基于肺泡屏障、肺内积液及肺细胞坏死为靶点的抗IAV中药筛选、归类及药效药理研究

Influenza A virus (IAV) induces acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), which is closely related with the disruption of alveolar barrier, dysfunction of alveolar fluid clearance (AFC) and pulmonary cell necrosis, releasing a huge number of ‘damage-associated molecular patterns (DAMP)’ and leading to excessive inflammation. Stablizing pulmonary alveolar barrier, enhancing AFC, and inhibiting excessive inflammation are three important measurements of ALI/ARDS rehabilitation. Traditional Chinese medicine (TCM) has a long history, with a lot of effective prescriptions, but the ambiguous mechanism of TCM hinders its further development, which is a long-standing problem in TCM research.. Basing on IAV pathogenic mechanisms: (1) IAV infection induces the disruption of alveolar barrier and dysfunction of AFC; (2) IAV infection induces "excessive" pulmonary cell necrosis, including: MPT-dependency necrosis/Mitoptosis, Necroptosis, Parthanatos and Pyroptosis, we will establish several high throughput screening (HTS) methods, and perform screening, classification and pharmacologic researches on our collected TCM.. This study may achieve these goals: (1) achieving new anti-IAV drugs or precursor compounds; (2) carrying out ‘the researches of classification of anti-IAV TCM by modern pharmacological mechanisms’, which may provide theoretical and practical foundations for clinical medication and internationalization of TCM; (3) providing a chance to verify the latest anti-IAV theories, methods and drug screening targets; (4) instantly publishing the screening results, which may enhance the independent research capacity and competitiveness of TCM industry.

甲型流感病毒（IAV）诱导ALI/ARDS与肺泡屏障破坏、肺内积液清除障碍、肺细胞大量坏死, 诱发过度炎症密切相关，抑制此三者是ALI/ARDS康复的重要条件。中药历史悠久、有大量经方验方，但药理机制不明或研究不深入。本研究基于IAV致病新机制：①IAV诱导肺泡屏障破坏、肺内积液清除障碍，②IAV诱导肺细胞过度坏死，包括：MPT依赖性坏死、Necroptosis、Parthanatos 和Pyroptosis，作为筛选靶点，对中药进行筛选、归类、药理研究。其结果：①筛选抗IAV新药；②开展抗IAV中药按现代药理机制归类研究，为中药用药、复方配伍、国际化提供基础；在综合现代药理机制和传统中药理论基础上，开展类似艾滋病鸡尾酒治疗的‘流感鸡尾酒疗法’的大型联合用药研究。③对基础医学最新的抗IAV理论、方法、药物筛选靶点进行验证。④即时公开筛选结果，为增强中医药行业自主研发能力和产业竞争力做贡献。

项目背景:.本研究以IAV诱导肺泡屏障破坏、肺内积液清除障碍、肺细胞坏死（MPT依赖性坏死、Necroptosis、Parthanatos 和Pyroptosis）作为靶点，对中药进行不同药理机制的筛选、归类及相关药效药理研究。..研究内容:.1：继续进行‘药物收集与处理’ .2：构建基于 RHOA-ROCK-MYLK，ENAC，NA,K-ATPase、Nrf2-ARE，线粒体稳定，坏死及焦亡信号通路的筛选模型，进行药物筛选。利用PARP-1， IL-1Β，ATP 水平商业化检测试剂盒进行药物筛选。 .3：活性化合物结构修饰及联合用药的药效研究 ..重要结果:.1. 已得到143种单体和活性部位。 .2. 基于 Nrf2-ARE-LUC 报告质粒的药物筛选工作已完成，活性药物的专利在申报中。.3. 成功构建TLR4、Gpx4，SIRT1、PGC-1α，ROCK 和 NA+,K+-ATPase等启动子荧光素酶报告质粒，完成药物筛选，活性药物的专利撰写中。.4. 基于线粒体分裂与融合及线粒体膜电位变化，进行了JC-1的药物筛选。.5.通过TFEB过表达和siRNA/慢病毒干扰，证明了TFEB 在IAV感染中有重要作用。 .6. 多个单体/活性部位与达菲的联合用药, 动物试验已完成。.7. 三个单体/活性部位联合用药（类中药复方）, 动物试验已完成，专利撰写中。.关键数据:.（1）基于SIRT1、PGC-1Α、ROCK及Gpx4高通量药物筛选，发现多个高活性中草药（如槟榔）及多个活性单体化合物（如三七总皂苷等）。 .（2）基于ENAC、 NA+,K+-ATPase等高通量药物筛选，发现多个芹菜素糖苷、龙胆苦苷脂及京尼平糖苷衍生物有高活性，正报专利。 .（3）基于线粒体的JC-1药物筛选结果，正在深入研究中。..科学意义: .严重IAV感染可诱导ALI/ ARDS及高死亡率。本课题以“肺泡屏障破坏”、“肺积液清除”、“肺细胞坏死”为靶点，构建模型，进行药学研究。其结果可：①筛选出抗 IAV 新药或潜在药物；②进一步开展“抗IAV中药按现代药理机制归类”的研究工作，开展 “流感鸡尾酒疗法”的联合用药方案的研究。③对基础医学研究领域的抗IAV新理论、新方法和新药物筛选靶点进行验证。为中医药发展做贡献。