全反式视黄醛诱导的视网膜色素上皮细胞死亡与 NLRP3 炎性小体的激活有关。
Retinal Pigment Epithelium Cell Death Is Associated With NLRP3 Inflammasome Activation by All-trans Retinal.
机构信息
Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, School of Medicine, Xiamen University, Xiamen City, China.
Department of Ophthalmology, Shenzhen Hospital, Southern Medical University, Shenzhen City, China.
出版信息
Invest Ophthalmol Vis Sci. 2019 Jul 1;60(8):3034-3045. doi: 10.1167/iovs.18-26360.
PURPOSE
Visual (retinoid) cycle anomalies induce aberrant build-up of all-trans retinal (atRAL) in the retinal pigment epithelium (RPE), which is a cause of RPE atrophy in Stargardt disease type 1 and age-related macular degeneration. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation is implicated in the etiology of age-related macular degeneration. Here, we elucidated the relationship between NLRP3 inflammasome activation and atRAL-induced death of RPE cells.
METHODS
Cellular toxicities were assessed by MTS or MTT assays. Expression levels of mRNAs and proteins were determined by quantitative reverse transcription-polymerase chain reaction, Western blotting, or enzyme-linked immunosorbent assay. Fluorescence microscopy was used to examine intracellular signals. Ultrastructural features of organelles were examined by transmission electron microscope.
RESULTS
Abnormal accumulation of atRAL was associated with a significant increase in the proportion of human ARPE-19 cells exhibiting features of apoptosis and Caspase-3/gasdermin E (GSDME)-mediated pyroptosis. These cells also exhibited elevated expression of NLRP3, ASC, cleaved Caspase-1/poly ADP-ribose polymerase (PARP)/Caspase-3/GSDME, interleukin-1β (IL-1β), and IL-18, as well as NLRP3 inflammasome-related genes (IL1B and IL18). After exposure of human ARPE-19 cells to excess atRAL, reactive oxygen species (ROS) (including mitochondrial ROS) and cathepsins released from lysosomes transmitted signals leading to NLRP3 inflammasome activation. Suppressing the production of ROS, NLRP3 inflammasome, Caspase-1, cathepsin B, or cathepsin D protected ARPE-19 cells against atRAL-associated cytotoxicity. Damage to mitochondria, lysosomes, and endoplasmic reticulum in atRAL-exposed ARPE-19 cells was partially alleviated by treatment with MCC950, a selective NLRP3 inflammasome inhibitor.
CONCLUSIONS
Aberrant build-up of atRAL promotes the death of RPE cells via NLRP3 inflammasome activation.
目的
视黄醇（retinoid）循环异常导致全反式视黄醛（all-trans retinal，atRAL）在视网膜色素上皮（retinal pigment epithelium，RPE）中的异常蓄积，这是 Stargardt 病 1 型和年龄相关性黄斑变性中 RPE 萎缩的原因。NLR 家族含有吡喃结构域蛋白 3（NLR family pyrin domain containing 3，NLRP3）炎性小体的激活与年龄相关性黄斑变性的发病机制有关。在这里，我们阐明了 NLRP3 炎性小体的激活与 atRAL 诱导的 RPE 细胞死亡之间的关系。
方法
通过 MTS 或 MTT 测定法评估细胞毒性。通过定量逆转录聚合酶链反应、Western blot 或酶联免疫吸附试验测定 mRNA 和蛋白质的表达水平。荧光显微镜用于检测细胞内信号。透射电子显微镜用于检查细胞器的超微结构特征。
结果
atRAL 的异常积累与人类 ARPE-19 细胞中表现出凋亡特征和半胱天冬酶-1/gasdermin E（GSDME）介导的细胞焦亡特征的细胞比例显著增加相关。这些细胞还表现出 NLRP3、ASC、裂解的 Caspase-1/多聚 ADP-核糖聚合酶（poly ADP-ribose polymerase，PARP）/Caspase-3/GSDME、白细胞介素-1β（interleukin-1β，IL-1β）和白细胞介素-18（IL-18）以及 NLRP3 炎性小体相关基因（IL1B 和 IL18）的表达升高。在人类 ARPE-19 细胞暴露于过量的 atRAL 后，来自溶酶体的活性氧（reactive oxygen species，ROS）（包括线粒体 ROS）和组织蛋白酶传递信号导致 NLRP3 炎性小体的激活。抑制 ROS、NLRP3 炎性小体、Caspase-1、组织蛋白酶 B 或组织蛋白酶 D 的产生可保护 ARPE-19 细胞免受 atRAL 相关的细胞毒性。MCC950（一种选择性 NLRP3 炎性小体抑制剂）治疗部分减轻了 atRAL 暴露的 ARPE-19 细胞中线粒体、溶酶体和内质网的损伤。
结论
atRAL 的异常蓄积通过 NLRP3 炎性小体的激活促进 RPE 细胞的死亡。
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