Ocular Immunology and Angiogenesis Lab, Department of Ophthalmology and Visual Sciences, Medical College of Wisconsin, Milwaukee, WI, 53226, USA.
Ophthalmology and Molecular Medicine, Mason Eye Institute, University of Missouri, Columbia, MO, 65211, USA.
Exp Eye Res. 2023 May;230:109461. doi: 10.1016/j.exer.2023.109461. Epub 2023 Apr 5.
Sulfur mustard (SM) is a chemical warfare agent (CWA) that causes severe eye pain, photophobia, excessive lacrimation, corneal and ocular surface defects, and blindness. However, SM's effects on retinal cells are relatively meager. This study investigated the role of SM toxicity on Müller glial cells responsible for cellular architecture, inner blood-retinal barrier maintenance, neurotransmitter recycling, neuronal survival, and retinal homeostasis. Müller glial cells (MIO-M1) were exposed to SM analog, nitrogen mustard (NM), at varying concentrations (50-500 μM) for 3 h, 24 h, and 72 h. Müller cell gliosis was evaluated using morphological, cellular, and biochemical methods. Real-time cellular integrity and morphological evaluation were performed using the xCELLigence real-time monitoring system. Cellular viability and toxicity were measured using TUNEL and PrestoBlue assays. Müller glia hyperactivity was calculated based on glial fibrillary acidic protein (GFAP) and vimentin immunostaining. Intracellular oxidative stress was measured using DCFDA and DHE cell-based assays. Inflammatory markers and antioxidant enzyme levels were determined by quantitative real-time PCR (qRT-PCR). AO/Br and DAPI staining further evaluated DNA damage, apoptosis, necrosis, and cell death. Inflammasome-associated Caspase-1, ASC, and NLRP3 were studied to identify mechanistic insights into NM toxicity in Müller glial cells. The cellular and morphological evaluation revealed the Müller glia hyperactivity after NM exposure in a dose- and time-dependent manner. NM exposure caused significant oxidative stress and enhanced cell death at 72 h. A significant increase in antioxidant indices was observed at the lower concentrations of NM. Mechanistically, we found that NM-treated MIO-M1 cells increased caspase-1 levels that activated NLRP3 inflammasome-induced production of IL-1β and IL-18, and elevated Gasdermin D (GSDMD) expression, a crucial component actuating pyroptosis. In conclusion, NM-induced Müller cell gliosis via increased oxidative stress results in caspase-1-dependent activation of the NLRP3 inflammasome and cell death driven primarily by pyroptosis.
硫芥（SM）是一种化学战剂（CWA），会引起严重的眼睛疼痛、畏光、过度流泪、角膜和眼表面缺陷以及失明。然而，SM 对视网膜细胞的影响相对较小。本研究探讨了 SM 毒性对负责细胞结构、内血视网膜屏障维持、神经递质回收、神经元存活和视网膜内稳态的 Müller 胶质细胞的作用。Müller 胶质细胞（MIO-M1）暴露于 SM 类似物氮芥（NM），浓度分别为 50-500 μM，暴露时间分别为 3 h、24 h 和 72 h。使用形态学、细胞和生化方法评估 Müller 细胞胶质增生。使用 xCELLigence 实时监测系统进行实时细胞完整性和形态评估。使用 TUNEL 和 PrestoBlue 测定法测量细胞活力和毒性。根据胶质纤维酸性蛋白（GFAP）和波形蛋白免疫染色计算 Müller 胶质细胞过度活跃。使用 DCFDA 和 DHE 细胞测定法测量细胞内氧化应激。通过定量实时 PCR（qRT-PCR）测定炎症标志物和抗氧化酶水平。AO/Br 和 DAPI 染色进一步评估 DNA 损伤、细胞凋亡、坏死和细胞死亡。研究了炎性小体相关的 Caspase-1、ASC 和 NLRP3，以确定 NM 对 Müller 胶质细胞毒性的机制见解。细胞和形态学评估显示，NM 暴露后 Müller 胶质细胞呈剂量和时间依赖性过度活跃。NM 暴露在 72 h 时会导致明显的氧化应激和细胞死亡增加。在 NM 的较低浓度下观察到抗氧化指数显著增加。从机制上讲，我们发现 NM 处理的 MIO-M1 细胞增加了 Caspase-1 水平，激活了 NLRP3 炎性小体诱导的 IL-1β 和 IL-18 的产生，并上调了 Gasdermin D（GSDMD）表达，这是激活细胞焦亡的关键组成部分。总之，NM 通过增加氧化应激诱导 Müller 细胞胶质增生，导致 caspase-1 依赖性 NLRP3 炎性小体激活和主要由细胞焦亡驱动的细胞死亡。
