Department of Cardiology, First Affiliated Hospital of Harbin Medical University, No. 199 Dazhi Street, Harbin 150001, China.
Department of Medical Oncology, The Fourth Affiliated Hospital of Harbin Medical University, No. 37 YiYuan Street, Harbin 150001, China.
Biochimie. 2018 May;148:55-62. doi: 10.1016/j.biochi.2018.02.017. Epub 2018 Mar 1.
Cyclosporin A (CsA) is an effective immunosuppressive agent, but its myocardial toxicity limits its widespread and long-term clinical application. In this study, CsA treatment led to damages in myocardial fiber structure, an increase in myocardial fibrosis, and changes in heart size and shape; moreover, the degree of damage was exacerbated with prolonged drug application and increases in dose. However, the mechanism is not clear; therefore, the purpose of this study was to reveal the mechanism of CsA-induced myocardial fibrosis and identify a new target for the prevention and treatment of CsA-induced myocardial injury. Cardiac fibroblasts were treated with CsA (5, 10, or 20 μg/mL) for 24 h. Autophagy was observed by electron microscopy and immunofluorescence. The expression of NRP-2/WDFY-1, autophagy-related proteins (Beclin1 and LC3B), fibrosis-related proteins (MMP2/9), and fibroblast phenotype conversion factor (α-SMA) was evaluated by Western blot. The expression of collagen I was determined by ELISA. Then, we used the gene interference technique to alter WDFY-1 expression with or without CsA or 3-MA treatment for 24 h, and the effects on autophagy and the expression of autophagy-related proteins, fibrosis-associated proteins, IFN-α, TNF-α, and IL-6 were determined. The results showed the following: (1) CsA induced fibrosis-related protein (MMP2/9), fibroblast phenotype conversion factor (α-SMA), and collagen I up-regulation in a dose-dependent manner. (2) CsA induced the formation of autophagosomes and up-regulated the expression of Beclin1, LC3B, and the ERK/MAPK pathway in cardiac fibroblasts. (3) CsA induced NRP-2 down-regulation and WDFY-1 up-regulation. (4) Depletion of WDFY-1 inhibited CsA-induced autophagy, TNF-α and IFN-α up-regulation, and fibrosis. (5) The autophagy inhibitor 3-MA inhibited CsA-induced TNF-α and IFN-α up-regulation and fibrosis. Overall, cyclosporin A induces autophagy in cardiac fibroblasts through the NRP-2/WDFY-1 axis, which promotes the progression of myocardial fibrosis.
环孢素 A（CsA）是一种有效的免疫抑制剂，但它的心肌毒性限制了其广泛和长期的临床应用。在本研究中，CsA 治疗导致心肌纤维结构损伤、心肌纤维化增加、心脏大小和形状改变；而且，随着药物应用时间的延长和剂量的增加，损伤程度加剧。然而，其机制尚不清楚；因此，本研究旨在揭示 CsA 诱导心肌纤维化的机制，并为预防和治疗 CsA 诱导的心肌损伤寻找新的靶点。用 CsA（5、10 或 20μg/ml）处理心肌成纤维细胞 24h。电镜和免疫荧光观察自噬。Western blot 检测 NRP-2/WDFY-1、自噬相关蛋白（Beclin1 和 LC3B）、纤维化相关蛋白（MMP2/9）和纤维母细胞表型转化因子（α-SMA）的表达。ELISA 法测定胶原 I 的表达。然后，我们采用基因干扰技术改变 WDFY-1 的表达，并用 CsA 或 3-MA 处理 24h，观察对自噬和自噬相关蛋白、纤维化相关蛋白、IFN-α、TNF-α和 IL-6 表达的影响。结果表明：（1）CsA 呈剂量依赖性诱导纤维化相关蛋白（MMP2/9）、纤维母细胞表型转化因子（α-SMA）和胶原 I 的表达上调。（2）CsA 诱导心肌成纤维细胞自噬小体形成，上调 Beclin1、LC3B 和 ERK/MAPK 通路的表达。（3）CsA 诱导 NRP-2 下调和 WDFY-1 上调。（4）WDFY-1 耗竭抑制 CsA 诱导的自噬、TNF-α和 IFN-α上调及纤维化。（5）自噬抑制剂 3-MA 抑制 CsA 诱导的 TNF-α和 IFN-α上调及纤维化。总之，环孢素 A 通过 NRP-2/WDFY-1 轴诱导心肌成纤维细胞自噬，促进心肌纤维化的进展。
