基于TLR4/NF-kappaB信号通路金粟兰属药用植物中抗炎免疫的物质基础和作用机制研究

炎症是肿瘤等多种重大疾病的诱发原因之一，其反应剧烈可引发炎症风暴导致患者猝死。当前抗炎药物多数具有毒副作用，亟待开发高效安全的新型抗炎药物。传统抗炎中草药及活性物质毒副作用低但多数药效有限且缺乏抗炎机制研究。项目组前期于金粟兰属植物银线草中提取发现乌药烷型倍半萜化合物CJB具有良好抗炎免疫调节作用，初步研究提示其抗炎作用与TLR4蛋白相关。本项目拟对具有抗炎活性金粟兰属药材开展系统分离及化合物结构修饰，以获得抗炎药效更佳的化合物；采用CRISPR/Cas9基因编辑技术构建敲除TLR4的RAW264.7细胞以验证TLR4是否为CJB的靶点蛋白，进一步通过内毒素血症和CIA两种炎症模型验证其对TLR4介导的NF-kappaB特异性免疫信号通路的影响。项目的实施将提供一类安全高效的乌药烷型倍半萜抗炎物质，并揭示其抗炎作用的潜在靶点及相关通路，为后续中草药中抗炎物质开发及作用机制研究提供理论依据。

Inflammation is one of the inducing causes of various serious diseases such as tumor. Intense inflammatory response causes "cytokine storm" which may led to sudden death. Nowadays treatment for this mostly can’t avoid toxic and side effects, so it is urgent to explore more high-effective and safe anti-inflammatory drugs. Traditional anti-inflammatory Chinese medicines and active compounds from them have low toxic and little side effects, but most of them performs limited effect and lacks basic mechanism studies. At the early stage, the project team extracted the onoseriolide sesquiterpenoid CJB from Chloranthus genus which is a potential drug-like compound with significant anti-Inflammatory and Immunomodulatory activities. The preliminary study suggested that its anti-inflammatory effect was related to the Toll-like receptor (TLR) 4. This project intends to carry out systematic extraction and separation on plants Chloranthus genus and further to modify compound structure for better anti-inflammatory effect. TLR4 genome knockout (GKO) RAW264.7 macrophage cell line using the third generation CRISPR/Cas9 technique is constructed to verify whether TLR4 is the target of CJB. The effect of CJB on TLR4-mediated NF-kappaB specific immune signaling pathway will be further verified by two inflammatory models, endotoxemia and CIA. The successful implementation of this project will provide a class of safe and effective onoseriolide sesquiterpenoid anti-inflammatory drug-like compounds, and reveal the potential targets and related pathways of their anti-inflammatory effects, so as to provide theoretical and scientific basis for the development of anti-inflammatory substances and the mechanism of Traditional Chinese herbal medicine.