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.

环孢菌素A（CsA）是一种有效的免疫抑制剂，但其心肌毒性限制了其广泛的长期临床应用。在这项研究中，CsA治疗导致心肌纤维结构受损，心肌纤维化增加以及心脏大小和形状改变。此外，延长药物使用时间和增加剂量会加剧损伤程度。但是，机制尚不明确。因此，本研究的目的是揭示CsA诱导的心肌纤维化的机制，并确定预防和治疗CsA诱导的心肌损伤的新目标。心脏成纤维细胞用CsA（5、10或20μg/ mL）处理24小时。通过电子显微镜和免疫荧光观察自噬。自噬相关蛋白（Beclin1和LC3B）NRP-2 / WDFY-1的表达，通过蛋白质印迹法评估纤维化相关蛋白（MMP2 / 9）和成纤维细胞表型转化因子（α-SMA）。通过ELISA确定胶原蛋白I的表达。然后，我们使用基因干扰技术来改变有无CsA或3-MA处理24小时的WDFY-1表达，以及对自噬的影响以及自噬相关蛋白，纤维化相关蛋白，IFN-α，测定TNF-α和IL-6。结果表明：（1）CsA诱导的纤维化相关蛋白（MMP2 / 9），成纤维细胞表型转化因子（α-SMA）和胶原I呈剂量依赖性上调。（2）CsA诱导心脏成纤维细胞中自噬体的形成，并上调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的耗竭抑制了CsA诱导的自噬，TNF-α和IFN-α的上调以及纤维化。（5）自噬抑制剂3-MA抑制CsA诱导的TNF-α和IFN-α的上调和纤维化。总体而言，环孢菌素A通过NRP-2 / WDFY-1轴诱导心脏成纤维细胞自噬，从而促进心肌纤维化的发展。