{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,29]],"date-time":"2026-10-29T17:47:04Z","timestamp":1793296024034,"version":"4.5.0"},"reference-count":32,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T00:00:00Z","timestamp":1768867200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>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)\u2014a potent driver of tissue damage\u2014might be a pathological feature and potential therapeutic target.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      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\n                      <jats:italic>in vivo<\/jats:italic>\n                      and in PMA-stimulated rat neutrophils\n                      <jats:italic>in vitro<\/jats:italic>\n                      . Bacterial burden were also assessed.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      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-\u03b1) 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.\n                      <jats:italic>In vitro<\/jats:italic>\n                      , ZQG reduced PMA-induced NETosis by 63.0% and superoxide production by 58.1%, comparable to Nox4 knockdown.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fimmu.2025.1730184","type":"journal-article","created":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T05:28:00Z","timestamp":1768886880000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["Pathological neutrophil extracellular traps hinder postoperative anal fistula wound healing and are attenuated by Zuoqing granule via suppression of the Nox4 pathway"],"prefix":"10.3389","volume":"16","author":[{"given":"Xiaoli","family":"Fang","sequence":"first","affiliation":[{"name":"Department of Anorectal Surgery, The First Affiliated Hospital of Anhui University of Chinese Medicine","place":["Hefei, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heng","family":"Deng","sequence":"additional","affiliation":[{"name":"Department of Anorectal Surgery, Second Affiliated Hospital, Anhui University of Chinese Medicine","place":["Hefei, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ming","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Anorectal Surgery, The First Affiliated Hospital of Anhui University of Chinese Medicine","place":["Hefei, China"]},{"name":"Department of Anorectal Surgery, The Third Affiliated Hospital of Anhui University of Chinese Medicine","place":["Hefei, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiang","family":"Gao","sequence":"additional","affiliation":[{"name":"Department of Anorectal Surgery, Second Affiliated Hospital, Anhui University of Chinese Medicine","place":["Hefei, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunrong","family":"He","sequence":"additional","affiliation":[{"name":"General Practice Department, Hefei Haiheng Health Service Center","place":["Hefei, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hui","family":"Liu","sequence":"additional","affiliation":[{"name":"General Practice Department, Anhui Provincial Second People\u2019s Hospital","place":["Hefei, Anhui, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,1,20]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.3389\/fsurg.2023.1235666","article-title":"Evaluation of the mechanical properties and clinical application of nickel-titanium shape memory alloy anal fistula clip","volume":"10","author":"Deng","year":"2023","journal-title":"Front Surg"},{"key":"B2","doi-asserted-by":"publisher","first-page":"7363006","DOI":"10.1155\/2022\/7363006","article-title":"The effects of phellodendron decoction on wound healing of anal fistula after anal fistulotomy","volume":"2022","author":"Deng","year":"2022","journal-title":"Evid Based Complement Alternat Med"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.3389\/fphar.2025.1599633","article-title":"Exosomal miR-93-3p targets EIF4EBP1 to regulate macrophage polarization and accelerate wound healing post-anal fistula surgery","volume":"16","author":"Zhang","year":"2025","journal-title":"Front Pharmacol"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1126\/science.1092385","article-title":"Neutrophil extracellular traps kill bacteria","volume":"303","author":"Brinkmann","year":"2004","journal-title":"Science"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1016\/j.tips.2024.09.007","article-title":"Neutrophil