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首页> 外文期刊>Cellular Physiology and Biochemistry >Oxidative Stress is the Principal Contributor to Inflammasome Activation in Retinal Pigment Epithelium Cells with Defunct Proteasomes and Autophagy
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Oxidative Stress is the Principal Contributor to Inflammasome Activation in Retinal Pigment Epithelium Cells with Defunct Proteasomes and Autophagy

机译：氧化应激是导致死亡的蛋白酶体和自噬的视网膜色素上皮细胞中炎症小体活化的主要因素。

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Background/Aims Previously, we demonstrated that blockade of the intracellular clearance systems in human retinal pigment epithelial (RPE) cells by MG-132 and bafilomycin A1 (BafA) induces NLRP3 inflammasome signaling. Here, we have explored the activation mechanisms behind this process. NLRP3 is an intracellular receptor detecting factors ranging from the endogenous alarmins and adenosine triphosphate (ATP) to ultraviolet radiation and solid particles. Due to the plethora of triggers, the activation of NLRP3 is often indirect and can be mediated through several alternative pathways. Potassium efflux, lysosomal rupture, and oxidative stress are currently the main mechanisms associated with many activators. Methods NLRP3 inflammasomes were activated in human RPE cells by blocking proteasomes and autophagy using MG-132 and bafilomycin A1 (BafA), respectively. P2X7 inhibitor A740003, potassium chloride (KCl), and glyburide, or N-acetyl-L-cysteine (NAC), ammonium pyrrolidinedithiocarbamate (APDC), diphenyleneiodonium chloride (DPI), and mito-TEMPO were added to cell cultures in order to study the role of potassium efflux and oxidative stress, respectively. IL-1β was measured using the ELISA method. ATP levels and cathepsin B activity were examined using commercial kits, and ROS levels using the fluorescent dye 2´,7´-dichlorodihydrofluorescein diacetate (DCFDA). Results Elevated extracellular potassium prevented the priming factor IL-1α from inducing the production of reactive oxygen species (ROS). It also prevented IL-1β release after exposure of primed cells to MG-132 and BafA. Inflammasome activation increased extracellular ATP levels, which did not appear to trigger significant potassium efflux. The activity of the lysosomal enzyme, cathepsin B, was reduced by MG-132 and BafA, suggesting that cathepsin B was not playing any role in this phenomenon. Instead, MG-132 triggered ROS production already 30 min after exposure, but treatment with antioxidants blocking NADPH oxidase and mitochondria-derived ROS significantly prevented IL-1β release after this activating signal. Conclusion Our data suggest that oxidative stress strongly contributes to the NLRP3 inflammasome activation upon dysfunctional cellular clearance. Clarification of inflammasome activation mechanisms provides novel options for alleviating pathological inflammation present in aggregation diseases, such as age-related macular disease (AMD) and Alzheimer’s disease.
机译：背景/目的以前，我们证明了MG-132和bafilomycin A1（BafA）对人视网膜色素上皮（RPE）细胞的细胞内清除系统的诱导可诱导NLRP3炎性体信号传导。在这里，我们探索了此过程背后的激活机制。 NLRP3是一种细胞内受体，可检测各种因子，范围从内源性警报蛋白和三磷酸腺苷（ATP）到紫外线辐射和固体颗粒。由于过多的触发因素，NLRP3的激活通常是间接的，可以通过几种替代途径介导。钾外流，溶酶体破裂和氧化应激是目前与许多活化剂相关的主要机制。方法MGRP-132和bafilomycin A1（BafA）分别通过封闭蛋白酶体和自噬作用，在人RPE细胞中激活NLRP3炎性体。为了研究，将P2X7抑制剂A740003，氯化钾（KCl）和格列本脲或N-乙酰基-L-半胱氨酸（NAC），吡咯烷二硫代氨基甲酸铵（APDC），二苯基碘化铵氯化物（DPI）和mito-TEMPO加入了细胞培养物中。钾外排和氧化应激的作用分别。使用ELISA方法测量IL-1β。使用商业试剂盒检测ATP水平和组织蛋白酶B活性，使用荧光染料2'，7'-dichlorodihydrofluorescein diacetate（DCFDA）检查ROS水平。结果升高的细胞外钾可阻止启动因子IL-1α诱导活性氧（ROS）的产生。在引发细胞暴露于MG-132和BafA后，它还阻止了IL-1β的释放。炎性体激活增加了细胞外ATP的水平，这似乎并未触发明显的钾外流。 MG-132和BafA降低了溶酶体酶组织蛋白酶B的活性，这表明组织蛋白酶B在此现象中没有任何作用。取而代之的是，MG-132在暴露30分钟后已经触发了ROS的产生，但是用抗氧化剂阻断NADPH氧化酶和线粒体衍生的ROS的处理显着阻止了该激活信号后IL-1β的释放。结论我们的数据表明，氧化应激在细胞清除功能异常时强烈促进NLRP3炎性体的活化。炎症小体激活机制的澄清为减轻聚集性疾病（例如年龄相关性黄斑疾病（AMD）和阿尔茨海默氏病）中存在的病理性炎症提供了新的选择。

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