BACKGROUND Fabry disease (FD) is an X-linked lysosomal storage disorder that leads to cellular globotriaosylceramide (Gb3) accumulation due to mutations in the gene encoding α-galactosidase A. Trigger-induced acral burning pain is an early FD symptom of unknown pathophysiology. We aimed at investigating the potential role of skin fibroblasts in nociceptor sensitization. PATIENTS AND METHODS We enrolled 40 adult FD patients and ten healthy controls, who underwent a 6-mm skin punch biopsy at the lower leg. Dermal fibroblasts were cultivated and analyzed for Gb3 load. Fibroblast electrical activity was assessed using patch-clamp analysis at baseline and upon incubation with agalsidase-α for 24 h. We investigated gene expression of CC motif chemokine ligand 2 (CCL2), Ca2+activated K+-channel 1.1 (KCa1.1), interferone-γ (IFN-γ), transforming growth factor-β1 (TGF-β1), and transmembrane receptor notch homolog 1 (Notch1) using quantitative real-time-PCR, and protein levels of KCa1.1 by ELISA. Gene expression was determined at baseline and after fibroblast stimulation with tumor necrosis factor-α (TNF), modeling inflammation as a common pain trigger in FD. RESULTS Total Gb3 load was higher in FD fibroblasts than in control fibroblasts (p < .01). Upon increase of intracellular Ca2+ concentrations, we detected differential electrical activity of KCa1.1 in fibroblasts obtained from patients with FD. Gene expression (p < .05) and protein levels of KCa1.1 (p < .05) were higher in fibroblasts from FD patients compared to control fibroblasts, whereas electric channel activity was lower in FD fibroblasts. After incubation with agalsidase-α, we observed an over-proportionate increase of KCa1.1 activity in FD fibroblasts reaching 7-fold the currents of control cells (p < .01). Gene expression studies revealed higher mRNA levels of CCL2, INF-γ, and Notch1 in FD fibroblasts compared to controls at baseline and after TNF incubation (p < .05 each), while TGF-β1 was higher in FD fibroblasts only after incubation with TNF (p < .05). CONCLUSIONS Gb3 deposition in skin fibroblasts may impair KCa1.1 activity and activate the Notch1 signaling pathway. The resulting increase in pro-inflammatory mediator expression may contribute to cutaneous nociceptor sensitization as a potential mechanism of FD-associated pain.

中文翻译：

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globotriaosylceramide诱导男性法布里疼痛患者的皮肤成纤维细胞中KCa1.1通道活性的降低和Notch1信号通路的激活。

背景技术法布里病（FD）是一种X连锁的溶酶体贮积病，由于编码α-半乳糖苷酶A的基因突变，导致细胞globotriaosylceramide（Gb3）积累。触发诱发的急性烧灼性疼痛是病理生理未知的早期FD症状。我们旨在研究皮肤成纤维细胞在伤害感受器敏化中的潜在作用。患者和方法我们招募了40名成人FD患者和10名健康对照，他们对小腿进行了6毫米皮肤打孔活检。培养真皮成纤维细胞并分析Gb3负荷。在基线和与阿糖苷酶-α孵育24小时后，使用膜片钳分析评估成纤维细胞的电活性。我们研究了CC基序趋化因子配体2（CCL2），Ca2 +激活的K +通道1.1（KCa1.1），干扰素-γ（IFN-γ），实时荧光定量PCR检测转化生长因子-β1（TGF-β1）和跨膜受体缺口同源物1（Notch1），并通过ELISA检测KCa1.1的蛋白水平。在基线和成纤维细胞用肿瘤坏死因子-α（TNF）刺激后确定基因表达，将炎症建模为FD中常见的疼痛触发因素。结果FD成纤维细胞的总Gb3负载高于对照成纤维细胞（p <.01）。随着细胞内Ca2 +浓度的增加，我们在从FD患者获得的成纤维细胞中检测到了KCa1.1的不同电活性。与对照成纤维细胞相比，FD患者的成纤维细胞的基因表达（p <.05）和KCa1.1的蛋白质水平（p <.05）较高，而FD成纤维细胞的电通道活性较低。与半乳糖苷酶-α孵育后，我们观察到FD成纤维细胞中KCa1.1活性过度增加，达到了对照细胞电流的7倍（p <0.01）。基因表达研究显示，与基线和TNF培养后的对照相比，FD成纤维细胞中的CCL2，INF-γ和Notch1的mRNA水平更高（每个p <.05），而FD成纤维细胞中的TGF-β1仅在与TNF培养后更高。 （p <.05）。结论Gb3在皮肤成纤维细胞中的沉积可能损害KCa1.1活性并激活Notch1信号通路。促炎性介质表达的增加可能有助于皮肤伤害感受器的致敏，这是FD相关性疼痛的潜在机制。在基线和TNF孵育后，FD成纤维细胞中的Notch1和Notch1与对照组相比（每个p <.05），而在FD纤维成纤维细胞中，仅在与TNF孵育后，TGF-β1更高（p <.05）。结论Gb3在皮肤成纤维细胞中的沉积可能损害KCa1.1活性并激活Notch1信号通路。促炎性介质表达的增加可能有助于皮肤伤害感受器的致敏，这是与FD相关的疼痛的潜在机制。在基线和TNF孵育后，FD成纤维细胞中的Notch1和Notch1与对照组相比（每个p <.05），而在FD纤维成纤维细胞中，仅在与TNF孵育后，TGF-β1更高（p <.05）。结论Gb3在皮肤成纤维细胞中的沉积可能损害KCa1.1活性并激活Notch1信号通路。促炎性介质表达的增加可能有助于皮肤伤害感受器的致敏，这是与FD相关的疼痛的潜在机制。