extracellular traps in wound healing","volume":"45","author":"Zhu","year":"2024","journal-title":"Trends Pharmacol Sci"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1097\/SLA.0000000000006196","article-title":"Netosis in surgery: pathophysiology, prevention and treatment","volume":"279","author":"James","year":"2024","journal-title":"Ann Surg"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1038\/nri.2017.105","article-title":"Neutrophil extracellular traps in immunity and disease","volume":"18","author":"Papayannopoulos","year":"2018","journal-title":"Nat Rev Immunol"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2023.1113948","article-title":"Murine scald models characterize the role of neutrophils and neutrophil extracellular traps in severe burns","volume":"14","author":"Elrod","year":"2023","journal-title":"Front Immunol"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.7150\/ijbs.97150","article-title":"Mesenchymal stem cell-derived extracellular vesicles accelerate diabetic wound healing by inhibiting NET-induced ferroptosis of endothelial cells","volume":"20","author":"Lu","year":"2024","journal-title":"Int J Biol Sci"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.3389\/fphar.2025.1646545","article-title":"Zuoqing granules attenuate ulcerative colitis via macrophage polarization modulation: involvement of the PPAR-gamma\/NF-kappaB\/STAT1 signaling axis","volume":"16","author":"Deng","year":"2025","journal-title":"Front Pharmacol"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1515\/jcim-2024-0435","article-title":"The active ingredients and mechanism of Zuoqing San in the treatment of sigmoid ulcerative colitis by retention enema","volume":"22","author":"Li","year":"2025","journal-title":"J Complement Integr Med"},{"key":"B12","doi-asserted-by":"publisher","first-page":"e3000410","DOI":"10.1371\/journal.pbio.3000410","article-title":"The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research","volume":"18","author":"Percie Du Sert","year":"2020","journal-title":"PloS Biol"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.12122\/j.issn.1673-4254.2024.07.05","article-title":"Tongyangxiao Lotion promotes postoperative wound healing in a rat model of anal fistula by downregulating inflammatory factors and suppressing inflammation","volume":"44","author":"Wang","year":"2024","journal-title":"Nan Fang Yi Ke Da Xue Xue Bao"},{"key":"B14","doi-asserted-by":"publisher","first-page":"e0291508","DOI":"10.1371\/journal.pone.0291508","article-title":"ICG-mediated fluorescence-assisted debridement to promote wound healing","volume":"18","author":"Zhu","year":"2023","journal-title":"PloS One"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.3390\/biomedicines13020508","article-title":"Honey, gellan gum, and hyaluronic acid as therapeutic approaches for skin regeneration","volume":"13","author":"Sousa","year":"2025","journal-title":"Biomedicines"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1021\/acs.nanolett.5c00014","article-title":"Hydrogel-based sequential photodynamic therapy promotes wound healing by targeting wound infection and inflammation","volume":"25","author":"Li","year":"2025","journal-title":"Nano Lett"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.19852\/j.cnki.jtcm.2025.03.015","article-title":"Effect of Neibu Huangqi Youhua formula on postoperative wound healing, inflammatory factors and pain mediators of anal fistula","volume":"45","author":"Yanyan","year":"2025","journal-title":"J Tradit Chin Med"},{"key":"B18","doi-asserted-by":"publisher","first-page":"112516","DOI":"10.1016\/j.intimp.2024.112516","article-title":"Nets in fibrosis: Bridging innate immunity and tissue remodeling","volume":"137","author":"Ma","year":"2024","journal-title":"Int Immunopharmacol"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2025.1595994","article-title":"Neutrophil extracellular traps and interleukin-1beta in cystic fibrosis lung disease","volume":"16","author":"Fantone","year":"2025","journal-title":"Front Immunol"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1038\/s41423-024-01137-x","article-title":"NLRP3 inflammasome activation and NETosis positively regulate each other and exacerbate proinflammatory responses: implications of NETosis inhibition for acne skin inflammation treatment","volume":"21","author":"Kim","year":"2024","journal-title":"Cell