Anti-inflammatory and anti-nociceptive activities of indigo through regulating IKKβ/IκB/NF-κB pathway in mice

This study was to investigate the anti-inflammatory and anti-nociceptive activities of indigo in mice and to explore the possible related mechanisms. Xylene-induced ear edema, carrageenan-induced paw edema and acetic acid-induced vascular permeability test were used to investigate the anti-inflammatory activities of indigo in mice. Anti-nociceptive effects of Indigo were assessed by using acetic acid-induced writhing, hot plate test and formalin test, as well as evaluation of spontaneous locomotor activity and motor performance. Mechanisms of indigo activities also were explored by evaluating expression levels of IκB kinase (IKK)β、p-IKKβ, inhibitor κB (IκB)α, p-IκBα, p65 nuclear factor (NF)-kB, p-p65 NF-κB, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) using Western blotting and expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, Prostaglandin E2 (PGE2) using enzyme-linked immunosorbent assay ( ELISA) kits. The results showed that indigo significantly reduced xylene-induced ear edema, carrageenan-induced paw edema, and acetic acid-induced vascular permeation. Additionally, indigo significantly inhibited the nociceptions induced by acetic acid and formalin. However, the nociceptions could not be decreased by indigo in the hot plate test, and indigo did not affect spontaneous locomotor activity and motor performance. Expression levels of p-IKKβ, p-IκBα, p65 NF-kB, p-p65 NF-κB, COX-2, iNOS, TNF-α, IL-1β, IL-6, PGE2 were decreased and the level of IκBα was increased obviously by indigo treatment. In conclusion, indigo exerts significant anti-inflammatory and analgesic activities in mice by inhibiting IKKβ phosphorylation, which leads to the reduction in the production of important pain mediators, such as PGE2 and COX-2, via the IKKβ/IκB/NF-κB pathway.