Dehydroandrographolide attenuates Toll-like receptor signaling by dual inhibition of MyD88- and TRIF-dependent pathways.

Toll-like receptors (TLRs) are key mediators of innate immune responses, and their dysregulation contributes to inflammatory diseases. Dehydroandrographolide (DAG), a diterpene lactone from Andrographis paniculata, is known for its anti-inflammatory activity, but its effects on individual branches of TLR signaling remain unclear. RAW264.7 and 293T cells were used to evaluate the effects of DAG on MyD88- and TRIF-dependent signaling pathways. Luciferase reporter assays, Western blotting, RT-PCR, and nitrite assays were employed to assess NF-κB and IRF3 activation and inflammatory mediator expression. Statistical analysis was performed using one-way ANOVA. DAG significantly inhibited activation of NF-κB and IRF3 induced by TLR agonists (LPS, MALP-2, and Poly[I:C]) and by overexpression of MyD88- and TRIF-associated downstream molecules. Correspondingly, DAG suppressed iNOS, IFNβ, and IP-10 expression, indicating dual inhibition of both TLR signaling branches. DAG exerts dual inhibitory effects on MyD88- and TRIF-dependent TLR signaling, attenuating inflammatory mediator expression. DAG may represent a promising molecular scaffold or mechanistic candidate for modulating TLR-mediated inflammatory signaling.