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

This study investigated the anti-inflammatory and analgesic activities of indigo in mice and explored the possible related mechanisms. Xylene-induced ear edema, carrageenan-induced paw edema, and acetic acid-induced vascular permeability tests were used in investigating the anti-inflammatory activities. The anti-nociceptive effects of indigo were assessed through acetic acid-induced writhing, hot plate test, and formalin test, and spontaneous locomotor activity and motor performance were evaluated. The mechanisms of activities of indigo were explored by evaluating the 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), and inducible nitric oxide synthase (iNOS) through western blotting and the expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and prostaglandin E₂ (PGE₂) through enzyme-linked immunosorbent assay. The results showed that indigo significantly reduced xylene-induced ear edema, carrageenan-induced paw edema, and acetic acid-induced vascular permeation. In addition, indigo significantly inhibited nociception induced by acetic acid and formalin. However, the level of nociception was not decreased by indigo in the hot plate test, and indigo did not affect spontaneous locomotor activity and motor performance. The expression levels of p-IKKβ, p-IκBα, p65 NF-kB, p-p65 NF-κB, COX-2, iNOS, TNF-α, IL-1β, IL-6, and PGE₂ decreased, whereas the expression level of IκBα increased obviously after indigo treatment. In conclusion, indigo exerts significant anti-inflammatory and analgesic activities in mice by inhibiting IKKβ phosphorylation and reducing the production of important pain mediators, such as PGE₂ and COX-2, via the IKKβ/IκB/NF-κB pathway.