Pathological neutrophil extracellular traps hinder postoperative anal fistula wound healing and are attenuated by Zuoqing granule via suppression of the Nox4 pathway.

Abstract

BACKGROUND: The impaired healing of postoperative anal fistula wounds often complicated by a contaminated environment and persistent inflammation. The key pathological immune events that sustain this chronic inflammatory milieu remain largely unknown. We hypothesized that dysregulated neutrophil extracellular trap (NET) formation (NETosis)-a potent driver of tissue damage-might be a pathological feature and potential therapeutic target. METHODS: Using a rat model of contaminated wounds akin to postoperative anal fistula, we characterized NETosis via immunofluorescence (CitH3/CD66b), transmission electron microscopy, and ELISA. The involvement of the Nox4/ROS/PI3K/Akt/PADI4 pathway was assessed. The therapeutic potential of Zuoqing Granule (ZQG), a clinically used traditional Chinese formulation, was evaluated bothand in PMA-stimulated rat neutrophils. Bacterial burden were also assessed. RESULTS: We identified pervasive NETosis as a pathological hallmark of non-healing anal fistula wounds, accompanied by a surge in pro-inflammatory cytokines (IL-2, IL-5, IL-6, IL-12, TNF-α) and a ~15.5-fold increase in bacterial load compared to controls. ZQG treatment dose-dependently accelerated wound closure, resolved inflammation, reduced bacterial burden, and suppressed NETosis by up to 75.1% at day 7. Mechanistically, ZQG inhibited the Nox4/ROS/PI3K/Akt/PADI4 axis., ZQG reduced PMA-induced NETosis by 63.0% and superoxide production by 58.1%, comparable to Nox4 knockdown. CONCLUSION: Our study establishes aberrant NETosis as a pathological feature and potential therapeutic target in anal fistula-like wounds. We further identify ZQG as a promising candidate therapy that alleviates this pathology by suppressing the Nox4/ROS/PI3K/Akt/PADI4 pathway, without compromising bacterial clearance.