Abstract
BACKGROUND: Periodontitis (PD) is a highly prevalent inflammatory disease associated with complex microbial infection in the subgingival cavity. We have previously shown that polybacterial periodontal infection led to aortic atherosclerosis and lipid profile modulation; however, the underlying mechanism(s) has not been yet demonstrated. METHODS: Primary human aortic endothelial cells (pHAECs) were infected periodically for 48 hours either with P. gingivalis (monobacterial infection) or polybacterial periodontal pathogens, P. gingivalis (Pg), T. forsythia (Tf), T. denticola (Td), and F. nucleatum (Fn), using HoxBan coculture technique. Cell viability was assessed using MTT assay. Nitric oxide synthesis was measured in the form of total nitrite released into the media after incubation period. Inflammatory and oxidative gene expression were evaluated in the cell lysate using quantitative real-time polymerase chain reaction (qRT-PCR) at each time point (12-48h). Statistical analysis was performed using two-way ANOVA. RESULTS: pHAEC cell viability was significantly (p<0.05) reduced in both mono and poly-bacterial infection when compared with non-infected cells in a time dependent manner. Nitrite levels in the media were significantly impaired in both infection groups. mRNA expression for cytokines (TLR-4, IL-6, and TNF-α,) and inducible nitric oxide synthase (iNOS) were significantly (p<0.05) elevated in both experimental groups. In contrast, endothelial (e) NOS, tetrahydrobiopterin (BH(4)) synthesis, GCH -1, Nrf2, and Nqo 1 were significantly (p<0.05) reduced in both experimental groups. Finally, polybacterial infection group showed greater changes in cell viability, nitrite levels, cytokines, eNOS/BH(4)/Nrf2 expression in a time dependent manner. CONCLUSIONS: Our study highlights the adverse effects of in vitro PD infection in NO mediated vascular endothelial cell function.