Integrated proteomics and metabolomics reveal NF-κB pathway modulation by Xiangpi Shengji ointment and Qingre Zaoshi compound in anal fistula wound healing.

This study investigates the molecular mechanisms by which Xiangpi Shengji Ointment (XPSJO), combined with a heat-clearing and dampness-drying compound, accelerates postoperative wound healing in an anal fistula (AF) rat model, employing integrated proteomic and metabolomic approaches. An AF rat model was established and treated topically with the herbal combination. Wound healing progression was monitored postoperatively. Histopathological assessment (hematoxylin and eosin [H&E] staining), enzyme-linked immunosorbent assay [ELISA] quantification of inflammatory markers (e.g., tumor necrosis factor-alpha [TNF-α], interleukin [IL]-6) and vascular endothelial growth factor [VEGF], and proteomic (liquid chromatography-tandem mass spectrometry [LC-MS/MS]) and metabolomic (gas chromatography-mass spectrometry [GC-MS]) analyses identified critical pathways and biomolecules involved. In vitro, lipopolysaccharide (LPS)-stimulated THP-1 macrophages and human umbilical vein endothelial cells (HUVECs) were co-treated with XPSJO and Coptis chinensis-Phellodendron amurense Rupr. Extract (CGE). Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway modulation was examined via reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blot, and functional assays (cell counting kit [CCK]-8, flow cytometry, scratch wound, tube formation). Lentiviral NF-κB overexpression validated mechanistic specificity in vitro and in vivo. Results showed that XPSJO combined with the compound accelerated AF wound closuresuppressed NF-κB signaling pathway activation (evidenced by downregulation of pathway-associated proteins in proteomics and reduced p65 phosphorylation), decreased pro-inflammatory cytokine secretion, and enhanced VEGF expression. Critically, it promoted M2 macrophage polarization (increased Arg-1/CD206, decreased iNOS/CD80) and stimulated HUVEC migration/angiogenesis. Multi-omics analysis further confirmed NF-κB, TNF, and IL-17 pathways as central therapeutic targets. The study concludes that XPSJO combined with a heat-clearing and dampness-drying compound promotes AF healing through pharmacological inhibition of the NF-κB pathway, macrophage reprogramming, and tissue remodeling.