基于“辛散”理论的臭灵丹倍半萜类挥发组分调控流感早期粘膜免疫与炎症机制研究

The new influenza virus is frequently emergence, TCM, which has the pharmacological characteristics of multi targets and immunoregulation, is an important resource for the development of new anti-influenza drugs. Ethnic medicine Choulingdan (Laggera pterodonta (DC.) Benth) is used for curing cold in clinic and has a good effect. Choulingdan has special TCM property and flavor (cold, bitter, pungent, having powerful taste). TCM pungent - disperse theory and ethnical clinical experience (infused fresh) inspires us, volatile components of Laggera Pterodonta may be an important active component in early influenza. Our previous research shows, sesquiterpene fraction, which isolated from Laggera Pterodonta volatile components, can suppress the flu virus directly, and inhibit p38/MAPK phosphorylation and mRNA expression of some inflammation in vitro. We speculates, sesquiterpene fraction Isolated from Laggera Pterodonta, has anti-influenza pharmacology mechanism targeting to mucosal immunity and inflammation regulation. In this project, the cell, mice model will be used for analysis the antiviral efficacy; ELISA、Western blot, gene-silence technology will be used for analyze effects of the components’ influence on p38/MAPK pathway, respiratory tract SIgA, respiratory tract and systemic inflammation and the balance of pro-inflammatory factor and anti-inflammatory factor. The results will definite the effective substance and mechanism of Laggera Pterodonta, and explains the connotation of modern medicine for the TCM theory such as " pungent - disperse "and so on.

新突发流感近年呈高发态势，中医药因多靶点协同、整体平衡等优势成为抗流感研究热点。民族名药臭灵丹防治感冒疗效确切，药性苦寒兼辛（气味浓烈），《内经》“辛散”“风淫于内…以辛散之”经典理论提示，臭灵丹挥发成分可能在病毒感染早期发挥功效。我们前期研究发现臭灵丹倍半萜类挥发组分在细胞模型能抑制流感病毒复制及宿主P38/MAPK通路活化。提出假说：臭灵丹倍半萜类组分在抑制病毒复制同时，还可在流感早期调控呼吸道粘膜免疫、p38/MAPK通路及Th1/Th2平衡，控制全身炎症。本项目拟通过体内外实验评价该组分抗流感药效，运用流感小鼠模型、ELISA、Western blot、基因沉默等方法分析其对呼吸道粘膜免疫（SIgA）、呼吸道与全身p38/MAPK通路蛋白、多种炎症因子及Th1/Th2漂移的动态影响，以期初步阐明其早期抗流感机制，为新药研发提供依据，并阐释“辛散”、臭灵丹“味辛”、“泡服”现代内涵。

新突发流感近年呈高发态势，中药因多靶点、调节免疫等优势成为研究热点。民族名药臭灵丹治疗感冒疗效确切，性味独特（苦寒清热而兼辛味），“辛散”理论和“单味泡服”民间经验提示，其挥发成分可能是流感早期的重要活性组分。依据前期研究提出假设：臭灵丹倍半萜类化合物能在流感早期调节呼吸道粘膜免疫、抑制局部炎症，进而减轻全身炎症损伤。本项目完成了臭灵丹倍半萜类组分的提取分离及化学成分分析、结构鉴定；运用ELISA、Western blot、流式细胞术等，通过体内外实验确认其抗流感谱及药效强度；探讨其对流感早期黏膜免疫的调控机制；依据民间泡服经验制备臭灵丹水提物，验证其体内抗甲流的药效及机制。.主要结论包括：.臭灵丹倍半萜类组分（低沸点成分）是其重要的药效部位，其中分离鉴别出倍半萜单体臭灵丹酸；提示臭灵丹“辛味”的主要物质基础与倍半萜类组分密切相关。.臭灵丹倍半萜组分、臭灵丹酸均在体外对A/PR/8/34 (H1N1)、A/GZ/GIRD07/09 (H1N1)、A/Aichi/2/1968 (H3N2) 有抑制作用；并能改善A/Aichi/2/1968 (H3N2小鼠的病毒性肺炎；提示其具有较广的抗流感谱和一定的病毒抑制作用。.臭灵丹倍半萜类组分在细胞实验、小鼠实验都对H3N2感染的SIgA表达有调节作用；流感小鼠实验中，臭灵丹倍半萜类组分能下调炎症相关因子IL-4、PIGRR、IFN-y、TNF-α；提示其具有独特的免疫调控作用。.臭灵丹水提物（依照“泡服”经验制备）在H3N2流感小鼠体内能改善病毒性肺炎肺炎，显著上调SIgA分布以及肺部辅助性T淋巴细胞；在体外实验中能抑制甲流H3N2病毒复制并上调pIgR，抑制病毒诱发宿主NF-κB通路以及Toll样受体的激活以及相应炎症因子（IL-4、IFN-y、TNF-α）产生。.臭灵丹倍半萜类组分、臭灵丹水提物的体外最佳抗病毒药效均为预防给药模式，佐证了温病防治要在早期运用“辛散”治法的中医经验。.综上，本着重从宿主因素（粘膜免疫、宿主信号通路调控）探讨臭灵丹倍半萜组分抗流感机制，为臭灵丹“辛味”“泡服”、外感早期“以辛散之”治法等传统中医理论提供现代研究依据，加深传统中医药理论与现代药物研究方法技术的融合。