Statins as Modulators of mPDL Response to Inflammatory Cytokines

Objectives: Statins inhibit HMG-CoA reductase which is the rate-limiting enzyme in cholesterol biosynthesis and are prescribed for the treatment of hypercholesterolemia. Statins also have immunomodulatory effects and are potent inhibitors of osteoclastogenesis. Since the extent of periodontal destruction is based on the host's inflammatory response, we hypothesized that statins decrease periodontal disease progression by modulating iNOS (inducible nitric oxide synthase) and COX2 (cyclooxygenase-2) expression, two enzymes implicated in the host response to periodontal pathogens. Methods: To determine the appropriate experimental dose, SV-40 transformed mouse periodontal ligament cells were stimulated with concentrations of simvastatin ranging from 1-500nM and apoptosis was assessed by Western blotting using an antibody to cleaved PARP (Asp214). Cells were pre-treated for 5h with 250nM simvastatin before stimulation with the following cytokines for an additional 18h: TNF-α (20ng/ml), IL-1β (1ng/ml), IFN-γ (102 IU/ml). To assess nitric oxide release, media was analyzed via the Griess assay. Western blotting was used to detect iNOS and COX-2 expression. For transcriptional assays, cells were transfected with an NF-kB-responsive promoter-reporter construct, stimulated 24h post transfection as detailed above and samples were processed for dual luciferase assays. Results: Apoptosis was observed at a 500nM concentration of simvastatin. Since the clinically relevant serum concentration in patients taking simvastatin is 300nM, we used 250nM for all experiments. Inflammatory cytokines induced iNOS and COX-2 expression and simvastatin potentiated this effect. Consistent with these findings, NO release in the media of cells stimulated with cytokines was increased in the presence of simvastatin. NF-kB activation, which is responsible for both iNOS and COX-2 transcriptional control, was also increased in the presence of simvastatin. Conclusion: These preliminary findings do not support a role for statin therapy in decreasing the inflammatory response of PDL cells. Further experiments using primary mouse and human PDL cells are necessary to confirm this finding.