Anti-inflammatory activities of amber extract in lipopolysaccharide-induced RAW 264.7 macrophages

Amber is a type of fossil tree resin with several bioactive properties and has been traced in traditional medicines used in Russia and China. However, its anti-inflammatory activities are poorly characterized. Here, the anti-inflammatory effects of the extract of amber mined from Kaliningrad, Russia was investigated in lipopolysaccharide (LPS)-induced RAW 264.7 cells. The effect of the amber extract on cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Further, its effects on the production of intracellular reactive oxygen species (ROS), NO, and inflammatory cytokines were assessed by 2',7'-dichlorodihydrofluorescein diacetate staining, Griess test, and cytokine enzyme-linked immunosorbent assays, respectively. Western blotting and real-time reverse transcription-polymerase chain reaction analysis were performed to assess the mRNA and protein expression levels of the inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). The translocation of the nuclear factor-kappa B (NF-kappa B) p65 subunit was observed by immunofluorescent staining. Amber extract negatively regulated the LPS-induced differentiation of RAW 264.7 cells to dendritic-like cells and reduced the LPS-induced increase in ROS and NO levels. It also reduced the level of mRNA and protein expressions of TNF-alpha, IL-6, COX-2, and iNOS in LPS-induced RAW 264.7 macrophages, in a dose-dependent manner. Furthermore, amber extract suppressed the nuclear translocation of the NF-kappa B p65 subunit. These findings suggest that the potent anti-inflammatory effect of the amber extract is mediated by the inhibition of the NF-kappa B p65 signaling pathway. Collectively, this study renders amber extract as a potential pharmacological alternative to treat inflammation-related diseases.