基于调节宿主干扰素信号通路的板蓝根3-吲哚醛抗流感病毒机制研究

Influenza endangers the public health continually, thus the demand for anti-influenza drug also continually. In the early research, we extracted one kind of single monomer compounds from radix isatidis named “3-indolal”, at the same time, it has been proved that 3-indolal has a broad-spectrum anti-virus effects (including influenza virus), which is different from the classical anti-flu drug. Yet the underlying broad-spectrum anti-virus mechanisms of 3-indolal were not clear. Meanwhile, studies have showed that the post-market drug “Arbidol”, which exhibits a similar drug structure and antiviral efficacy with 3-indolal, can regulate the interferon signaling of host, inducing the production of interferon antiviral factor protein. Therefore, we hypothesized that 3-indolal may presented an anti-influenza efficacy by regulating the interferon signaling of host. To clarify the scientific hypothesis, the influenza virus infection cell model will be used to verify the anti-influenza efficacy of 3-indolal was connected with the regulation of host interferon signaling. Next, gene chip method will be used to screen the target genes of host interferon signaling which regulated by 3-indolal, protein immunoblot (Western-bloting) and siRNA silence methods will be used to verify the related target proteins; further study will be conducted in the influenza virus-induced pneumonia mouse mode to verified the antiviral efficacy and target proteins. It can help to expound our scientific hypothesis that 3-indolal, and clear the target protein. Furthermore, it will provide the primary basis for drug structure modification of 3-indolal, as well as for developing the novel antiviral drug from radix isatidis.

流感持续危害大众健康，对抗流感药物的需求不断。而我们从板蓝根中追踪到活性单体“3-吲哚醛”，证实其具有体外广谱抗病毒活性，包括流感病毒，这特征有别于经典抗流感药，但其具体的抗病毒机制尚未明确。同时，我们发现与3-吲哚醛的结构和药效类似的已上市药“阿比多尔”通过调节宿主干扰素信号通路诱导抗病毒因子“干扰素蛋白”的产生而发挥广谱抗病毒药效。因此，我们推测板蓝根3-吲哚醛可能通过有效的调节宿主干扰素信号通路而发挥抗流感病毒的药效。为了验证本假设，拟采用流感病毒感染细胞模型验证3-吲哚醛的抗流感药效与调控宿主干扰素信号通路相关，进一步用基因芯片法筛选目标基因，蛋白免疫印迹检测目标蛋白以及siRNA沉默目标蛋白表达等方法验证相关靶点蛋白，最后在小鼠感染模型中验证其药效和靶点，从而阐明板蓝根3-吲哚醛抗流感的作用机制，明确其靶点，为后续药物结构改造和从板蓝根中挖掘具有自主知识产权的抗流感新药提供依据。

临床对新型抗流感药物的需求不断，特别是对抗流感病毒介导的炎症反应的药物需求量大。从板蓝根中分离提取获得的吲哚生物碱单体化合物“3-吲哚醛”被我们证实不仅具有抑制多种流感病毒复制的药效，包括甲型流感H1N1，H3N2和H9N2以及乙型流感，SI值范围为2.03~6.08；此外，对多种常见呼吸道病毒也有抑制作用。同时，3-吲哚醛被证实可以有效的抑制流感病毒介导的炎症反应，表现为在药物浓度为200μg/mL，100μg/mL和50μg/mL是可以明显抑制IP-10，IL-6，MIG等蛋白水平，呈现明显的药物浓度依赖性。随后，我们通过转录组学检测发现流感病毒感染后宿主干扰素信号通路的RIG-i（DDX58）基因表达量明显升高，但3-吲哚醛干预24和48小时后，RIG-i的基因表达量明显下调，也呈现明显的药物浓度依赖性。同时，我们通过蛋白免疫印迹技术也证实3-吲哚醛（200μg/mL和100μg/mL）干预后，病毒感染介导的RIG-i的蛋白表达水平可被明显抑制，STAT1的磷酸化水平被明显的抑制。另外，我们对项目的研究内容进行拓展发现3-吲哚醛可有效的抑制流感病毒的唾液酸受体的表达和RNA聚合酶的活性，从而有效的抑制流感病毒的复制。本项目的研究结果表明板蓝根3-吲哚醛可通过干预宿主干扰素信号通路的RIG-i的表达从而有效的抑制流感病毒介导的过度炎症反应，为后续研发新型抗流感药物提供科学的参考依据。