A novel limonin derivate modulates inflammatory response by suppressing the TLR4/NF-κB signalling pathway

In our previous studies, we have demonstrated that a novel water-soluble derivative of limonin, (12S,12aS,Z)--8((2-(diethylamino)ethoxy)imino)-12-(furan-3-yl)-6,6,8a,12a-tetramethyldodecahydro-1H,3H-oxireno[2,3-d]pyrano[4', 3': 3,3a]isobenzofuro[ 5,4-f] isochromene-3,10(9aH)-dione (V-A-4), exhibited strong anti-inflammatory activity both in vitro and in vivo. The purpose of this study was to further explore the underlying mechanisms of such activity demonstrated by V-A-4. The protective effect of V-A-4 on the alleviation of xylene-induced ear swelling and carrageenan-induced subcutaneous air pouch model was detected in vivo. Furthermore, the in vitro effects of V-A-4 and its mechanisms of action were determined by colorimetric COX (ovine) inhibitor-screening assay and in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. This study showed that V-A-4 does not exert anti-inflammatory effect through the inhibition of COX-1 or COX-2. Rather, it is exerted through the suppression of the secretion of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), as well as through the infiltration of inflammatory cells. V-A-4 demonstrated strong inhibition of NF-kappa B activation through repression of IKK alpha and IKK beta phosphorylations, which in turn leads to the phosphorylation and degradation of I kappa B alpha in LPS-induced RAW264.7 cells. Moreover, toll-like receptor 4 (TLR4) pathway was involved in the anti-inflammatory effect of V-A-4, which also played an important role in the down-regulation of LPS-mediated miR-146a and miR-155 expressions. These results encourage further development of V-A-4 as a potential candidate for the treatment of inflammatory diseases.