Nayak Mukti Kant, Agrawal Anurodh S, Bose Sudeshna, Naskar Shaon, Bhowmick Rahul, Chakrabarti Saikat, Sarkar Sagartirtha, Chawla-Sarkar Mamta
Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road Scheme-XM, Beliaghata, Kolkata, West Bengal 700010, India.
J Antimicrob Chemother. 2014 May;69(5):1298-310. doi: 10.1093/jac/dkt534. Epub 2014 Jan 23.
Baicalin, a flavonoid, has been shown to have antiviral and anti-inflammatory activities, although the mechanism of action has been unknown. Therefore, attempts were made to analyse the mechanism behind the antiviral effects of baicalin using an influenza A virus (IAV) model in vitro and in vivo.
Baicalin's anti-influenza activity was elucidated (in vitro and in vivo) utilizing pandemic influenza strain A/H1N1/Eastern India/66/pdm09 (H1N1-pdm09). Anti-influenza activity was measured by plaque inhibition, fluorescent focus-forming units (ffu) and quantifying viral transcripts using quantitative real-time PCR following treatment with baicalin in a dose- and time-dependent manner. The role of the IAV non-structural protein 1 (NS1) gene in modulating host responses was measured by immunoblotting, co-immunoprecipitation and molecular docking.
Baicalin treatment following IAV infection revealed up-regulation of interferon (IFN)-induced antiviral signalling and decreased phosphoinositide 3-kinase/Akt (PI3K/Akt) activation compared with infected, untreated controls. Baicalin exerts its antiviral effects by modulating the function of the IAV-encoded NS1 protein. NS1 has been shown to counteract cellular antiviral responses by down-regulating IFN induction and up-regulating PI3K/Akt signalling. Baicalin disrupted NS1-p85β binding. Molecular docking predicted the binding site of baicalin in the RNA binding domain (RBD) of NS1. Site-directed mutagenesis within the RBD region of NS1 and the difference in the fluorescence quenching pattern of full-length NS1 and mutant NS1 proteins in the presence of baicalin confirmed the interaction of baicalin with the NS1 RBD. Amino acid residues 39-43 of the NS1 RBD were found to be crucial for the baicalin-NS1 interaction.
Overall, this study highlights that baicalin exerts its anti-influenza virus activity by modulating viral protein NS1, resulting in up-regulation of IFN-induced antiviral signalling and a decrease in PI3K/Akt signalling in cells.
黄芩苷作为一种黄酮类化合物，已显示出具有抗病毒和抗炎活性，但其作用机制尚不清楚。因此，本研究试图利用甲型流感病毒（IAV）体外和体内模型分析黄芩苷抗病毒作用的机制。
利用大流行性甲型H1N1/印度东部/66/pdm09（H1N1-pdm09）毒株在体外和体内阐明黄芩苷的抗流感活性。用黄芩苷以剂量和时间依赖性方式处理后，通过噬斑抑制、荧光灶形成单位（ffu）以及使用定量实时PCR定量病毒转录本来测量抗流感活性。通过免疫印迹、免疫共沉淀和分子对接来检测IAV非结构蛋白1（NS1）基因在调节宿主反应中的作用。
与感染但未处理的对照组相比，IAV感染后用黄芩苷处理显示干扰素（IFN）诱导的抗病毒信号上调，磷酸肌醇3-激酶/蛋白激酶B（PI3K/Akt）激活降低。黄芩苷通过调节IAV编码的NS1蛋白的功能发挥其抗病毒作用。NS1已被证明通过下调IFN诱导和上调PI3K/Akt信号来对抗细胞抗病毒反应。黄芩苷破坏了NS1与p85β的结合。分子对接预测了黄芩苷在NS1的RNA结合结构域（RBD）中的结合位点。NS1的RBD区域内的定点诱变以及在黄芩苷存在下全长NS1和突变NS1蛋白荧光猝灭模式的差异证实了黄芩苷与NS1 RBD的相互作用。发现NS1 RBD的39-43位氨基酸残基对黄芩苷与NS1的相互作用至关重要。
总体而言，本研究强调黄芩苷通过调节病毒蛋白NS1发挥其抗流感病毒活性，导致细胞中IFN诱导的抗病毒信号上调以及PI3K/Akt信号降低。
