NO production in RAW264 cells stimulated with Porphyromonas gingivalis extracellular vesicles

Abstract

This experiment was carried out in order to prove the inducible nitric oxide synthase (iNOS) expression and the nitric oxide (NO) production in mouse macrophage cells (RAW264) which were stimulated by vesicles released from Porphyromonas gingivalis, and discussed about the role of vesicles in advance periodontal diseases. Production of NO(2) (-) in RAW264 cells was investigated after 0, 1, 3, 6 and 12h of stimulation with P. gingivalis vesicles. NO was analyzed by HPLC-based flow reactor system with Griess reagent. The cells stained by the enzyme-labeled antibody method, after being stimulated with vesicles for 12h. The iNOS proteins, which were expressed in RAW264 cells after 12h of stimulation with vesicles, were detected by western blot. When stimulated with vesicles from W83 and from ATCC33277, the RAW264 cells produced NO, but cell proteins that came in contact with the vesicles were degraded by protease activities in vesicles. When stimulated with vesicles from gingipain-deficient mutant strain KDP136, the RAW264 cells produced NO, but the quality was about 60%, compared with the vesicles from ATCC33277. The results suggest that vesicles are not only just a part of bacterial component, but also are a toxic complex of lipopolysaccharide and protease, and one of the putative virulence factor for periodontal diseases that continue inflammation and cause chronic conditions.