BACKGROUND Influenza A virus (IAV) is still a major health threat. The clinical manifestations of this infection are related to immune dysregulation, which causes morbidity and mortality. The usage of traditional medication with immunomodulatory properties against influenza infection has been increased recently. Our previous study showed antiviral activity of quercetin-3-O-α-L-rhamnopyranoside (Q3R) isolated from Rapanea melanophloeos (RM) (L.) Mez (family Myrsinaceae) against H1N1 (A/PR/8/34) infection. This study aimed to confirm the wider range of immunomodulatory effect of Q3R on selective pro- and anti-inflammatory cytokines against IAV in vitro, to evaluate the effect of Q3R on apoptosis pathway in combination with H1N1, also to assess the physical interaction of Q3R with virus glycoproteins and RhoA protein using computational docking. METHODS MDCK cells were exposed to Q3R and 100CCID50/100 μl of H1N1 in combined treatments (co-, pre- and post-penetration treatments). The treatments were tested for the cytokines evaluation at RNA and protein levels by qPCR and ELISA, respectively. In another set of treatment, apoptosis was examined by detecting RhoA GTPase protein and caspase-3 activity. Molecular docking was used as a tool for evaluation of the potential anti-influenza activity of Q3R. RESULTS The expressions of cytokines in both genome and protein levels were significantly affected by Q3R treatment. It was shown that Q3R was much more effective against influenza when it was applied in co-penetration treatment. Q3R in combination with H1N1 increased caspase-3 activity while decreasing RhoA activation. The molecular docking results showed strong binding ability of Q3R with M2 transmembrane, Neuraminidase of 2009 pandemic H1N1, N1 and H1 of PR/8/1934 and Human RhoA proteins, with docking energy of - 10.81, - 10.47, - 9.52, - 9.24 and - 8.78 Kcal/mol, respectively. CONCLUSIONS Quercetin-3-O-α-L-rhamnopyranoside from RM was significantly effective against influenza infection by immunomodulatory properties, affecting the apoptosis pathway and binding ability to viral receptors M2 transmembrane and Neuraminidase of 2009 pandemic H1N1 and human RhoA cellular protein. Further research will focus on detecting the detailed specific mechanism of Q3R in virus-host interactions.

中文翻译：

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来自南非本土药用植物 Rapanea melanophloeos 的槲皮素-3-O-α-L-吡喃鼠李糖苷的抗流感作用的实验验证和计算模型。


背景技术甲型流感病毒(IAV)仍然是主要的健康威胁。这种感染的临床表现与免疫失调有关，导致发病和死亡。最近，具有免疫调节特性的传统药物对抗流感感染的使用有所增加。我们之前的研究表明，从 Rapanea melanophloeos (RM) (L.) Mez（紫金牛科）中分离出的槲皮素-3-O-α-L-吡喃鼠李糖苷 (Q3R) 对 H1N1 (A/PR/8/34) 感染具有抗病毒活性。本研究旨在体外证实 Q3R 对 IAV 的选择性促炎和抗炎细胞因子的更广泛的免疫调节作用，评估 Q3R 与 H1N1 结合对细胞凋亡途径的影响，并评估 Q3R 与 H1N1 的物理相互作用。使用计算对接分析病毒糖蛋白和 RhoA 蛋白。方法 将 MDCK 细胞暴露于 Q3R 和 100CCID50/100 μl H1N1 联合治疗（共同治疗、渗透前治疗和渗透后治疗）。分别通过 qPCR 和 ELISA 对治疗方法进行 RNA 和蛋白质水平的细胞因子评估测试。在另一组治疗中，通过检测 RhoA GTPase 蛋白和 caspase-3 活性来检查细胞凋亡。分子对接被用作评估 Q3R 潜在抗流感活性的工具。结果 Q3R 处理显着影响基因组和蛋白质水平中细胞因子的表达。结果表明，当Q3R应用于共渗透治疗时，其对抗流感的效果要好得多。 Q3R 与 H1N1 结合可增加 caspase-3 活性，同时降低 RhoA 激活。 分子对接结果显示，Q3R与M2跨膜、2009年H1N1流感神经氨酸酶、PR/8/1934的N1和H1以及人RhoA蛋白具有较强的结合能力，对接能分别为- 10.81、- 10.47、- 9.52、- 9.24和- 分别为 8.78 Kcal/mol。结论 RM 中的槲皮素-3-O-α-L-吡喃鼠李糖苷通过免疫调节特性显着有效对抗流感感染，影响细胞凋亡途径以及与 2009 年大流行 H1N1 病毒受体 M2 跨膜和神经氨酸酶以及人 RhoA 细胞蛋白的结合能力。进一步的研究将集中于检测Q3R在病毒-宿主相互作用中的具体具体机制。