Dehydroandrographolide succinate alleviates ulcerative colitis via regulating RAB9A/NF-κB axis-mediated macrophage polarization and remodeling the gut microbiota.

Abstract

BACKGROUND: Dehydroandrographolide succinate (DAS), isolated from Andrographis paniculata, exhibits potent anti-inflammatory activity, yet its therapeutic potential and precise mechanism in ulcerative colitis (UC) remain unexplored. PURPOSE: This study aims to investigate the efficacy and molecular basis that is responsible for the amelioration of DAS against UC. METHODS: Effect of DAS against colitis was studied in a DSS-induced colitis model, and the critical role of macrophage was verified by the macrophage depletion and adoptive macrophage transfer (AMT) model. The anti-inflammation activity of DAS was investigated in the LPS/IFN-γ-stimulated THP-1-derived macrophage model in vitro, followed by DARTS, CETSA, molecular docking/dynamics, and transcriptomics to elucidate the underlying mechanism. The effect of DAS on gut microbiota was analyzed with metagenomic sequencing. RESULTS: DAS attenuated the colitis features, including weight loss, diarrhea, rectal bleeding, and colon shortening, together with reduced inflammatory infiltrates and restored crypt architecture. DAS down-regulated pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and up-regulated anti-inflammatory mediators (IL-10, IL-13), meanwhile restoring tight-junction proteins (ZO-1, Occludin) and goblet-cell mucins. Macrophage depletion abolished DAS's benefit, while AMT with DAS-treated macrophages relieved the colitis features, confirming the macrophage-dependency of DAS. Transcriptomics and the following verification revealed that the anti-inflammatory activity of DAS mainly relied on the NF-κB signaling pathway by suppressing p65 phosphorylation and downstream targets. DAS inhibited M1 polarization and protected epithelial monolayers from macrophage-mediated damage. Moreover, DAS exhibited high-affinity binding to RAB9A, and RAB9A knockdown abolished DAS-mediated suppression of TLR4/NF-κB signaling pathway in macrophages. Metagenomic analysis revealed that DAS treatment enriched Lachnospiraceae bacterium, Duncaniella freteri, Lachnospiraceae bacterium 10-1, Bacterium 1XD8-76, Schaedlerella arabinosiphila, while depleted Muribaculaceae bacterium, Bacteroides intestinalis and Clostridiaceae bacterium. Functional gene profiling indicated that DAS upregulated genes related to butyrate metabolism, amino sugar and nucleotide sugar metabolism, and starch and sucrose metabolism. CONCLUSION: DAS alleviates DSS-colitis by targeting RAB9A to block the NF-κB signaling pathway-driven M1 macrophage polarization, and is accompanied by gut microbiota remodeling, highlighting the promising application of DAS against UC.