DataSheet_1_Dihydroartemisinin Exerts Antifibrotic and Anti-Inflammatory Effects in Graves’ Ophthalmopathy by Targeting Orbital Fibroblasts.docx

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.

Graves' 视神经炎（GO）是一种常见的眼眶疾病，严重威胁视力功能与外观。眼眶成纤维细胞（OFs）被视为GO中的关键靶细胞和效应细胞。此外，透明质酸（HA）的生成、炎症和眼眶纤维化与GO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GO来源的原代OFs的治疗效果。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OF的迁移能力，而qRT-PCR、Western blotting、ELISA和免疫荧光技术被用于检测这些细胞中纤维化相关和促炎标志物的表达。此外，通过RNA测序技术鉴定了DHA处理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，以探讨DHA在GO来源OFs中抗纤维化作用的潜在机制。结果表明，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诱导的纤维化标志物，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GO来源OFs中HA的产生。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的产生，显著缓解GO的致病表现。这些数据表明，DHA可能是一种治疗GO的具有潜力的候选药物。