Porphyromonas gingivalis lipopolysaccharides affect gingival stem/progenitor cells attributes through NF-κB, but not Wnt/β-catenin, pathway

Abstract

Aim: This study investigates for the first time the effect of Porphyromonas gingivalis lipopolysaccharides (Pg-LPS) on proliferative/regenerative aptitudes of gingival stem/progenitor cells (G-MSCs).

Material and methods: G-MSCs (n = 5) were treated by 0, 10 ng/ml, 100 ng/ml, 1 μg/ml or 10 μg/ml Pg-LPS. At 1 hour, Toll-like receptor 4 (TLR-4) expression and NF-κB and Wnt/β-catenin signalling pathways were examined. Colony-forming unit assay was conducted at day 12. At 24 and 48 hours, MTT test, ALP activity, mRNA for tumour necrosis factor-α (TNF-α), interleukin-6, collagen-I (Col-I), collagen-III, RUNX-2, alkaline phosphatase (ALP), osteonectin and protein expression of interleukin-6 and TNF-α were analysed.

Results: With increasing Pg-LPS, TLR-4 was upregulated, pNF-κB-p65 rose from median (Q25/Q75) 6.56% (4.19/7.90) to 13.02% (8.90/16.50; p = 0.002) and pNF-κB-p65/tNF-κB-p65 from 0.14(0.10/0.17) to 0.30(0.21/0.42; p = 0.002). pβ-Catenin, tβ-catenin and pβ-catenin/tβ-catenin showed no differences. Increasing Pg-LPS concentration increased cell numbers from 288.00(72.98/484.32) to 861.39 (540.41/1599.94; p = 0.002), ALP mRNA from 0.00(0.00/0.01) to 0.56(0.00/1.90; p = 0.004) and TNF-α from 32.47(12.11/38.57) to 45.32(28.68/48.65; p = 0.036). Over time, ALP activity increased from 0.89(0.78/0.95) to 1.90(1.83/2.09; p < 0.001), mRNA for TNF-α from 0.00(0.00/0.12) to 0.01(0.00/0.06; p = 0.007), mRNA for Col-I from 82.70(0.03/171.50) to 124.00(52.85/232.50; p = 0.019), while mRNA for RUNX-2 decreased from 1.73(0.92/3.20) to 0.84(0.48/1.47; p = 0.005).

Conclusions: Pg-LPS upregulated G-MSCs' proliferation, without attenuation of their regenerative potential. The effects were NF-κB, but not Wnt/β-catenin, pathway dependent.

Keywords: Porphyromonas gingivalis; Lipopolysaccharides; NF-κB; Wnt/β-catenin; stem cells.