贵州省民族药苦参主要成分氧化苦参碱调节IFN-γ改善致死性人呼吸道合胞病毒感染小鼠的实验研究

Human Respiratory Syncytial Virus is the most critical pathogen that leads to child viral pneumonia and is one of the most important pathogen that causes lower respiratory tract infection for elderly either. Albeit the infection of human Respiratory Syncytial Virus annually led to several hundred thousand people died directly or indirectly, no licensed vaccine of human Respiratory Syncytial Virus was approved. The recent researches showed there was a possibility for IFN-γ, in behalf of the immune factors, which involved in the pathological immunity caused by human Respiratory Syncytial Virus infection. Such pathological immunity would lead to severer inflammation, even to death, but the exact mechanism was urgent to be uncovered. Oxymatrine, the main constituent of Guizhou traditional herb Sophora flavescens, had inhibitory effects for many DNA/RNA viruses, such as Hepatitis B Virus, Coxsackie Virus, and also human Respiratory Syncytial Virus. Therefore, the current study would investigate the anti-human Respiratory Syncytial Virus effect of Oxymatrine in a lethal mouse model for human Respiratory Syncytial Virus infection. Such lethal mouse model was first established by our group and it should be a great platform for selecting and screening anti-human Respiratory Syncytial Virus agents. Not only would the anti-viral effect of Oxymatrine be defined, but the novel antiviral mechanism of Oxymatrine via regulating the IFN-γ to ameliorate lethal human Respiratory Syncytial Virus infection in mice would be elucidated. This study combined with the abundant traditional medicine resources in Guizhou, revealing the new antiviral mechanism of Chinese herbal medicine will provide scientific basis for the rational utilization of Sophora flavescens, and it was of great social significance to further exert the advantages of Guizhou characteristic traditional herbal resources, integrate the extra value of traditional herb, and promote the modernization of traditional medicine.

人呼吸道合胞病毒是引起儿童病毒性肺炎的最重要病原体，对长者下呼吸道感染亦为重要病原体之一，该病毒每年全球直接或间接引起数十万例儿童或长者死亡，迄今却尚未有疫苗上市。近年来的研究认为以IFN-γ为主的免疫因子可能参与了人呼吸道合胞病毒感染引起的免疫病理作用，导致炎症加重，其确切机制仍亟待阐明。贵州省民族药苦参主要成分氧化苦参碱通过对免疫因子的调节对多种病毒具有抑制作用，因此本课题拟以前期研究已初步建立的致死性人呼吸道合胞病毒小鼠动物模型为平台，评价氧化苦参碱抗人呼吸道合胞病毒的活性，并深入阐明氧化苦参碱通过调节IFN-γ等免疫因子改善人呼吸道合胞病毒感染的新抗病毒机制。本研究结合贵州省丰沛的民族药资源，揭示新的中草药抗病毒机制，将为苦参的合理利用和深层次系统研究开发提供科学依据，对进一步发挥贵州民族医药特色资源优势，整合中药民族药附加值，促进民族药研究现代化具有重要的社会意义。

人呼吸道合胞病毒（hRSV）作为导致呼吸系统感染性疾病的重要病因，对新生儿、长者和免疫抑制病人危害很大，目前除帕利殊单抗外，尚没有疫苗及特异性药物上市，其重要原因之一是该病毒特殊的病理免疫机制，针对这一特点，以中草药来源的小分子药物很可能通过调控宿主免疫系统拮抗病毒感染。本项目依托致死性hRSV感染小鼠模型，评价苦参类生物碱主要成分氧化苦参碱（oxymatrine, OMT）的体内体外抗hRSV活性，同时力图阐明OMT的抗感染机制。.我们在小鼠肺内连续传代hRSV，获得数株高毒力hRSV小鼠适应株，可感染小鼠致死，在该研究领域首次发展具可操作性的致死性hRSV感染小鼠模型，依托该模型评价了纳米结构脂质载体包被OMT的抗hRSV活性，发现纳米结构脂质载体包被OMT和甲壳素纳米结构脂质载体包被OMT均能提高hRSV感染小鼠存活率，鼠肺病毒滴度显著下降，鼠肺病理切片示感染减轻。我们在细胞内观察到苦参类生物碱具多种抗hRSV活性，其中槐果碱和槐定碱显著抑制病毒滴度并具较理想治疗指数，对抗感染机制探索发现苦参类生物碱均能抑制病毒感染导致的细胞凋亡，western blot发现NF-κB由胞浆到胞核的转位增多，并导致NF-κB下游抗凋亡蛋白表达增多。我们还在一株高毒力hRSV小鼠适应株（GZ08-18）感染的细胞中发现细胞凋亡被显著抑制，GZ08-18感染能够上调BCL-2表达，并抑制促凋亡相关蛋白表达，抑制细胞线粒体功能，随后发现鱼藤酮（一种特异性内始性细胞凋亡诱导剂）可以显著抑制GZ08-18的体内外感染，说明高毒力hRSV毒株可能通过调控宿主细胞的内始性细胞凋亡，获得更多的病毒复制机会。.本项目的科学意义主要体现在依托致死性hRSV感染动物模型，从不同的机制角度筛选并发现OMT和鱼藤酮的抗感染活性。由于贵州省具有丰富的中草药和民族药资源，因此本项目对于贵州省中草药和民族药现代化具较重大意义，同时也为临床病毒感染治疗提供新理论支撑和防治策略，为创新药物研制提供切实可靠的科学依据。.本项目取得以下成果：SCI论文2篇，北图核心期刊论文2篇，统计源期刊论文1篇，授权和在审发明专利各1项。