The LasB inhibitor decreases the disruption of the nasal epithelial barrier through the extracellular matrix-focal adhesion pathway during the early phase of Pseudomonas aeruginosa infection.

Elastase (LasB) activity in clinical isolates of Pseudomonas aeruginosa (PA) is strongly correlated with the severity of chronic rhinosinusitis (CRS). However, the early molecular targets of LasB and the protective potential of LasB inhibitors have not been clearly defined. To determine how a LasB inhibitor modulates PA or exoprotein-induced damage to the nasal epithelial barrier and to elucidate the underlying mechanism. Differentiated human nasal epithelial air-liquid-interface cultures (HNEC-ALI) were exposed to PA bacteria (1 × 10^7 CFU) or exoproteins (20 μg/μL) for 1-5 h, with or without a LasB inhibitor (2-8 mM). Barrier integrity was assessed using transepithelial electrical resistance (TEER), FITC-dextran flux, and hematoxylin and eosin (H&E) staining. Each group included three independent cultures. Global transcriptional responses were assessed by RNA sequencing and validated by RT-qPCR and immunoblotting. Enriched pathways were identified using KEGG analysis. PA and its exoproteins reduced TEER within 2 h, reaching the lowest point at 4 h (P < 0.01). The addition of the inhibitor after exposure partially restored TEER and reduced FITC-dextran permeability (P < 0.05), without affecting bacterial load. RNA-seq revealed significant upregulation of extracellular-matrix/Focal-adhesion genes (ITGA2, FLNB, COL1A1, LAMA2), while tight-junction genes(TJP1、OCLN) were minimally affected. The inhibitor reversed Focal-adhesion gene expression and shifted pathway activity from activation to repression. While tight-junction transcripts were minimally affected, immunoblotting revealed post-transcriptional degradation of ZO-1 and occludin, which was prevented by inhibitor treatment. Pro-inflammatory transcripts (IL-6, IL-8, TNF, IL-1β) increased significantly, but corresponding protein levels remained unchanged at 4 h, indicating a delay between transcription and translation. Early LasB-mediated injury primarily targets the ECM-Focal adhesion genes rather than tight junction genes transcription. LasB inhibition rapidly restores epithelial-barrier structure and function by modulating Focal-adhesion signaling, highlighting this pathway as a promising therapeutic target for PA-induced CRS.