1,6-O,O-diacetylbritannilactones inhibits IkappaB kinase beta-dependent NF-kappaB activation

To determine the chemical constituents responsible for pharmacological effects of Inula britannica-F., three specific sesquiterpene lactones in Inula britannica were isolated from chloroform extract and identified, including britannilactone (BL), 1-O-acetylbritannilactone (ABLO), and 1,6-O,O-diacetylbritannilactone (ABLOO). Electrophoretic mobility shift assay (EMSA) was performed to detect the nuclear translocation of nuclear factor-kappaB (NF-kappaB) p65. The expressions of IkappaBalpha, pIkappaBalpha, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IkappaB kinase alpha/beta (IKKalpha/beta) and NF-kappaB kinase (NIK) were detected by Western blot and RT-PCR. We found that acetyl side groups enhanced the inhibitory action of the agents on LPS/IFN-gamma-induced iNOS and COX-2 expression. Their inhibiting activity was positive correlation with the acetyl side group number. The effects of LPS/IFN-gamma were reversed by ABLOO, and BL without acetyl side groups showed only a weak inhibitory action. Further study indicated that ABLOO markedly inhibited the phosphorylation of IKKbeta down to based level, but not IKKalpha, corresponding with decreased in IkappaBalpha degradation and phosphorylation induced by LPS/IFN-gamma, resulting in the suppression of NF-kappaB nuclear translocation and activity. These results suggest that the acetyl moieties add to the lipophilicity, and consequently enhance cellular penetration, so that ABLOO possess the most anti-inflammatory effect and may be a potent lead structure for the development of therapeutic and cytokine-suppressing remedies valuable for the treatment of various inflammatory diseases.