Aqp5-mediated Lacrimal Gland Epithelial Cell Pyroptosis And Mechanism Via The ROS/NLRP3 Inflammasome Study

PurposeThe pathogenesis of the lacrimal gland（LG）is facilitated by inflammation mediated by NLRP3 inflammasomes in dry eye disease（DED）.Aqp5 is expressed in LG epithelial cells,involved in tear secretion,Aqp5 expression is considerably abnormal in DED in Sjogren’s syndrome patients.This research aimed to investigate the protective effects and mechanism of Aquaporin5（Aqp5）in regulating NLRP3 inflammasome activation by regulating intracellular reactive oxygen species（ROS）levels of the LG.MethodsWild-type(Aqp5^+/+)and Aqp5 knockout(Aqp5^-/-)mice were used to evaluate pathological changes in LGs.ROS generation was detected with a dichlorodihydro-fluorescein diacetate assay.Oil Red O staining was used to assess lipid metabolism.The reversal of the mitochondrial membrane potential was detected using a JC-1 fluorescent probe kit.DNA breaks were detected using TUNEL staining.Corneal epithelial punctate defects were evaluated using a sodium fluorescein solution staining.Tear production was assessed using phenol red cotton thread.The expression of Aqp5,NLRP3,Caspase-1,and GSDMD were determined by immunofluorescence.Expression of inflammation-related factors（NLRP3,Caspase-1,IL-1β）and pyroptosis-related factors（GSDMD）were determined by Western blot.A powerful and specific NLRP3 inflammasome inhibitor is MCC950.N-acetyl-L-cysteine（NAC）is a powerful antioxidant that effectively removes ROS.The effect of Aqp5 on NLRP3/Caspase-1/GSDMD-mediated cell pyroptosis was examined using pharmacological treatment of MCC950 or NAC.Results（1）Cultivation and identification of the primary LG epithelial cells（2）Mitochondrial dysfunction and lipid accumulation in the LG epithelial cells of Aqp5^-/-mice（3）Enhanced pyroptosis in the acinar epithelial cells of the LG of the Aqp5^-/-mice（4）Enhanced pyroptosis in the primary LG epithelial cells of the Aqp5^-/-mice（5）MCC950 reducuated pyroptosis in the acinar epithelial cells of the LG of Aqp5^-/-mice（6）MCC950 reducuated pyroptosis in the primary LG epithelial cells of Aqp5^-/-mice（7）NAC inhibited NLRP3 inflammasome activation,which reduced pyroptosis in LG epithelial cells.ConclusionsLoss of Aqp5 elicited the accumulation of ROS in the LG epithelial cells and promoted the activation of the NLRP3 inflammasome.Activation of caspase-1/GSDMD was increased in LGs and primary LG epithelial cells,while pyroptosis was increased in Aqp5^-/-mice.Inhibition of NLRP3 inflammasome or ROS significantly alleviate injury and pyroptosis in Aqp5-deficient LG epithelial cells,which provides evidence that Aqp5 is involve in the cellular pyroptosis process and triggers DED,and suggests that Aqp5 might be a fresh target for DED treatment.