Andrographis paniculata diterpenoids and ethanolic extract inhibit TNFα-induced ICAM-1 expression in EA.hy926 cells

Andrographis paniculata (A. paniculata), a traditional herb in Southeastern Asia, is used to treat inflammation-mediated diseases. Purpose: The two major bioactive diterpenoids in A. paniculata are andrographolide (AND) and 14-deoxy-11,12-didehydroandrographolide (deAND). Because of the anti-inflammatory evidence for AND, we hypothesized that deAND might possess similar potency for inhibiting monocyte adhesion to the vascular endothelium, which is a critical event for atherosclerotic lesion formation.In the present study, we used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine cell viability. We evaluated the production of intracellular reactive oxygen species (ROS) by using DCFDA assay. We assayed the protein expression by using Western blot analysis, the mRNA expression by using RT-PCR, and the nuclear protein-DNA binding activity by using EMSA.We showed that pretreatment of EA.hy926 cells with A. paniculata ethanolic extract (APE), deAND, and AND significantly inhibited TNFα-induced ICAM-1 protein and mRNA expression, ICAM-1 promoter activity, and monocyte adhesion. TNFα-stimulated IKKβ phosphorylation, IκBα phosphorylation and degradation, p65 nuclear translocation, and NFκB nuclear protein-DNA binding activity were attenuated by pretreatment with APE, deAND, and AND. APE, deAND, and AND attenuated TNFα-induced Src phosphorylation and membrane translocation of the NOX subunits p47phox and p67phox. Both APE and AND induced protein expression of heme oxygenase 1 and the glutamate cysteine ligase modifier subunit and enhanced glutathione content. Pretreatment with AND and deAND inhibited TNFα-induced ROS generation.These results suggest that the mechanism by which APE, deAND, and AND down-regulates TNFα-induced ICAM-1 expression in EA.hy926 cells is via attenuation of activation of the IKK/IκB/NFκB pathway.