Table_2_Dihydroartemisinin Exerts Antifibrotic and Anti-Inflammatory Effects in Graves’ Ophthalmopathy by Targeting Orbital Fibroblasts.xls

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 via 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.

格莱斯氏眼病（GO）是一种常见的眼眶疾病，威胁着视觉功能与外观。眼眶成纤维细胞（OFs）被视为格莱斯氏眼病中的关键靶细胞和效应细胞。此外，透明质酸（HA）的生成、炎症反应以及眼眶纤维化与格莱斯氏眼病的发病机制密切相关。在本研究中，我们探讨了抗疟药物双氢青蒿素（DHA）对由格莱斯氏眼病衍生的原代眼眶成纤维细胞（OFs）的疗效。通过CCK8和EdU实验评估了DHA对OFs的抗增殖作用。伤口愈合实验用于评估OFs的迁移能力，而实时荧光定量PCR、蛋白质印迹、ELISA和免疫荧光技术被用于检测这些细胞中与纤维化相关的和促炎症标记物的表达。此外，通过RNA测序识别了DHA处理后的OFs中差异表达基因（DEGs），并对DEGs进行了基因本体（GO）和京都基因与基因组百科全书（KEGG）通路富集分析，以探索DHA在格莱斯氏眼病衍生的OFs中抗纤维化作用的潜在机制。结果显示，DHA剂量依赖性地抑制了OFs的增殖，并在mRNA和蛋白质水平下调了TGF-β1诱导的纤维化标记物，包括α-平滑肌肌动蛋白（α-SMA）和结缔组织生长因子（CTGF）的表达。此外，DHA抑制了TGF-β1诱导的细胞外信号调节激酶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抑制了促炎细胞因子和趋化因子，包括IL-6、IL-8、CXCL-1、MCP-1和ICAM-1的表达，并减轻了由IL-1β诱导的格莱斯氏眼病衍生的OFs中HA的生成。总之，我们的研究首次提供了证据，表明DHA可能通过抑制OFs的增殖、纤维化和炎症相关基因的表达以及HA的生成，显著缓解格莱斯氏眼病的病理性表现。这些数据表明，DHA可能成为治疗格莱斯氏眼病的潜在候选药物。