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

## TL;DR

This study shows that excessive neutrophil extracellular traps (NETs) hinder healing in anal fistula wounds, and a traditional Chinese medicine called Zuoqing Granule can reduce these NETs and inflammation.

## Contribution

The study identifies NETosis as a key pathological feature in anal fistula wounds and introduces Zuoqing Granule as a novel therapeutic agent targeting the Nox4 pathway.

## Key findings

- NETosis is a pathological hallmark in non-healing anal fistula wounds, accompanied by high levels of pro-inflammatory cytokines and bacterial load.
- Zuoqing Granule reduces NETosis, inflammation, and bacterial burden in both in vivo and in vitro models.
- Zuoqing Granule suppresses the Nox4/ROS/PI3K/Akt/PADI4 pathway, which is central to NET formation.

## Abstract

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.

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 both in vivo and in PMA-stimulated rat neutrophils in vitro. Bacterial burden were also assessed.

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. In vitro, ZQG reduced PMA-induced NETosis by 63.0% and superoxide production by 58.1%, comparable to Nox4 knockdown.

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.