Guo Yan, Li Hai, Chen Xueying, Yang Huasheng, Guan Hongyu, He Xiaoying, Chen Yuxin, Pokharel Sunil, Xiao Haipeng, Li Yanbing
Department of Endocrinology and Diabetes Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
J Clin Endocrinol Metab. 2020 Jun 1;105(6):1906-17. doi: 10.1210/clinem/dgaa161.
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.
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 μM), and subsequently examined in vitro.
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.
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 μM) induced GO-OF apoptosis without aggravating oxidative stress.
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.
格雷夫斯眼眶病（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产生，导致浸润性突眼。干扰OF自噬是治疗眼球突出的一种潜在疗法。
在此，我们旨在评估临床实践中常用的自噬抑制剂氯喹（CQ）和羟氯喹（HCQ）对OFs的影响。
设计/地点/参与者：从GO患者（GO-OFs）或对照个体（非GO-OFs）分离的OFs在增殖培养基（PM）中培养或接受分化培养基处理。OFs用CQ或HCQ（0、0.5、2和10μM）处理，随后进行体外检测。
采用CCK-8、EdU掺入和流式细胞术检测评估细胞活力。通过蛋白质免疫印迹分析、实时聚合酶链反应（PCR）和油红O染色评估脂肪生成。通过实时PCR和酶联免疫吸附测定法测定透明质酸产生。通过红色荧光蛋白（RFP）-绿色荧光蛋白（GFP）-LC3荧光染色和蛋白质免疫印迹分析检测自噬通量。
CQ/HCQ通过阻断自噬以浓度依赖性方式阻止GO-OFs的增殖和脂肪生成，在非GO-OFs中未检测到这些表型。CQ/HCQ对GO-OFs透明质酸分泌的抑制作用也是浓度依赖性的，由透明质酸合酶2的下调而非透明质酸酶介导。此外，CQ（10μM）诱导GO-OF凋亡而不加重氧化应激。
抗疟药CQ/HCQ通过抑制自噬影响GO-OFs的增殖、脂肪生成和透明质酸生成，为它们作为自噬抑制剂用于治疗GO提供了证据。
