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Updated: Oct 28, 2025
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Published on: May 2, 2020
Calcitriol Alleviates Hyperosmotic Stress-Induced Corneal Epithelial Cell Damage via Inhibiting the
Jing Zhang1, Yiqin Dai1, Yujing Yang1
1Eye 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.
Purpose:
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.
Insights
Pyroptosis, a cell death process, is elevated in dry eye disease. Calcitriol effectively reduces hyperosmotic stress-induced pyroptosis in corneal cells by inhibiting the NLRP3-ASC-caspase-1-GSDMD pathway, showing promise for dry eye treatment.
Area of Science:
- Ophthalmology
- Cell Biology
- Immunology
Background:
- Dry eye disease is linked to corneal inflammation initiated by inflammasome activation due to elevated tear osmolarity.
- Pyroptosis, a lytic form of programmed cell death, is a key consequence of inflammasome activation, mediated by gasdermin D (GSDMD).
Purpose of the Study:
- To investigate the role of pyroptosis in dry eye pathogenesis.
- To determine if calcitriol can mitigate hyperosmotic stress (HS)-induced pyroptosis in human corneal epithelial cells (iHCECs).
- To elucidate the underlying molecular mechanisms of calcitriol's action against HS-induced pyroptosis.
Main Methods:
- Assessed GSDMD expression in dry eye patients' tears via western blotting.
- Induced hyperosmotic stress in iHCECs in vitro to mimic dry eye conditions.
- Quantified pyroptosis using scanning electron microscopy, flow cytometry (caspase-1/PI staining), and ASC speck formation assays.
- Evaluated cell viability using CCK-8 and LDH release assays.
- Tested the effects of calcitriol and the pyroptosis inhibitor disulfiram.
Main Results:
- Pyroptosis markers, including elevated N-GSDMD, were detected in the tears of dry eye patients.
- HS significantly promoted pyroptosis in iHCECs, which was reduced by disulfiram.
- Calcitriol effectively suppressed HS-induced pyroptosis in iHCECs, mirroring the effects of disulfiram.
- Calcitriol's mechanism involves inhibiting the NLRP3-ASC-caspase-1-GSDMD pathway.
Conclusions:
- This study provides direct evidence of increased pyroptosis in dry eye patients.
- Calcitriol demonstrates efficacy in alleviating HS-induced corneal epithelial cell damage by inhibiting the pyroptosis pathway.
- Calcitriol presents potential as a therapeutic agent for dry eye due to its multifaceted therapeutic targets.
