Aquaporin5 Deficiency Aggravates ROS/NLRP3 Inflammasome-Mediated Pyroptosis in the Lacrimal Glands

PURPOSE: The pathogenesis of the lacrimal glands (LGs) is facilitated by inflammation mediated by the NACHT, LRR, and NLRP3 inflammasomes in dry eye disease. This research aimed to explore the protective effects of Aquaporin 5 (AQP5) on LGs by inhibiting reactive oxygen species (ROS) and the NLRP3 inflammasome. METHODS: AQP5 knockout (AQP5(−/−)) mice were used to evaluate pathological changes in LGs. ROS generation was detected with a dichlorodihydro–fluorescein diacetate assay. Lipid metabolism was assessed by Oil Red O staining. The reversal of the mitochondrial membrane potential was detected using a JC-1 fluorescent probe kit. The effect of AQP5 on NLRP3/caspase-1/Gasdermin-D (GSDMD)-mediated pyroptosis was examined using pharmacological treatment of N-acetyl L-cysteine or MCC950. RESULTS: AQP5 loss significantly increased ROS generation, lipid metabolism disorders, TUNEL-positive cells, and reversal of the mitochondrial membrane potential in the AQP5(−/−) LGs. NLRP3 upregulation, increased caspase-1 and GSDMD activity, and enhanced IL-1β release were detected in the AQP5(−/−) mouse LGs and primary LG epithelial cells. MCC950 significantly suppressed NLRP3 inflammasome-related pyroptosis induced by AQP5 deficiency in LGs and primary LG epithelial cells. Furthermore, we discovered that prestimulating the AQP5(−/−) primary LG epithelial cells with N-acetyl L-cysteine decreased NLRP3 expression, caspase-1 and GSDMD activity levels, and IL-1β release. CONCLUSIONS: Our results revealed that AQP5 loss promoted NLRP3 inflammasome activation through ROS generation. Inhibiting the ROS or NLRP3 inflammasome significantly alleviated the damage and pyroptosis of AQP5-deficient LG epithelial cells, which could provide new insights into dry eye disease.