Circulating fibrocytes are an important source of fibroblasts and myofibroblasts, which are involved in fibrotic processes, including systemic sclerosis (SSc). The study aimed to investigate the effect of nintedanib (a tyrosine kinase inhibitor) in inhibiting the in vitro transition of circulating SSc fibrocytes into myofibroblasts and their pro-fibrotic activity. Circulating fibrocytes were obtained from 18 SSc patients and 5 healthy subjects (HSs). Cultured SSc fibrocytes were maintained in growth medium (untreated cells) or treated with nintedanib 0.1 and 1 μM for 3 and 24 h. Fibroblast-specific protein-1 (S100A4) and α-smooth muscle actin (αSMA), as markers of fibroblast/myofibroblast phenotype, together with type I collagen (COL1) and fibronectin (FN), were investigated by qRT-PCR and Western blotting. Non-parametric tests were used for statistical analysis. Significantly elevated gene and protein expressions of αSMA, S100A4, COL1, and FN were observed in SSc fibrocytes compared to HS fibrocytes (gene: αSMA p < 0.001; others p < 0.0001; protein: all p < 0.05). Interestingly, an increased gene and protein expression of αSMA and S100A4 was found in fibrocytes from SSc patients positive for anti-Scl70 and with interstitial lung disease (ILD) (Scl70+ILD+) compared to Scl70−ILD− patients (S100A4: gene: p < 0.01; protein: p < 0.05), whereas no differences were observed for COL1 and FN. Nintedanib reduced gene and protein expression of αSMA, S100A4, COL1, and FN in SSc fibrocytes compared to untreated ones with different statistical significance. Noteworthy, nintedanib significantly downregulated gene and protein expression of αSMA, S100A4, COL1, and FN in Scl70+ILD+ fibrocytes (all p < 0.05), whereas only that of S100A4 and FN was significantly downregulated (p < 0.05) in Scl70−ILD− fibrocytes compared to the related untreated cells. Nintedanib seems to downregulate in vitro the transition of fibrocytes into myofibroblasts and their pro-fibrotic activity, particularly in cells isolated from Scl70+ILD+ SSc patients.

中文翻译：

---

尼达尼布下调培养的系统性硬化症纤维细胞向肌成纤维细胞的转变及其促纤维化活性

循环纤维细胞是成纤维细胞和肌成纤维细胞的重要来源，参与纤维化过程，包括系统性硬化症（SSc）。该研究旨在探讨尼达尼布（一种酪氨酸激酶抑制剂）在体外抑制循环 SSc 纤维细胞向肌成纤维细胞转变及其促纤维化活性的作用。循环纤维细胞取自 18 名 SSc 患者和 5 名健康受试者 (HS)。将培养的 SSc 纤维细胞维持在生长培养基（未处理的细胞）中或用 nintedanib 0.1 和 1 μM 处理 3 和 24 小时。通过 qRT-PCR 和蛋白质印迹法研究了成纤维细胞特异性蛋白 1 (S100A4) 和 α-平滑肌肌动蛋白 (αSMA) 作为成纤维细胞/肌成纤维细胞表型的标记物，以及 I 型胶原 (COL1) 和纤连蛋白 (FN) 。使用非参数检验进行统计分析。与 HS 纤维细胞相比，SSc 纤维细胞中 αSMA、S100A4、COL1 和 FN 的基因和蛋白表达显着升高（基因：αSMA p < 0.001；其他 p < 0.0001；蛋白：所有 p < 0.05）。有趣的是，与 Scl70−ILD− 患者相比，抗 Scl70 阳性且患有间质性肺疾病 (ILD) (Scl70+ILD+) 的 SSc 患者的纤维细胞中发现 αSMA 和 S100A4 的基因和蛋白表达增加（S100A4：基因：p < 0.01；蛋白质：p < 0.05），而 COL1 和 FN 没有观察到差异。与未治疗的相比，尼达尼布降低了 SSc 纤维细胞中 αSMA、S100A4、COL1 和 FN 的基因和蛋白表达，具有不同的统计显着性。值得注意的是，尼达尼布显着下调 Scl70+ILD+ 纤维细胞中 αSMA、S100A4、COL1 和 FN 的基因和蛋白表达（均 p < 0.05），而在 Scl70−ILD− 中仅 S100A4 和 FN 的基因和蛋白表达显着下调（p < 0.05）。纤维细胞与相关的未处理细胞相比。尼达尼布似乎在体外下调纤维细胞向肌成纤维细胞的转变及其促纤维化活性，特别是在从 Scl70+ILD+ SSc 患者分离的细胞中。