Chen Qian, Pan Yuan, Hu Yunwei, Chen Guanyu, Chen Xiaoqing, Xie Yanyan, Wang Minzhen, Li Zhuang, Huang Jun, Shi Yuxun, Huang Haixiang, Zhang Te, Wang Mei, Zeng Peng, Wang Sha, Chen Rongxin, Zheng Yongxin, Zhong Liuxueying, Yang Huasheng, Liang Dan
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology Visual Science, Guangzhou, 510060, China.
Department of Ophthalmology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510630, China.
Eye Vis (Lond). 2024 Sep 6;11(1):37. doi: 10.1186/s40662-024-00401-5.
Thyroid eye disease (TED) is a vision-threatening autoimmune disorder. Orbital tissue fibrosis leading to intractable complications remains a troublesome issue in TED management. Exploration of novel therapeutic targets and agents to ameliorate tissue fibrosis is crucial for TED. Recent work suggests that Ca signaling participates in tissue fibrosis. However, whether an alteration of Ca signaling has a role in fibrogenesis during TED remains unclear. In this study, we aimed to investigate the role of Ca signaling in the fibrogenesis process during TED and the potential therapeutic effects of a highly selective inhibitor of the L-type calcium channel (LTCC), nimodipine, through a TGF-β1 induced in vitro TED model.
Primary culture of orbital fibroblasts (OFs) were established from orbital adipose connective tissues of patients with TED and healthy control donors. Real-time quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing were used to assess the genes expression associated with LTCC in OFs. Flow cytometry, RT-qPCR, 5-ethynyl-2'-deoxyuridine (EdU) proliferation assay, wound healing assay and Western blot (WB) were used to assess the intracellular Ca response on TGF-β1 stimulation, and to evaluate the potential therapeutic effects of nimodipine in the TGF-β1 induced in vitro TED model. The roles of Ca/calmodulin-dependent protein kinase II (CaMKII) and signal transducer and activator of transcription 1 (STAT1) in fibrogenesis during TED were determined by immunohistochemistry, WB, flow cytometry and co-immunoprecipitation assay. Selective inhibitors were used to explore the downstream signaling pathways.
LTCC inhibitor nimodipine blocked the TGF-β1 induced intracellular Ca response and further reduced the expression of alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 (Col1A1) and collagen type I alpha 2 (Col1A2) in OFs. Besides, nimodipine inhibited cell proliferation and migration of OFs. Moreover, our results provided evidence that activation of the CaMKII/STAT1 signaling pathway was involved in fibrogenesis during TED, and nimodipine inhibited the pro-fibrotic functions of OFs by down-regulating the CaMKII/STAT1 signaling pathway.
TGF-β1 induces an LTCC-mediated Ca response, followed by activation of CaMKII/STAT1 signaling pathway, which promotes the pro-fibrotic functions of OFs and participates in fibrogenesis during TED. Nimodipine exerts potent anti-fibrotic benefits in vitro by suppressing the CaMKII/STAT1 signaling pathway. Our work deepens our understanding of the fibrogenesis process during TED and provides potential therapeutic targets and alternative candidate for TED.
甲状腺眼病（TED）是一种威胁视力的自身免疫性疾病。眼眶组织纤维化导致难以处理的并发症，仍然是TED治疗中的一个棘手问题。探索新的治疗靶点和药物以改善组织纤维化对TED至关重要。最近的研究表明，钙信号参与组织纤维化。然而，在TED期间钙信号的改变是否在纤维化发生中起作用仍不清楚。在本研究中，我们旨在通过转化生长因子-β1（TGF-β1）诱导的体外TED模型，研究钙信号在TED纤维化过程中的作用以及L型钙通道（LTCC）高度选择性抑制剂尼莫地平的潜在治疗效果。
从TED患者和健康对照供体的眼眶脂肪结缔组织中建立眼眶成纤维细胞（OFs）原代培养。采用实时定量聚合酶链反应（RT-qPCR）和RNA测序评估OFs中与LTCC相关的基因表达。采用流式细胞术、RT-qPCR、5-乙炔基-2'-脱氧尿苷（EdU）增殖试验、伤口愈合试验和蛋白质印迹法（WB）评估TGF-β1刺激后的细胞内钙反应，并评估尼莫地平在TGF-β1诱导的体外TED模型中的潜在治疗效果。通过免疫组织化学、WB、流式细胞术和免疫共沉淀试验确定钙/钙调蛋白依赖性蛋白激酶II（CaMKII）和信号转导子与转录激活子1（STAT1）在TED纤维化过程中的作用。使用选择性抑制剂探索下游信号通路。
LTCC抑制剂尼莫地平阻断了TGF-β1诱导的细胞内钙反应，并进一步降低了OFs中α-平滑肌肌动蛋白（α-SMA）、I型胶原α1（Col1A1）和I型胶原α2（Col1A2）的表达。此外，尼莫地平抑制了OFs的细胞增殖和迁移。此外，我们的结果表明，CaMKII/STAT1信号通路的激活参与了TED的纤维化过程，尼莫地平通过下调CaMKII/STAT1信号通路抑制了OFs的促纤维化功能。
TGF-β1诱导LTCC介导的钙反应，随后激活CaMKII/STAT1信号通路，促进OFs的促纤维化功能并参与TED的纤维化过程。尼莫地平通过抑制CaMKII/STAT1信号通路在体外发挥强大的抗纤维化作用。我们的工作加深了我们对TED纤维化过程的理解，并为TED提供了潜在的治疗靶点和替代候选药物。
