidins (AC-PACs) have recently been reported to be beneficial for human health, especially urinary tract health. The effect of these proanthocyanidins on periodontitis, a destructive disease of tooth-supporting tissues, needs to be investigated. The purpose of this study was to investigate the effects of AC-PACs on various virulence determinants of Porphyromonas gingivalis as well as on the inflammatory response of oral epithelial cells stimulated by this periodontopathogen. We examined the effects of AC-PACs on P. gingivalis growth and biofilm formation, adherence to human oral epithelial cells and protein-coated surfaces, collagenase activity, and invasiveness. We also tested the ability of AC-PACs to modulate the P. gingivalis-induced inflammatory response by human oral epithelial cells. Our results showed that while AC-PACs neutralized all the virulence properties of P. gingivalis in a dose-dependent fashion, they did not interfere with growth. They also inhibited the secretion of interleukin-8 (IL-8) and chemokine (C-C motif) ligand 5 (CCL5) but did not affect the secretion of IL-6 by epithelial cells stimulated with P. gingivalis. This anti-inflammatory effect was associated with reduced activation of the nuclear factor-kappaB (NF-kappaB) p65 pathway. AC-PACs may be potentially valuable bioactive molecules for the development of new strategies to treat and prevent P. gingivalis-associated periodontal diseases.

FIG. 1.

¹³C nuclear magnetic resonance (NMR) spectrum of cranberry proanthocyanidins, showing the presence of A-type linkages.

FIG. 2.

Effect of AC-PACs on the growth of and biofilm formation by P. gingivalis. AC-PACs (0, 25, 50, and 100 μg/ml) were added to the growth medium, and the cultures were incubated under anaerobic conditions at 37°C for 24 h. Bacterial growth was assessed by measuring the OD₆₅₅ using a microplate reader. Biofilm formation was assessed by staining with 0.4% crystal violet and measuring the A₅₅₀. Assays were run in quadruplicate, and the means ± SD from three independent assays were calculated. A value of 100% was assigned to growth and biofilm formation in the absence of AC-PACs. *, significantly lower than the value for the control (no AC-PACs) (P < 0.05).

FIG. 3.

Effect of AC-PACs on the adherence of P. gingivalis to human oral epithelial cells and a Matrigel-coated polystyrene surface. ¹⁴C-labeled P. gingivalis cells in the presence of AC-PACs (0, 25, 50, and 100 μg/ml) were added to wells coated with epithelial cells or Matrigel. After 30 min of incubation at 37°C, the wells were extensively washed and the quantity of adhered ¹⁴C-labeled bacteria was determined by gamma counting using a multipurpose scintillation counter. Assays were run in triplicate, and the means ± SD from three independent assays were calculated. A value of 100% was assigned to the amount of ¹⁴C bound in the absence of AC-PACs. *, significantly lower than the value for the untreated control (P < 0.05).

FIG. 4.

Effect of AC-PACs on the degradation of type I collagen by extracellular proteases of P. gingivalis. Assays were run in quadruplicate, and the means ± SD from three independent assays were calculated. A value of 100% was assigned to the degradation obtained after a 4-h incubation at room temperature in the absence of AC-PACs (A). The inhibition of collagen degradation was also measured as function of time (B). *, significantly lower than the value for the untreated control (P < 0.05).

FIG. 5.

Effect of AC-PACs on the migration of P. gingivalis through a reconstituted basement membrane model (Matrigel). ¹⁴C-labeled P. gingivalis cells were preincubated with AC-PACs (0, 25, 50, and 100 μg/ml) for 30 min. They were then placed on the Matrigel in the top of a double-chamber system. After a 48-h incubation at 37°C under anaerobic conditions, the number of ¹⁴C-labeled P. gingivalis organisms recovered in the lower chamber was determined by gamma counting. Assays were run in triplicate, and the means ± SD from three independent assays were calculated. A value of 100% was assigned to the amount of radioactivity counted in the absence of AC-PACs. *, significantly lower than the value for the untreated control (P < 0.05).

FIG. 6.

Effect of AC-PACs on the P. gingivalis-induced inflammatory response in oral epithelial cells. Oral epithelial cells were pretreated with AC-PACs (0, 25, 50, and 100 μg/ml) for 2 h (1 h for the NF-κB p65 assay). They were then stimulated with P. gingivalis at an MOI of 125 for 24 h (30 min for the NF-κB p65 assay). Cell-free supernatants were collected to determine IL-6, IL-8, and CCL5 concentrations by ELISA (A). The activation of NF-κB p65 in the cellular extract was determined by an ELISA-based assay (B). Assays were run in triplicate, and the means ± SD from three independent assays were calculated. *, significantly higher than the value for the unstimulated (P. gingivalis) control (P < 0.05); †, significantly lower than the value for the untreated (AC-PACs) control (P < 0.05).