Zhang Jing, Dai Yiqin, Yang Yujing, Xu Jianjiang
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.
J Inflamm Res. 2021 Jul 5;14:2955-2962. doi: 10.2147/JIR.S310116. eCollection 2021.
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.
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.
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.
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.
响应泪液渗透压升高的炎性小体激活在干眼相关角膜炎症中起初始信号的作用。细胞焦亡是炎性小体激活的另一个显著后果，其特征是由gasdermin D（GSDMD）驱动的细胞裂解。本研究旨在探讨细胞焦亡在干眼中的作用，并验证骨化三醇（一种潜在的干眼治疗药物）是否对人角膜上皮细胞（iHCECs）中高渗应激（HS）诱导的细胞焦亡有一定作用及其潜在机制。
使用蛋白质免疫印迹法检测干眼患者泪液中细胞焦亡执行者GSDMD的表达。将iHCECs培养在高渗培养基（450 mOsM）中，以在体外模拟干眼时泪液渗透压升高的特征。使用外源性骨化三醇或细胞焦亡抑制剂双硫仑。通过扫描电子显微镜、流式细胞术检测半胱天冬酶 -1和碘化丙啶（PI）双染色、ASC斑点形成的免疫荧光染色以及蛋白质免疫印迹法，检测不同处理下iHCECs的细胞焦亡程度。通过CCK-8法和乳酸脱氢酶（LDH）释放试验测量细胞活力。
我们发现干眼患者存在细胞焦亡，表现为患者泪液中其效应物GSDMD N端结构域（N-GSDMD）升高。进一步的体外结果表明，HS促进人角膜上皮细胞的细胞焦亡，而外源性补充双硫仑可减少具有细胞焦亡标记的iHCECs数量。更重要的是，我们证明，与双硫仑的作用一致，骨化三醇也可通过抑制NLRP3-ASC-半胱天冬酶 -1-GSDMD细胞焦亡途径减轻HS诱导的细胞焦亡。
本研究提供了直接证据表明干眼患者的细胞焦亡增加。我们证明骨化三醇能够通过抑制NLRP3-ASC-半胱天冬酶 -1-GSDMD细胞焦亡途径有效减轻HS诱导的角膜上皮细胞损伤。鉴于其多个治疗靶点，本研究强调骨化三醇是一种有前景的干眼治疗药物。
Zotero 插件
