Anti-inflammatory and anti-atherosclerosis mechanisms of Neovestitol, from Brazilian red propolis

Method: neovestitol was obtained from ethanolic extract of Brazilian red propolis after bio-guided fractionation coupled with GC-MS analysis. Different concentrations of Neovestitol were added to RAW 264.7 macrophages after activation with LPS. After 48 hours, NO production and cell viability were determined. Production of TNF-α, IL1β, TGF-β, IL-4, IL-6, IL-10, IL-12, GM-CSF, IFN-Ɣ and expression of genes related to cytokines production, nitric oxide , PI3K-AKT and signal transduction pathways were evaluated by ELISA and RT-qPCR, respectively.  Differences were determined by one-way ANOVA followed by Tukey-Kramer

Result: Neovestitol inhibited NO production by 63% without affecting cell viability at the concentration of 60 µg/ml when compared with control cells (treated with vehicle) (p<0.05).  Levels of GM-CSF, IFN- Ɣ, IL-1 β, IL-4, TNF- α and IL-6 in cell supernatant were reduced and of  IL-10 increased with the addition of neovestitol (p<0.05). Neovestitol induced alteration in the expression of 43 genes. Genes involved in Toll-like response;  nitric oxide sintetase production (all types), Nf-KB and Il-1 signaling pathways such as Bad, Chuk, Elk1, Ifnb1,  Il1b, Lif, Calm1, Capns1, Egr1, Nox1, Scd2 and Scd3 were negatively regulated  by neovestitol (fold-change rate > 5; p<0.05). Furthermore, its role in inhibition of leukocyte transmigration was suggested by inhibition of expression of Nox1 and in the regulation of fatty acid associated genes such as Scd2 e Scd3 

Conclusion: Neovestitol may represent a new antiinflammatory agent by inhibiting signaling pathways and consequently reducing the production of pro-inflammatory factors and by interfering in leukocyte transmigration