Anti-inflammatory activity of Khayandirobilide A from Khaya senegalensis via NF-κB, AP-1 and p38 MAPK/Nrf2/HO-1 signaling pathways in lipopolysaccharide-stimulated RAW 264.7 and BV-2 cells

Background: Immunocytes-involved inflammation is considered to modulate the damage in various diseases. Herein, novel therapeutics suppressing over-activation of immunocytes could prove an effective strategy to prevent inflammation-related diseases. Purpose: The objective of this study is to evaluate the anti-inflammatory activity of Khayandirobilide A (KLA), a new andirobin-type limonoid with modified furan ring isolated from the Khaya senegalensis (Desr.) A. Juss., and to explore its potential underlying mechanisms in LPS-stimulated inflammatory models. Methods: The structure of KLA was elucidated on the basis of 1D- and 2D-NMR spectroscopic data as well as HR-ESI-MS. As for its anti-inflammatory effect, the production of pro-inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 and BV-2 cells were measured by Griess reagent, ELISA and qRT-PCR. The relevant proteins including nuclear factor kappa B (NF-kappa B), p-AKT, p-p38 and Nrf2/HO-1 were investigated by western blot. Nuclear localisations of NF-kappa B, activator protein-1 (AP-1) and Nrf2 were also examined by western blot and immunofluorescence. Results: KLA could inhibit the production of LPS-induced NO with IC50 values of 5.04 +/- 0.14 mu M and 4.97 +/- 0.5 mu M in RAW 264.7 and BV-2 cells, respectively. KLA also attenuated interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels. Further mechanistic studies demonstrated the activation of NF-kappa B and AP-1 were reduced by KLA. Moreover, KLA elevated expression of heme oxygenase-1(HO-1) via inducing Keap1 autophagic degradation and promoting Nrf2 nuclear translocation. Despite KLA induced the phosphorylation of mitogen-activated protein kinases (MAPKs) family, inhibiting the phosphorylation of p38 by its specific inhibitor SB203580 attenuated the degradation of KLA-induced Keap1, and then reduced KLA-induced Nrf2 nuclear translocation and HO-1 expression. Furthermore, SB203580, Brusatol (a Nrf2 specific inhibitor) and ZnPP (a HO-1 specific inhibitor) could partly reverse the suppressive effects of KLA on LPS-induced NO production and mRNA levels of pro-inflammatory genes. Conclusion: These data displayed that KLA possessed anti-inflammatory activity, which was attributed to inhibit the release of LPS-stimulated inflammatory mediators via suppressing the activation of NF-kappa B, AP-1, and upregulating the induction of p38 MAPK/Nrf2-mediated HO-1.