Calcitriol Alleviates Hyperosmotic Stress-Induced Corneal Epithelial Cell Damage via Inhibiting the NLRP3–ASC–Caspase-1–GSDMD Pyroptosis Pathway in Dry Eye Disease

Abstract

Jing Zhang,* Yiqin Dai,* Yujing Yang, Jianjiang Xu Eye Institute and Department of Ophthalmology, Eye & ENT Hospital, Fudan University; Shanghai Key Laboratory of Visual Impairment and Restoration; NHC Key Laboratory of Myopia, Fudan University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jianjiang XuDepartment of Ophthalmology and Visual Science, Eye, and ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, People’s Republic of ChinaEmail [email protected]: Inflammasome activation in response to elevated tear osmolarity behaves as an initial signal in dry eye-related corneal inflammation. Pyroptosis is another prominent consequence of inflammasome activation, which is featured by gasdermin D (GSDMD)-driven cell lysis. This study aims to explore the role of pyroptosis in dry eye, and also to verify if calcitriol, a potential therapeutic agent for dry eye, has certain effects against hyperosmotic stress (HS)-induced pyroptosis in human corneal epithelial cells (iHCECs) and the underlying mechanism.Methods: The expression of pyroptosis executor GSDMD in tears from dry eye patients was examined using western blotting. iHCECs were grown in hyperosmotic medium (450 mOsM) to mimic the feature of elevated tear osmolality of dry eye in vitro. Exogenous calcitriol or pyroptosis inhibitor disulfiram was used. The extent of pyroptosis of iHCECs under various treatments was examined by scanning electron microscopy, caspase-1 and propidium iodide (PI) double staining by flow cytometry, immunofluorescent staining for ASC speck formation, and western blotting. Cell viability was measured by a CCK-8 assay and an LDH release assay.Results: We found that pyroptosis was presented in dry eye patients, shown as the elevation of its effector GSDMD N-terminal domain (N-GSDMD) in patients’ tears. Further in vitro results showed that HS promoted pyroptosis in human corneal epithelial cells, while exogeneous supplementation of disulfiram could reduce the number of iHCECs with pyroptotic markers. More importantly, we demonstrated that, in line with the effect of disulfiram, calcitriol could also alleviate HS-induced pyroptosis, through inhibiting the NLRP3–ASC–caspase-1–GSDMD pyroptosis pathway.Conclusion: The current study provided direct evidence showing increased pyroptosis in dry eye patients. We demonstrated that calcitriol was able to effectively alleviate HS-induced corneal epithelial cell damage through inhibiting the NLRP3–ASC–caspase-1–GSDMD pyroptosis pathway. This study underlined calcitriol as a promising therapeutic agent for dry eye given its multiple therapeutic targets.Keywords: calcitriol, dry eye, pyroptosis, GSDMD

Keywords

• calcitriol
• dry eye
• pyroptosis
• gsdmd