mTOR/Autophagy//NF-κB Axis Drives Benzalkonium Chloride-Induced Inflammatory Response in Corneal Epithelial Cells

Benzalkonium chloride (BAK), a quaternary ammonium antimicrobial agent widely utilized in ophthalmic formulations, is associated with chronic ocular surface toxicity. BAK induces corneal epithelial inflammation and autophagic impairment, yet their interrelationship remains unclear.

Mouse corneal epithelial cell lines (MCECs) were subjected to short-term BAK exposure (10-20 μg/mL, 15 min + 8 h recovery). TLR4/MyD88/NF-κB pathway activation and proinflammatory cytokines (Il-1α, Il-1β, Il-6) were assessed. Autophagic flux was evaluated via LC3-II/I ratio, p62 accumulation, GFP-RFP-LC3 autolysosome formation assay, and lysosomal function. Pharmacological interventions with rapamycin (mTOR blockade) and TAK-242 (TLR4 inhibition) were used to dissect pathway interactions.

Short-term BAK exposure dose-dependently activated the TLR4/MyD88/NF-κB pathway and elevated proinflammatory cytokines. Concurrently, BAK inhibited autophagic flux through mTOR hyperactivation, evidenced by reduced LC3-II/I ratio, p62 accumulation, impaired autolysosome formation, and lysosomal dysfunction. Both rapamycin and TAK-242 significantly attenuated BAK-induced MyD88/NF-κB activation and inflammation. Critically, BAK activates parallel TLR4-MyD88 and mTOR pathways that synergistically amplify NF-κB signaling.

These results unveil mTOR as a therapeutic target for mitigating BAK-induced corneal damage, thereby proposing mTOR suppression or autophagic activation as viable interventions for BAK-associated ocular surface disorders. Particularly at lower concentration (0.001%) and shorter exposure (15 min) of BAK.