AQP9 in Neutrophils Promotes the Formation of NETs by Regulating JAK2-STAT3 Pathway-Mediated Pyroptosis to Aggravate Intestinal Epithelial Cell Injury in Ulcerative Colitis.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory disorder primarily affecting the rectum and colon. This study aimed to identify potential therapeutic targets that may inhibit UC progression and mitigate patient suffering.

METHODS: UC-related datasets were retrieved from the Gene Expression Omnibus database. Differential expression analysis, weighted gene co-expression network analysis, immunoinfiltration analysis, and pyroptosis scoring were employed to identify key pyroptosis-related genes implicated in UC pathogenesis. A dextran sulfate sodium salt (DSS)-induced mouse model of UC was established, and neutrophil extracellular traps (NETs) were induced in neutrophils by stimulation with phorbol 12-myristate 13-acetate (PMA). Histopathological changes in mouse colon tissues were assessed by hematoxylin-eosin staining, and NET formation was evaluated via immunofluorescence. The expression of aquaporin 9 (AQP9), peptidylarginine deiminase 4 (PAD4), zonula occludens 1 (ZO-1), occludin, and proteins related to pyroptosis and the JAK2-STAT3 pathway was determined by Western blotting. Levels of inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA), production of reactive oxygen species was assessed using fluorescent probes, and intestinal epithelial cell viability and death were evaluated using the cell counting kit-8 (CCK-8) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, respectively.

RESULTS: Five hub genes (AQP9, S100A8, S100A9, S100A12, and VNN2) were identified through bioinformatics analysis, with AQP9 selected for further investigation. Single-cell analysis and immunofluorescence revealed that AQP9 was predominantly expressed in neutrophils and upregulated in the colon tissues of mice with UC and PMA-stimulated neutrophils. Knockdown of AQP9 in PMA-treated neutrophils led to suppression of the JAK2-STAT3 pathway, reduced pyroptosis, and decreased NET formation. Upon co-culture with intestinal epithelial cells, AQP9 knockdown resulted in enhanced epithelial cell viability, reduced apoptosis, and upregulation of ZO-1 and occludin. Conversely, treatment of neutrophils from the PMA+si-AQP9 with a JAK2-STAT3 pathway agonist increased pyroptosis, enhanced the formation of NETs, and induced epithelial cell injury. Similarly, treatment with a pyroptosis agonist enhanced both pyroptosis and the formation of NETs, further aggravating epithelial damage.

CONCLUSION: Knockdown of AQP9 inhibits JAK2-STAT3 pathway-mediated pyroptosis, thereby reducing the formation of NETs and attenuating intestinal epithelial cell injury.