Introduction: We assessed the influences of NCX 2121, a nitric oxide (NO)-releasing derivative of indomethacin, upon the generation of proinflammatory mediators using murine macrophages stimulated with LPS prepared from Prevotella intermedia, which is one of the pathogens implicated in periodontal diseases.
Methods: LPS from P. intermedia strain ATCC 25611 was prepared employing the conventional phenol-water procedure. Conditioned culture media were analyzed for the levels of NO, interleukin-1β (IL-1β) and IL-6. Real-time PCR analysis was carried out to determine the mRNA levels of inducible NO synthase (iNOS), IL-1β, IL-6, HO-1 and SOCS1. Protein expression levels were evaluated by immunoblot test. NF-κB-dependent SEAP reporter assay was performed using a reporter cell line. DNA-binding activities of NF-κB subunits were analyzed utilizing the ELISA-based kits.
Results: iNOS-derived NO, IL-1β and IL-6 as well as their relevant mRNA were significantly attenuated by NCX 2121 in RAW264.7 cells activated by P. intermedia LPS. Importantly, NCX 2121 showed inhibitory activities superior to its parent compound indomethacin. NCX 2121 triggered induction of HO-1 in cells exposed to P. intermedia LPS, and its inhibitory influence upon P. intermedia LPS-elicited NO generation was notably blocked by SnPP treatment. NCX 2121 attenuated NF-κB-dependent SEAP release induced by P. intermedia LPS. NCX 2121 did not display significant inhibitory action towards IκB-α degradation triggered by LPS. Instead, it significantly diminished nuclear translocation as well as DNA-binding action of NF-κB p50 subunit elicited by P. intermedia LPS. Further, NCX 2121 significantly up-regulated SOCS1 mRNA expression in cells challenged with P. intermedia LPS. NCX 2121 had no influences upon phosphorylation levels of p38, JNK and STAT1/3.
Conclusions: NCX 2121 may have potential benefits as a host immunomodulatory agent for the therapy of periodontal disease, though preclinical and clinical studies are needed to confirm this.