阿魏酸、槲皮素、甘草酸组合物拮抗禽流感病毒H5N1血凝素蛋白介导急性肺损伤的药理学作用及机制研究

Infection of the highly pathogenic avian influenza A virus H5N1 has been recognized by its high morbidity and mortality. Acute lung injury (ALI) elicited in severe patient with H5N1 infection is associated with the fatal outcome. “Cytokine storm” as a consequence of an exaggerated immune response has been proposed to contribute to rapid progression to ALI. Studies demonstrated that exposure to hemaglutinin (HA), the surface glycoprotein of H5N1, induced dysregulated innate immune response through activation of Janus tyrosine kinase(JAK) 3 and led to direct lung damage by the immunopathology. Simultaneously, also induced the inhibition of epithelial inflammatory regulator CFTR with the function of host tolerance, thereby exaggerated lung injury. Previously work found that the combination of Ferulic Acid, Quercetin and Glycyrrhizic Acid possessed the property of inhibition of JAK3 activation as well as an activation of CFTR which was reproduced from extract of Chinese herbal formula TianLong. We therefore speculated that the combination of Ferulic Acid, Quercetin and Glycyrrhizic Acid possessed the effects on acute lung injury induced by H5N1 HA, the protective effects could achieved by many ways including inhibition of JAK3 activation and activation of epithelial CFTR through elevating intracellular cAMP level. In order to investigate the effects and associated pharmacologic mechanisms of the combination, human epithelial cell line NHBE, mouse tracheal tissues and HA-induced ALI mouse model were challenged by applying techniques such as flow cytometry, LiquidChip assay, chamber recording (Short-Circuit Current), et al. The present study aims to find new research and development ideas and potential drugs for the treatment of H5N1-caused disease.

过度免疫应答引起“炎症级联瀑布效应”导致的急性肺损伤（ALI）是H5N1高病死率的主要原因。研究发现，H5N1血凝素蛋白（HA）通过活化气道上皮JAK3激酶，既可直接引起过强的固有免疫炎症损伤，又可同时干扰气道上皮CFTR的炎症调节功能，抑制机体免疫耐受机制，进而扩大免疫炎症损伤，最终导致严重ALI。前期研究发现阿魏酸、槲皮素、甘草酸三者协同既能抑制JAK3激酶活化，又能拟合中药复方天龙粗提物激活气道上皮CFTR的活性。据此我们推测，该组合物通过抑制异常活化JAK3激酶同时激活气道上皮CFTR，能够有效拮抗H5N1 HA介导的严重ALI。为此，本研究采用NHBE细胞系，小鼠气管组织、H5N1 HA介导小鼠急性肺损伤模型，应用短路电流、液相芯片、流式细胞术等技术，从细胞、组织以及动物水平多方面验证该组合物药理学活性并揭示其机制，为开发治疗H5N1药物提供新的思路与选择。

过度免疫应答反应引起“炎症级联瀑布效应”而致急性肺损伤是禽流感病毒H5N1导致高病死率的主要原因。前期研究表明，高致病性禽流感病毒H5N1血凝素蛋白HA通过激活TLR4/JAK3，同时干扰气道上皮cAMP依赖的CFTR通道介导严重的急性肺损伤。JAK3激酶是关键的靶点。阿魏酸、槲皮素、甘草酸组合物既能抑制JAK3激酶活化，又能激活气道上皮CFTR，但是该组合物拮抗H5N1 HA介导急性肺损伤的药理学作用及机制不明。.本项目通过短路电流、免疫荧光、Real-time PCR，液相芯片等方法从体内外证实了该组合物通过协同作用抑制JAK3激酶活性同时提高细胞内 cAMP 浓度，激活气道上皮 CFTR，发挥拮抗 H5N1 HA介导严重急性肺损伤的药理学作用，其中阿魏酸起主要的作用。并进一步发现该组合物可能还通过抑制JAK3激酶活性同时激活气道上皮 CFTR进而抑制或减缓H5N1 HA促进上皮细胞衰老进程，进而拮抗H5N1 HA介导的肺纤维化。.阿魏酸、槲皮素、甘草酸组合物来自于临床常用组方“天龙咳喘灵”二次开发。对该组合物药理学作用及机制的深入研究既可以为临床治疗H5N1提供新的方法，又可以为传统中药国际化，现代化提供新的思路。。