Effect of smokers'sera on Porphyromonas gingivalis internalizing KB cells and the expression of matrix metalloproteinase-1, -9 and tissue inhibitor of metalloproteinase-1

Abstract

To investigate the effects of serum from smoking individuals or non-smoking individuals with periodontitis on Porphyromonas gingivalis (Pg) internalizing KB cells, and the expression of matrix metalloproteinase(MMP)-1, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) in the culture supernatant of KB cells. The venous blood of 20 periodontitis patients' (10 smoking and 10 non-smoking) was extracted under the informed consent and centrifuged for serum. The smoking-individual serum (Y group) and non-smoking-individual (N group) serum were added to the model of Pg internalizing KB cells for 12 hours, plated on brain-heart infusion (BHI) and incubated anaerobically at 37 °C for 5 days. The colony forming units (CFU) of cell-invasive bacteria were estimated by colony counting. MMP-1, MMP-9 and TIMP-1 protein levels in culture supernatant were determined by enzyme-linked immunosorbent assay(ELISA) in the two groups following co-culture of Pg with KB cells for 12 hours. The CFU were (11.2 ± 1.1)×10(4), (12.6 ± 1.2)×10(4), (44.7 ± 1.3)×10(4) CFU/ml when adding 200, 400, 800 µl Y-group serum to the model of Pg co-culture with KB cells and when the serum was extracted from N group, the CFU were (33.6 ± 1.4)×10(4),(38.9 ± 1.1)×10(4), (11.2 ± 1.2)×10(4) CFU/ml respectively. When 200, 400, 800 µl Y group-serum was added to co-culture fluid of Pg internalizing KB cells, the concentrations of MMP-1 secreted from KB cells were (107.2 ± 21.5), (165.9 ± 20.2), (434.4 ± 48.0) µg/L respectively, the concentrations of MMP-9 were (3.99 ± 0.29), (4.21 ± 0.61), (5.62 ± 0.47) µg/L respectively, the concentrations of TIMP-1 were (401.3 ± 12.7), (418.3 ± 28.5), (637.3 ± 37.3) µg/L. When the serum (200, 400, 800 µl) extracted from N group, the concentration of MMP-1 and MMP-9 secreted by KB cell were (77.6 ± 10.8), (84.7 ± 10.2) and (98.2 ± 9.7) µg/L and (3.84 ± 0.52), (4.02 ± 0.68), (4.25 ± 0.37) µg/L, respectively. The concentration of TIMP-1 were (67.3 ± 26.9) , (89.4 ± 22.7) and (78.2 ± 16.5) µg/L secreted by KB cells in the course of Pg internalized KB cell. With the increasing of Y group-serum, the more MMP-1, MMP-9 and TIMP-1 were secreted by KB cells(P < 0.05). When 800 µl Y group-serum was added compared with N group-serum to the Pg co-culture with KB model, the more MMP-1, MMP-9 and TIMP-1 were secreted by KB cells(P < 0.05), when 400 µl Y group-serum was added compared with N group-serum to the Pg co-culture with KB model, the more MMP-1 and TIMP-1 were secreted by KB cells (P < 0.05). The smoking-serum might enhance Pg internalizing KB cells and enhance the expression of MMP-1, MMP-9 and TIMP-1 secreted from KB cells. The local microenvironment of smoking individual may contribute to the recurrence and progression of chronic periodontitis.