State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China.
Front Endocrinol (Lausanne). 2022 May 17;13:891922. doi: 10.3389/fendo.2022.891922. eCollection 2022.
Graves' ophthalmopathy (GO) is a common orbital disease that threatens visual function and appearance. Orbital fibroblasts (OFs) are considered key target and effector cells in GO. In addition, hyaluronan (HA) production, inflammation, and orbital fibrosis are intimately linked to the pathogenesis of GO. In this study, we explored the therapeutic effects of dihydroartemisinin (DHA), an antimalarial drug, on GO-derived, primary OFs. CCK8 and EdU assays were applied to evaluate the antiproliferative effect of DHA on OFs. Wound healing assays were conducted to assess OF migration capacity, while qRT-PCR, western blotting, ELISA, and immunofluorescence were used to determine the expression of fibrosis-related and pro-inflammatory markers in these cells. Moreover, RNA sequencing was conducted to identify differentially expressed genes (DEGs) in DHA-treated OFs, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs was performed to explore potential mechanisms mediating the antifibrotic effect of DHA on GO-derived OFs. Results showed that DHA dose-dependently inhibited OF proliferation and downregulated, at the mRNA and protein levels, TGF-β1-induced expression of fibrosis markers, including alpha smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF). Furthermore, DHA inhibited TGF-β1 induced phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3), which suggested that DHA exerted antifibrotic effects suppression of the ERK and STAT3 signaling pathways. In addition, DHA suppressed the expression of pro-inflammatory cytokines and chemokines, including IL-6, IL-8, CXCL-1, MCP-1, and ICAM-1, and attenuated HA production induced by IL-1β in GO-derived OFs. In conclusion, our study provides first-time evidence that DHA may significantly alleviate pathogenic manifestations of GO by inhibiting proliferation, fibrosis- and inflammation-related gene expression, and HA production in OFs. These data suggest that DHA may be a promising candidate drug for treatment of GO.
格雷夫斯眼病（GO）是一种常见的眼眶疾病，威胁着视觉功能和外观。眼眶成纤维细胞（OFs）被认为是 GO 的关键靶细胞和效应细胞。此外，透明质酸（HA）的产生、炎症和眼眶纤维化与 GO 的发病机制密切相关。在这项研究中，我们探讨了抗疟药物二氢青蒿素（DHA）对 GO 来源的原代 OFs 的治疗作用。CCK8 和 EdU 检测用于评估 DHA 对 OFs 的增殖抑制作用。划痕愈合实验用于评估 OF 迁移能力，同时 qRT-PCR、western blot、ELISA 和免疫荧光用于测定这些细胞中纤维化相关和促炎标志物的表达。此外，进行 RNA 测序以鉴定 DHA 处理的 OFs 中的差异表达基因（DEGs），并对 DEGs 进行基因本体论（GO）和京都基因与基因组百科全书（KEGG）通路富集分析，以探讨介导 DHA 对 GO 来源的 OFs 的抗纤维化作用的潜在机制。结果表明，DHA 呈剂量依赖性抑制 OF 增殖，并在 mRNA 和蛋白水平下调 TGF-β1 诱导的纤维化标志物表达，包括α平滑肌肌动蛋白（α-SMA）和结缔组织生长因子（CTGF）。此外，DHA 抑制 TGF-β1 诱导的细胞外信号调节蛋白激酶 1/2（ERK1/2）和信号转导和转录激活因子 3（STAT3）的磷酸化，这表明 DHA 通过抑制 ERK 和 STAT3 信号通路发挥抗纤维化作用。此外，DHA 抑制促炎细胞因子和趋化因子的表达，包括 IL-6、IL-8、CXCL-1、MCP-1 和 ICAM-1，并减弱 GO 来源的 OFs 中由 IL-1β 诱导的 HA 产生。总之，本研究首次提供证据表明，DHA 通过抑制 OFs 增殖、纤维化和炎症相关基因表达以及 HA 产生，可能显著减轻 GO 的致病表现。这些数据表明，DHA 可能是治疗 GO 的有前途的候选药物。
