Natural Product Chemistry Laboratory, Aquatic Biotechnology Center, Chemistry and Biology Faculty, Environmental Sciences Department, University of Santiago of Chile, Av. Bernardo O'Higgins, 3363, Santiago, Chile.
Immunology Laboratory, Aquatic Biotechnology Center, Biology Department, Chemistry and Biology Faculty, University of Santiago of Chile, Av. Bernardo O'Higgins, 3363, Santiago, Chile.
Dev Comp Immunol. 2022 Jan;126:104262. doi: 10.1016/j.dci.2021.104262. Epub 2021 Sep 17.
Alpinone is a flavonoid obtained from the resinous exudate of Heliotropium huascoense. This flavonoid shows antiviral activity against the infectious salmon anemia virus (ISAV), which causes severe disease in farmed Atlantic salmon. Here, we aim to elucidate mechanisms underlying the antiviral effects of the flavonoid. In this regard, we evaluated whether Alpinone can act upregulating the pattern-recognition receptor genes, i.e., the RIG-I-like, TLR3, and TLR9 genes, and the genes of the downstream signaling pathways. Transcriptional expression of the genes was analyzed using real-time PCR after 8, 24, and 48 h treatment of salmon kidney adherent cells with 15 μg/mL of Alpinone. First, we showed that Alpinone induced IFNa expression in the kidney adherent cells, indicating that this type of salmon cells is in part responsible for the effects previously reported in vivo. Upregulation of the IFN-induced myxovirus resistance (Mx) gene was also observed in the head kidney cells in response to the treatment. Overexpression reached a maximum level at 24 h post-treatment. Interestingly, Alpinone also induced upregulation of the cytosolic receptors of ssRNA, named Retinoic acid-inducible gene I (RIG-I) and Melanoma Differentiation-Associated protein 5 (MDA5), but there were no effects on the transcriptional expression of the TLR3 and TLR9 endosomal receptors. In addition, Alpinone upregulated the expression of genes encoding the main components of the RIG-I/MDA5 signaling pathways, such as the mitochondrial antiviral-signaling protein (MAVS), TNF Receptor Associated Factor 3 (TRAF3), TANK-binding kinase 1 (TBK1), I-kappaB kinase ε (IKKε), the transcription factors IRF-3, and IRF7. The increased expression of all these genes is consistent with the upregulation of IFNa and Mx mRNAs. Because BX795 completely prevents Alpinone-dependent upregulation of IFNa and IRF3, the flavonoid targets seem to be upstream of the kinases TBK1 and IKKε. Altogether, this study contributes to elucidating the mechanisms involved in Alpinone antiviral activity in fish. Alpinone can be used to counteract virus mechanisms of evasion where the onset of interferon-mediated response is prevented or delayed.
阿尔皮酮是一种从希罗普托姆·瓦斯科（Heliotropium huascoense）树脂状分泌物中提取的类黄酮。这种类黄酮对传染性鲑鱼贫血病毒（ISAV）表现出抗病毒活性，ISAV 会导致养殖大西洋鲑鱼严重患病。在这里，我们旨在阐明类黄酮抗病毒作用的机制。在这方面，我们评估了阿尔皮酮是否可以通过上调模式识别受体基因，即 RIG-I 样、TLR3 和 TLR9 基因，以及下游信号通路的基因来发挥作用。在用 15μg/mL 阿尔皮酮处理鲑鱼肾贴壁细胞 8、24 和 48 小时后，使用实时 PCR 分析基因的转录表达。首先，我们表明阿尔皮酮诱导了肾贴壁细胞中 IFNa 的表达，表明这种类型的鲑鱼细胞部分负责先前在体内报告的作用。在头肾细胞中，也观察到干扰素诱导的粘液病毒抗性（Mx）基因的上调。过表达在治疗后 24 小时达到最大值。有趣的是，阿尔皮酮还诱导了 ssRNA 胞质受体的上调，称为视黄酸诱导基因 I（RIG-I）和黑色素瘤分化相关蛋白 5（MDA5），但对内体受体 TLR3 和 TLR9 的转录表达没有影响。此外，阿尔皮酮上调了 RIG-I/MDA5 信号通路主要成分的基因表达，如线粒体抗病毒信号蛋白（MAVS）、TNF 受体相关因子 3（TRAF3）、TANK 结合激酶 1（TBK1）、I-κB 激酶ε（IKKε）、转录因子 IRF-3 和 IRF7。所有这些基因的表达增加与 IFNa 和 Mx mRNA 的上调一致。由于 BX795 完全阻止了阿尔皮酮依赖的 IFNa 和 IRF3 的上调，因此类黄酮的靶标似乎位于 TBK1 和 IKKε 激酶的上游。总之，这项研究有助于阐明阿尔皮酮在鱼类中的抗病毒活性的机制。阿尔皮酮可用于对抗病毒逃避干扰素介导的反应的机制，其中干扰素介导的反应的起始被阻止或延迟。
