Arctigenin derivatives form an elite class of naturally occurring compounds that possess promising antiviral therapeutic perspectives. In a previous study, we design and synthesize a arctigenin derivative, 4-(8-(2-ethylimidazole)octyloxy)-arctigenin (EOA), to evaluate its antiviral activity on infectious hematopoietic necrosis virus (IHNV). In this study, we find that the half maximal inhibitory concentrations (IC₅₀) of EOA on IHNV nucleoprotein (N), phosphoprotein (P), matrix protein (M), nonvirion protein (NV) and polymerase (L) mRNA expression is 0.92, 0.80, 0.98, 0.89 and 0.87 μM, respectively. Mechanistically, our results show that EOA do not damage the viral particles directly, indicating EOA does not possess antiviral activity by destroying virions. Viral binding assays reveal that EOA do not interfere with IHNV adsorption. Because rapamycin has been shown to exhibit anti-IHNV activity by inducing autophagy of epithelioma papulosum cyprini (EPC) cells, we further investigate the relationship between EOA and autophagy in EPC cells. Autophagy fluorescence detection shows that EPC cells have a strong autophagy body after being treated with derivative EOA. The electron microscopy results show that EOA could induce typical autophagosomes which are representative structures of autophagy activation. Moreover, the punctate accumulation of green fluorescence-tagged microtubule-associate protein 1 light chain 3 (LC3) and the protein conversion from LC3-I to LC3-II are respectively confirmed by confocal fluorescence microscopy and western blotting. Overall, these findings demonstrate that EOA plays an anti-IHNV role via inducing autophagy in EPC cells.

牛蒡子苷衍生物形成了一类具有有希望的抗病毒治疗前景的天然化合物。在之前的一项研究中，我们设计并合成了牛蒡子苷元衍生物 4-(8-(2-乙基咪唑)辛氧基)-牛蒡子苷元 (EOA)，以评估其对传染性造血坏死病毒 (IHNV) 的抗病毒活性。在这项研究中，我们发现半数最大抑制浓度（IC ₅₀) EOA 对 IHNV 核蛋白 (N)、磷蛋白 (P)、基质蛋白 (M)、非病毒粒子蛋白 (NV) 和聚合酶 (L) 的 mRNA 表达分别为 0.92、0.80、0.98、0.89 和 0.87 μM。从机制上讲，我们的结果表明 EOA 不会直接破坏病毒颗粒，这表明 EOA 不通过破坏病毒粒子而具有抗病毒活性。病毒结合测定表明 EOA 不干扰 IHNV 吸附。由于雷帕霉素已被证明通过诱导鲤鱼上皮瘤 (EPC) 细胞的自噬表现出抗 IHNV 活性，我们进一步研究 EOA 与 EPC 细胞自噬之间的关系。自噬荧光检测表明EPC细胞经衍生EOA处理后具有较强的自噬体。电镜结果表明EOA可以诱导典型的自噬体，这是自噬激活的代表结构。此外，通过共聚焦荧光显微镜和蛋白质印迹分别证实了绿色荧光标记的微管相关蛋白 1 轻链 3 (LC3) 的点状积累和从 LC3-I 到 LC3-II 的蛋白质转化。总体而言，这些发现表明 EOA 通过诱导 EPC 细胞自噬发挥抗 IHNV 作用。