Xiangpi Shengji Ointment Accelerates Anal Fistula Healing by Regulating Macrophage-Fibroblast Crosstalk Through the Nuclear Factor Kappa B/Hypoxia-Inducible Factor Alpha/Vascular Endothelial Growth Factor Signaling Axis.

Abstract

This study elucidates the molecular mechanism by which Xiangpi Shengji ointment (Xiangpi Shengji gao, XPSJG) promotes anal fistula wound healing. Integrated network pharmacology and transcriptomic analyses (GSE28914, GSE203244) revealed the involvement of the NF-κB/HIF-α/VEGF axis, with elevated expression of NF-κB, HIF1A, and VEGFA observed during the early healing phase (days 3 and 7). Single-cell RNA sequencing further indicated that activation of this signaling axis may drive early macrophage M1 polarization. In vitro experiments confirmed early treatment with the aqueous extract of XPSJG powder significantly enhanced macrophage M1 polarization and upregulated VEGF, COL1A1, and α-SMA, promoting fibroblast proliferation and migration (assessed via CCK-8, ELISA, WB, RT-qPCR). In vivo, using a murine anal fistula model, XPSJG accelerated wound closure, improved tissue architecture, and reduced inflammation and apoptosis through modulation of the NF-κB/HIF-α/VEGF axis. These effects were partially reversed by an NF-κB inhibitor, further verifying pathway involvement. Collectively, the findings demonstrate that early application of XPSJG facilitates anal fistula healing by inducing macrophage M1 polarization and enhancing fibroblast function via the NF-κB/HIF-α/VEGF signaling axis, thereby providing a mechanistic rationale for its clinical use in chronic wound management.