Abstract:
Periodontitis is initiated by specific groups of Gram-negative bacteria. Among them, P. gingivalis and A. actinomycetemcomitans have been strongly supported as etiological agents to induce host responses leading to destruction of periodontium. Therefore, the present study attempted to investigate the role of P. gingivalis and A. actinomycetemcomitans product on MMP-2 activation and RANKL-OPG expression in human periodontal ligament (HPDL) cells. The first part of the study was to determine the effect of P. gingivalis and A. actinomycetemcomitans supernatant on MMP-2 activation and its mechanism. Gelatin zymography, RT-PCR and Western analysis were used to detect the activation of MMP-2, expression of MT1-MMP and TIMP-2 mRNA and protein, respectively. The results showed that P. gingivalis and A. actinomycetemcomitans supernatant could activate MMP-2 in HPDL cells. Phenanthroline, an MMP inhibitor, could block the effect of supernatant from both bacteria on MMP-2 activation. In addition, A. actinomycetemcomitans supernatant induced the decreased amount of TIMP-2 protein while P. gingivalis supernatant up-regulated MT1-MMP both at mRNA and protein level. These results suggested that the MMP-2 activation by P. gingivalis and A. actinomycetemcomitans supernatant occurred via MMP-dependent pathway. The second part focuses on the effect of A. actinomycetemcomitans lipopolysaccharide (LPS) on both MMP-2 activation and the expression of RANKL and OPG. The results revealed that A. actinomycetemcomitans LPS could activate MMP-2 but in a different mechanism from the effect of A. actinomycetemcomitans supernatant, since Phenanthroline could not abolish this effect. In addition, serine protease did completely. A. actinomycetemcomitans LPS also up-regulated RANKL expression both at mRNA and protein levels while OPG mRNA level did not change. Additionally, indomethacin and serine protease inhibitor could abrogate this effect. These results suggested the activity of serine protease of A. actinomycetemcomitans LPS induced MMP-2 activation and up-regulated RANKL expression. In conclusion, the present study demonstrated the roles of P. gingivalis and A. actinomycetemcomitans virulences on periodontal tissue destruction represented here by MMP-2 activation and RANKL up-regulation.
