背景:甲状腺眼病(TED)是一种威胁视力的自身免疫性疾病。导致顽固性并发症的眼眶组织纤维化仍然是TED管理中的一个棘手问题。探索改善组织纤维化的新型治疗靶标和药物对于TED至关重要。最近的研究表明,Ca2+信号参与组织纤维化。然而,在TED期间,Ca2+信号的改变是否在纤维发生中起作用尚不清楚.在这项研究中,我们的目的是研究在TED期间Ca2+信号在纤维发生过程中的作用,以及高选择性L型钙通道(LTCC)抑制剂的潜在治疗作用,尼莫地平,通过TGF-β1诱导的体外TED模型。
方法:从患有TED的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)的原代培养。使用实时定量聚合酶链反应(RT-qPCR)和RNA测序来评估OF中与LTCC相关的基因表达。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(WB)用于评估细胞内Ca2+对TGF-β1刺激的反应,并评价尼莫地平在TGF-β1诱导的体外TED模型中的潜在治疗作用。通过免疫组织化学确定了Ca2/钙调蛋白依赖性蛋白激酶II(CaMKII)和信号转导和转录激活因子1(STAT1)在TED期间纤维发生中的作用。WB,流式细胞术和免疫共沉淀测定。选择性抑制剂用于探索下游信号通路。
结果:LTCC抑制剂尼莫地平阻断TGF-β1诱导的细胞内Ca2+反应,并进一步降低α-平滑肌肌动蛋白(α-SMA)的表达,OFs中的I型胶原α1(Col1A1)和I型胶原α2(Col1A2)。此外,尼莫地平抑制OFs细胞增殖和迁移。此外,我们的结果提供了证据,即在TED期间,CaMKII/STAT1信号通路的激活与纤维发生有关,尼莫地平通过下调CaMKII/STAT1信号通路抑制OFs的促纤维化功能。
结论:TGF-β1诱导LTCC介导的Ca2+反应,随后激活CaMKII/STAT1信号通路,在TED期间促进OFs的促纤维化功能并参与纤维发生。尼莫地平通过抑制CaMKII/STAT1信号通路在体外发挥有效的抗纤维化作用。我们的工作加深了我们对TED期间纤维发生过程的理解,并为TED提供了潜在的治疗靶标和替代候选物。
BACKGROUND: 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 Ca2+ signaling participates in tissue fibrosis. However, whether an alteration of Ca2+ signaling has a role in fibrogenesis during TED remains unclear. In this study, we aimed to investigate the role of Ca2+ 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.
METHODS: 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 Ca2+ 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 Ca2+/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.
RESULTS: LTCC inhibitor nimodipine blocked the TGF-β1 induced intracellular Ca2+ 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.
CONCLUSIONS: TGF-β1 induces an LTCC-mediated Ca2+ 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.