板蓝根中抗流感病毒新糖肽的分离鉴定和抑制流感病毒血凝素活性的机理研究

Isatis indigotica root（IIR, Radix Isatidis）has been traditionally used for antiviral treatment. However, its antiviral effective components and underlying mechanisms are yet to be elucidated. In previous study, total polysaccharides from this medicinal plant showed obvious anti-viral effects. Furthermore we have found a glycopeptide derived from this extract could inhibit influenza virus and its membrane protein hemagglutinin (HA). Based on these findings, we hypothesize that IIR glycopeptides may be main antiviral active substance of this herb. To test this hypothesis, this project is undertaken to isolate and purify glycopeptides from this plant and identify its primary structure. Furthermore, its antiviral effects and affection on the attachment of influenza virus by interfering HA will be evaluated using different influenza viruses subtypes, including human and avian influenza viruses in vitro. We will further detect effect and intensity of conjugation between glycopeptides and HA using HAI assay and competitive ELISA. Additionally, flow cytometry and high-resolution mass spectrometer were used to verify selective inhibition to HA1/HA2. Moreover, the corresponding oligosaccharides and peptides will be prepared by enzyme hydrolysis of parent glycopeptides. Their binding affinities with HA and antiviral effect will be subsequently determined to clarify the structure-activity relationship of parent glycopeptides. The in vivo efficacy of the extract will be evaluated in mice.Finally, the anti-influenza mechanism of IIR,particlualr the inhibition of HA, will be explored extensively, and the findings from this project are expected to provide new theoretical basis for the research of antiviral substance and mechanism of IIR.

板蓝根是传统的抗病毒中药，强极性部位为其主要活性部位，但确切的抗流感病毒成分和药理机制尚未明确。本课题组前期从板蓝根中分离出对流感病毒血凝素（HA）具有直接抑制作用的一种糖肽，因此提出该糖肽可能为板蓝根主要活性物质之一。本课题将对板蓝根中的糖肽类物质进行系统分离和活性检测，综合多糖化学和蛋白组学方法获取其结构信息，深入探索糖肽抑制流感病毒血凝素的活性及机制：包括利用血凝抑制、竞争性ELISA法、高分辨质谱法分析糖肽与HA的结合作用和强度；以流式细胞术鉴别糖肽对HA亚单位（HA1/HA2）的选择性抑制作用。最后利用小鼠流感模型明确板蓝根糖肽体内药效。本研究将揭示糖肽干预HA与宿主细胞受体结合和侵入的抗病毒机制，为板蓝根抗流感药效物质基础及作用机理的阐释提供新的理论基础。

流感病毒，尤其是新突发人感染禽流感病毒，如H7N9，H5N6等，死亡率高，严重威胁公众健康。现有抗流感病毒化学药物存在靶点单一，耐药性及毒副作用等特点，而传统抗流感中药具有多靶点协同作用优势，且临床应用广泛。板蓝根（Isatis infigotica root）为临床常用的抗病毒中草药，药理研究显示其各种成分具有抗菌、抗病毒、抗肿瘤以及免疫调节作用。我们前期从板蓝根强极性部位分离出一个新糖肽，其主要作用于流感病毒表面的血凝素蛋白。因此，本课题主要分离提取板蓝根药材中的活性糖肽，并阐明糖肽的化学组成、抗流感病毒作用及其抑制血凝素的作用机制。.本实验从板蓝根中分离得到多个多糖，体外抗流感病毒谱筛选结果显示板蓝根70%及80%醇沉多糖体外对多种流感病毒亚型均有抑制作用，其中70%醇沉物对流感病毒H1/PR8，09H1N1，H6N2，H9N2的IC50为6.78 mg/mL-35.26mg/mL；80%醇沉物对H1/PR8，09H1N1，H6N2，H9N2，H3N2的IC50为4.35mg/mL -28.2 mg/mL。通过包被流感病毒H1N1/PR8-HA蛋白于ELISA板，观察板蓝根糖肽与HA蛋白结合力，结果显示板蓝根80%醇沉多糖在0.078125mg/mL-5mg/mL范围内与PR8-HA蛋白具有直接结合能力，且呈剂量依赖关系。进一步对板蓝根80%醇沉多糖进行系统分离纯化，从中分离得到2种均一板蓝根多糖（IRPS1A 和IRPS1B）和2 种均一板蓝根糖蛋白（IRPS2A 和IRPS3A），测得4 种均一多糖（糖蛋白）的MW分别为18 000、31 000、36 000 和82 000，并完成板蓝根均一多糖的单糖组成和氨基酸组成测定。.传统中药除抗病毒作用外，常具有一定的抗炎作用，结果显示板蓝根多糖在30mg/mL, 15 mg/mL 和7.5 mg/mL能显著抑制PR8诱导的16HBE细胞中促炎因子IL-6，趋化因子（ IP-10, MIG 以及CCL-5）的过表达， H9N2诱导的IL-6， IP-10, CCL-5的过表达。此外，板蓝根多糖可显著抑制宿主细胞Toll样受体3（TLR3）的蛋白表达。小鼠体内药效结果显示板蓝根80%醇沉多糖（浓度1g/kg）可有效抑制肺组织中炎症因子（如IL-6，IP-10，MIG，MCP-1等）mRNA的表达水平。