中药复方升降散通过RIG1-JAK1-STAT1/2信号通路调控流感病毒宿主固有免疫的药效机制及其物质基础研究

Changing the expression of interferon stimulant gene (ISGs) by regulating the innate immunity of the host has become a novel research strategy to investigate the action mechanism of anti-influenza virus in recent years. Our previous results showed that phenolic extracts from SJS specifically suppress different subtypes of influenza viruses and are associated with regulating the expression of IRF3, ISG15, Mx and RIG-1. However, the extract bioactive components and the specific action mechanism are not clear up till now. In this research, the inhibition ability on 16-human bronchial epithelial (16HBE) cell lines infected with H7N9 was taken as the evaluation index to compare the activity of different groups of phenolics. The chemical structure of each component in the selected bioactive combinatorial phenolics will be characterized. The phosphorylation of JAK1 and STAT1/2 in 16HBE cells infected with H7N9 were analyzed. The expression of RIG1, junction protein MAVS and ISGs were evaluated. The expression of related molecules in each other's pathways were discussed by transient transfection. The mechanism of the interaction between the two was clarified by regulating RIG1 or STAT1/2 pathway. Transcription and proteomics were used to analyze the differences between mRNA and protein in the lung after viral infection in mice. The effective network mechanism for regulating the innate immunity of anti-influenza virus will be explained focusing on the centre on the RIG1-JAK1/STAT1/2 signal pathway. The results of the study enrich the source of traditional Chinese medicine anti-influenza virus drug research and provide theory basis for the development of deep and comprehensive utilization.

通过调控干扰素刺激基因（ISGs）的表达影响宿主固有免疫成为近年来探讨抗流感病毒生物活性机制的重要方向。前期研究显示升降散酚类提取物可抑制不同亚型流感病毒，且与调节IRF3, ISG15, Mx和RIG-1的表达有关，但药效物质、作用机制等问题尚未明确。本项目拟以对H7N9感染16HBE细胞的抑制能力为评价指标，比较不同酚类成分群的活性，确定其有效成分群，并表征其化学结构；分析其对细胞内JAK1、STAT1/2的磷酸化的影响；RIG1、接头蛋白MAVS以及ISGs表达的变化；采用瞬时转染技术，建立RIG1或JAK1-STAT1/2抑制的16HBE细胞模型，明确二者发挥交互作用的体外分子机制；采用转录组和蛋白组学技术，分析其对小鼠病毒感染后肺组织的差异mRNA和蛋白，阐释其调控流感病毒固有免疫的药效网络机制和物质基础。研究结果将丰富中药来源的抗流感病毒药物研究，为升降散精深开发提供科学依据。

（1）升降散体外抗病毒谱药效筛选显示其对A/Aichi/2/68(H3N2)、A/PR/8/34(H1N1)、A/Chicken/Guangdong/1996 (H9N2)、respiratory syncytial virus (RSV)具有一定的抗病毒作用，IC50分别是0.26 mg/ml、1.25 mg/ml、1.26 mg/ml、1.98mg/ml；（2）进一步研究升降散对甲型流感病毒诱导的细胞因子mRNA表达水平影响。实时荧光定量PCR结果显示，升降散能不同程度下调甲型流感病毒诱导的宿主细胞炎症因子CCL5、IL-6、IL-8、TNF-α、IP-10、MIP-1β、MIP-1a、MCP-1、SOCS3和TRAIL的mRNA水平的表达，且呈现一定的剂量依赖关系。（3）动物实验结果表明升降散提取物能够明显提高A/PR/8/34 H1N1流感病毒感染小鼠的存活率，体重；降低肺指数，肺病毒滴度水平；且对流感病毒感染小鼠肺组织病理炎症性改变较病毒对照组有一定程度的减轻。（4）鉴定出姜黄中倍半萜类物质的结构，且明确了其药效机制是通过靶向调控宿主固有免疫信号通路，进而调节NF-κB/MAPK 和 RIG-1/STAT-1/2 信号通路及炎症因子，从而改善炎症损伤。