Novel Roles of Chloroquine and Hydroxychloroquine in Graves' Orbitopathy Therapy by Targeting Orbital Fibroblasts
Novel Roles of Chloroquine and Hydroxychloroquine in Graves' Orbitopathy Therapy by Targeting Orbital Fibroblasts
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氯喹和羟氯喹在针对眼眶成纤维细胞的格雷夫斯眼眶病治疗中的新作用
DOI：
10.1210/clinem/dgaa161
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发表时间：
2020-06-01
影响因子：
5.8
通讯作者：
Li, Yanbing
中科院分区：
文献类型：
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作者：
Guo, Yan;Li, Hai;Li, Yanbing
背景：Graves眼病（GO）通过诱导眼眶成纤维细胞（OFs）过度增殖、脂肪生成和糖胺聚糖生成引起浸润性突出眼。干扰OF细胞自噬是治疗前列腺增生的一种潜在方法。目的：本研究旨在评价临床常用的自噬抑制剂氯喹（CQ）和羟氯喹（HCQ）对OFs的影响。设计/环境/参与者：从GO-OFs患者（GO-OFs）或对照个体（非GO-OFs）分离的OFs在增殖培养基（PM）或分化培养基中培养。用CQ或HCQ（0、0.5、2和10 μ M）处理OFs，随后进行体外检测。主要观察指标：CCK-8、EdU掺入和流式细胞术检测用于评估细胞活力。采用Western blot分析、实时聚合酶链反应（PCR）和油红O染色评估脂肪生成。采用实时荧光定量PCR和酶联免疫吸附法测定透明质酸的产量。通过红色荧光蛋白(RFP)-绿色荧光蛋白(GFP)-LC3荧光染色和Western blot检测自噬通量。结果：CQ/HCQ通过阻断自噬以浓度依赖的方式阻止GO-OFs的增殖和脂肪形成，这种表型在非GO-OFs中未检测到。CQ/HCQ对GO-OFs透明质酸分泌的抑制作用也是浓度依赖性的，其介导机制是下调透明质酸合成酶2而不是下调透明质酸酶。CQ （10 μ M）可诱导GO-OF凋亡，但不加重氧化应激。结论：抗疟疾药CQ/HCQ通过抑制氧化石墨烯细胞自噬来影响氧化石墨烯细胞的增殖、脂肪形成和透明质酸的产生，证明其可作为自噬抑制剂治疗氧化石墨烯。
Context: Graves' orbitopathy (GO) causes infiltrative exophthalmos by inducing excessive proliferation, adipogenesis, and glycosaminoglycan production in orbital fibroblasts (OFs). Interference with OF autophagy is a potential therapy for proptosis.Objectives: Here, we aimed to evaluate the effects of chloroquine (CQ) and hydroxychloroquine (HCQ), the autophagy inhibitors commonly used in clinical practice, on OFs.Design/Setting/Participants: OFs isolated from patients with GO (GO-OFs) or control individuals (non-GO-OFs) were cultured in proliferation medium (PM) or subjected to differentiation medium. OFs were treated with CQ or HCQ (0, 0.5, 2, and 10 mu M), and subsequently examined in vitro.Main Outcome Measures: CCK-8, EdU incorporation, and flow cytometry assays were used to assess cellular viability. Adipogenesis was assessed with Western blot analysis, real-time polymerase chain reaction (PCR), and Oil Red O staining. Hyaluronan production was determined by real-time PCR and enzyme-linked immunosorbent assay. Autophagy flux was detected through red fluorescent protein (RFP)-green fluorescent protein (GFP)-LC3 fluorescence staining and Western blot analyses.Results: CQ/HCQ halted proliferation and adipogenesis in GO-OFs in a concentration-dependent manner through blockage of autophagy, phenotypes that were not detected in non-GO-OFs. The inhibitory effect of CQ/HCQ on hyaluronan secretion of GO-OFs was also concentration dependent, mediated by downregulation of hyaluronan synthase 2 rather than hyaluronidases. Moreover, CQ (10 mu M) induced GO-OF apoptosis without aggravating oxidative stress.Conclusions: The antimalarials CQ/HCQ affect proliferation, adipogenesis, and hyaluronan generation in GO-OFs by inhibiting autophagy, providing evidence that they can be used to treat GO as autophagy inhibitors.