Cytotoxicity and potential anti-inflammatory activity of velutin on RAW 264.7 cell line differentiation: Implications in periodontal bone loss

Objectives: Hypoxia-inducible factor-1 alpha (HIF-1 alpha) has been implicated in periodontal tissue inflammation and possibly in osteoclast differentiation, while polyphenols are known to be anti-inflammatory natural compounds that are capable of regulating the NF-kappa B protein complex pathway. The objective of this study was to investigate cytotoxicity and HIF-1 alpha expression through the NF-kappa B pathway by polyphenol velutin (Euterpe oleracea Mart.), found in the pulp of acai fruit, during inflammatory RAW 264.7 differentiation. Design: RAW 264.7 mouse monocyte macrophage cells were stimulated with RANKL (30 ng/mL) and Porphyromonas gingivalis lipopolysaccharide (1 mu g/mL). Cells were treated with various concentrations of velutin (0.5-2 mu M) to check for viability, morphology, osteoclast differentiation, and HIF-1 alpha expression (Western blot). Results: Alamar blue cell viability assay showed no toxicity to RAW cells with the use of velutin in all concentrations tested (p > 0.05). Velutin did not induce cell apoptosis based on caspase 3/7 assay (p > 0.05). Fluorescence images stained by DAPI showed no alteration in the morphology of RAW cell nuclei (p > 0.05) treated with velutin. TRAP assays demonstrated a dose-dependent reduction in osteoclast formation by velutin when compared with control (p < 0.05). Velutin showed a reduction in HIF-1 alpha expression related to I kappa B phosphorylation when compared with control (p < 0.001). Conclusions: At the tested concentrations, velutin was not cytotoxic to RAW 264.7 and differentiated cells. Velutin reduced osteoclast differentiation-and downregulated HIF-1 alpha through the NF-kappa B pathway.