Retinal degenerative diseases significantly contribute to visual impairment and blindness. Microglia reactivity is a hallmark of neurodegenerative diseases including retinal cell death and immunomodulation emerges as a therapeutic option. Indole-3-carbinol (I3C) is a natural ligand of aryl hydrocarbon receptor (AhR), with potent immunomodulatory properties. Here, we hypothesized that I3C may inhibit microglia reactivity and exert neuroprotective effects in the light-damaged murine retina mimicking important immunological aspects of retinal degeneration. BV-2 microglia were treated in vitro with I3C followed by lipopolysaccharide (LPS) stimulation to analyze pro-inflammatory and anti-oxidant responses by quantitative real-time PCR (qRT-PCR) and Western blots. Nitric oxide (NO) secretion, caspase 3/7 levels, phagocytosis rates, migration, and morphology were analyzed in control and AhR knockdown cells. I3C or vehicle was systemically applied to light-treated BALB/cJ mice as an experimental model of retinal degeneration. Pro-inflammatory and anti-oxidant responses in the retina were examined by qRT-PCR, ELISA, and Western blots. Immunohistochemical staining of retinal flat mounts and cryosections were performed. The retinal thickness and structure were evaluated by in vivo imaging using spectral domain-optical coherence tomography (SD-OCT). The in vitro data showed that I3C potently diminished LPS-induced pro-inflammatory gene expression of I-NOS, IL-1ß, NLRP3, IL-6, and CCL2 and induced anti-oxidants gene levels of NQO1, HMOX1, and CAT1 in BV-2 cells. I3C also reduced LPS-induced NO secretion, phagocytosis, and migration as important functional microglia parameters. siRNA-mediated knockdown of AhR partially prevented the previously observed gene regulatory events. The in vivo experiments revealed that I3C treatment diminished light-damage induced I-NOS, IL-1ß, NLRP3, IL-6, and CCL2 transcripts and also reduced CCL2, I-NOS, IL-1ß, p-NFkBp65 protein levels in mice. Moreover, I3C increased anti-oxidant NQO1 and HMOX1 protein levels in light-exposed retinas. Finally, I3C therapy prevented the accumulation of amoeboid microglia in the subretinal space and protected from retinal degeneration. The AhR ligand I3C potently counter-acts microgliosis and light-induced retinal damage, highlighting a potential treatment concept for retinal degeneration.

中文翻译：

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Indole-3-carbinol 调节小胶质细胞稳态并保护视网膜免于变性


视网膜退行性疾病会严重导致视力障碍和失明。小胶质细胞反应性是神经退行性疾病的标志，包括视网膜细胞死亡，免疫调节成为一种治疗选择。 Indole-3-carbinol (I3C) 是芳基烃受体 (AhR) 的天然配体，具有有效的免疫调节特性。在这里，我们假设 I3C 可能抑制小胶质细胞反应性，并在光损伤的小鼠视网膜中发挥神经保护作用，模仿视网膜变性的重要免疫学方面。在体外使用 I3C 处理 BV-2 小胶质细胞，然后进行脂多糖 (LPS) 刺激，通过定量实时 PCR (qRT-PCR) 和蛋白质印迹分析促炎和抗氧化反应。分析对照细胞和 AhR 敲除细胞中的一氧化氮 (NO) 分泌、半胱天冬酶 3/7 水平、吞噬率、迁移和形态。 I3C 或媒介物被系统地应用于光处理的 BALB/cJ 小鼠作为视网膜变性的实验模型。通过 qRT-PCR、ELISA 和蛋白质印迹检查视网膜的促炎和抗氧化反应。对视网膜平片和冷冻切片进行免疫组织化学染色。使用谱域光学相干断层扫描（SD-OCT）通过体内成像评估视网膜厚度和结构。体外数据显示，I3C 有效降低 BV 中 LPS 诱导的 I-NOS、IL-1ß、NLRP3、IL-6 和 CCL2 促炎基因表达，并诱导 NQO1、HMOX1 和 CAT1 抗氧化剂基因水平-2个细胞。 I3C 还减少 LPS 诱导的 NO 分泌、吞噬作用和迁移，这些都是重要的功能性小胶质细胞参数。 siRNA 介导的 AhR 敲低部分阻止了之前观察到的基因调控事件。 体内实验表明，I3C 治疗可减少小鼠中光损伤诱导的 I-NOS、IL-1ß、NLRP3、IL-6 和 CCL2 转录本，并降低 CCL2、I-NOS、IL-1ß、p-NFkBp65 蛋白水平。此外，I3C 增加了暴露在光下的视网膜中抗氧化剂 NQO1 和 HMOX1 蛋白的水平。最后，I3C 疗法可防止变形虫小胶质细胞在视网膜下腔积聚，并防止视网膜变性。 AhR 配体 I3C 能有效对抗小胶质细胞增生和光诱导的视网膜损伤，凸显了视网膜变性的潜在治疗概念。