Hyperosmotic stress-induced NLRP3 inflammasome activation via the mechanosensitive PIEZO1 channel in dry eye corneal epithelium

The activation of the NLRP3 inflammasome by hyperosmotic stress is a critical pathophysiological response in dry eye disease (DED), driving the chronic cycle of inflammation on the ocular surface. The specific mechanism underlying hyperosmotic mechanical stimulation activates the NLRP3 inflammasome remains unclear. This study provides evidence that PIEZO1, a mechanosensitive ion channel, functions as the primary receptor for corneal epithelial cells in sensing mechanical stimulation induced by tear hyperosmolarity. Inhibition of PIEZO1 significantly reduces NLRP3 inflammasome-associated pyroptosis in corneal epithelial cells. These findings suggest a therapeutic strategy targeting mechanosensitive ion channels to manage chronic ocular surface inflammation in DED patients. Structured Abstract. Purpose: PIEZO1 modulates the inflammatory response by translating mechanical signals from osmotic pressure into biological processes. This study investigates the functional role of PIEZO1 in activating the NLRP3 inflammasome in corneal epithelial cells under hyperosmotic stress and evaluates its contribution to the pathogenesis of dry eye disease (DED). Methods: In the in vitro experiments, immortalized human corneal epithelial cells (HCECs) were cultured under hyperosmotic conditions (450mOsm). For in vivo studies, a dry eye disease mouse model was established by subcutaneous injection of scopolamine (SCOP) in C57BL/6 mice. After successfully inducing the dry eye model, corneal epithelial cell damage was assessed through corneal fluorescein staining scores and TUNEL assays. Protein expression levels were examined via western blotting and immunofluorescence staining, while mRNA expression was analyzed using quantitative RT-PCR. Activation of the NLRP3 inflammasome was evaluated by measuring IL-1 beta protein cleavage and the formation of ASC speckles. Results: In the DED model, activation of the NLRP3 inflammasome was detected in corneal epithelial cells, along with increased expression of PIEZO1. The PIEZO1-specific agonist Yoda1 induced upregulation of NLRP3 inflammasome-related gene expression and triggered NLRP3 inflammasome activation. Conversely, silencing PIEZO1 using siRNA or inhibiting its activity suppressed hyperosmotic stress-induced changes in NLRP3 inflammasome-related gene expression and activation. In vivo, PIEZO1 inhibition effectively prevented NLRP3 inflammasome activation in corneal epithelial cells and restored the damaged phenotype associated with dry eye disease. Conclusion: Hyperosmotic stress-induced activation of the NLRP3 inflammasome in corneal epithelial cells is mediated through PIEZO1 activation. The identification of PIEZO1's role in this DED-related pathophysiological response highlights its potential as a therapeutic target for mitigating inflammation in clinical settings.