Mol Immunol"},{"key":"B21","doi-asserted-by":"publisher","first-page":"2559","DOI":"10.1038\/s41467-025-57348-x","article-title":"Reprogramming aerobic metabolism mitigates Streptococcus pyogenes tissue damage in a mouse necrotizing skin infection model","volume":"16","author":"Xu","year":"2025","journal-title":"Nat Commun"},{"key":"B22","doi-asserted-by":"publisher","first-page":"e2411524","DOI":"10.1002\/advs.202411524","article-title":"NETs-CD44-IL-17A feedback loop drives th17-mediated inflammation in behcet\u2019s uveitis","volume":"12","author":"Wu","year":"2025","journal-title":"Adv Sci (Weinh)"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-07820-3","article-title":"Recognition and control of neutrophil extracellular trap formation by MICL","volume":"633","author":"Malamud","year":"2024","journal-title":"Nature"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.3390\/pharmaceutics17070809","article-title":"CitH3, a druggable biomarker for human diseases associated with acute NETosis and chronic immune dysfunction","volume":"17","author":"Chen","year":"2025","journal-title":"Pharmaceutics"},{"key":"B25","doi-asserted-by":"publisher","first-page":"8822728","DOI":"10.1155\/mi\/8822728","article-title":"Baicalin modulates glycolysis via the PKC\/raf\/MEK\/ERK and PI3K\/AKT signaling pathways to attenuate IFN-I-induced neutrophil NETosis","volume":"2025","author":"Wei","year":"2025","journal-title":"Mediators Inflammation"},{"key":"B26","doi-asserted-by":"publisher","first-page":"14166","DOI":"10.1038\/s41598-018-32576-y","article-title":"Respiratory Syncytial Virus induces the classical ROS-dependent NETosis through PAD-4 and necroptosis pathways activation","volume":"8","author":"Muraro","year":"2018","journal-title":"Sci Rep"},{"key":"B27","doi-asserted-by":"publisher","first-page":"103750","DOI":"10.1016\/j.redox.2025.103750","article-title":"EMAP-II from macrophage-derived extracellular vesicles drives neutrophil extracellular traps formation via PI3K\/AKT\/mtROS in lung ischemia\/reperfusion injury","volume":"85","author":"Zhuang","year":"2025","journal-title":"Redox Biol"},{"key":"B28","doi-asserted-by":"publisher","first-page":"7023","DOI":"10.1038\/s41467-024-51211-1","article-title":"Piezo1 expression in neutrophils regulates shear-induced NETosis","volume":"15","author":"Baratchi","year":"2024","journal-title":"Nat Commun"},{"key":"B29","doi-asserted-by":"publisher","first-page":"e2310320","DOI":"10.1002\/adma.202310320","article-title":"Neutrophil function conversion driven by immune switchpoint regulator against diabetes-related biofilm infections","volume":"36","author":"Guo","year":"2024","journal-title":"Adv Mater"},{"key":"B30","doi-asserted-by":"publisher","first-page":"102143","DOI":"10.1016\/j.mtbio.2025.102143","article-title":"Ginseng-derived nanoparticles accelerate diabetic wound healing by modulating macrophage polarization and restoring endothelial cell function","volume":"34","author":"Fan","year":"2025","journal-title":"Mater Today Bio"},{"key":"B31","doi-asserted-by":"publisher","first-page":"102867","DOI":"10.1016\/j.redox.2023.102867","article-title":"Lactate-upregulated NADPH-dependent NOX4 expression via HCAR1\/PI3K pathway contributes to ROS-induced osteoarthritis chondrocyte damage","volume":"67","author":"Huang","year":"2023","journal-title":"Redox Biol"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.1111\/jth.15313","article-title":"PAD4 takes charge during neutrophil activation: Impact of PAD4 mediated NET formation on immune-mediated disease","volume":"19","author":"Liu","year":"2021","journal-title":"J Thromb Haemost"}],"updated-by":[{"DOI":"10.3389\/fimmu.2026.1792671","type":"correction","label":"Correction","source":"publisher","updated":{"date-parts":[[2026,1,28]],"date-time":"2026-01-28T00:00:00Z","timestamp":1769558400000}}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1730184\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,24]],"date-time":"2026-02-24T07:46:36Z","timestamp":1771919196000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1730184\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,20]]},"references-count":32,"alternative-id":["10.3389\/fimmu.2025.1730184"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2025.1730184","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,20]]},"article-number":"1730184"}}