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

Objectives: Hypoxia-inducible factor-1α (HIF-1α) 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-κB protein complex pathway. The objective of this study was to investigate cytotoxicity and HIF-1α expression through the NF-κB 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 (30ng/mL) and Porphyromonas gingivalis lipopolysaccharide (1μg/mL). Cells were treated with various concentrations of velutin (0.5-2μM) to check for viability, morphology, osteoclast differentiation, and HIF-1α 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-1α expression related to IκB 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-1α through the NF-κB pathway.