斑马鱼内毒素炎症模型的建立及其在中药抗内毒素炎症活性筛选中的应用研究

Today, modern medical science does not have very effective method to treatment of systemic inflammation reaction syndrome (SIRS) induced by endotoxin, more and more Chinese medicine and compounds with anti-endotoxin activities are constantly being discovered, which become the focus of attention and show great prospects in the treatment of SIRS by Chinese herb medicines. In this project, with the aims of screening of anti-endotoxin activity from Chinese medicine, we take advantage of using zebrafish as a model system in drug screening. A model of zebrafish larvae Induced by lipopolysaccharide(LPS) for screening anti-endotoxic traditional Chinese medicine (TCM) has been established, in which macrophages(NR staining) and neutrophils(mpo-GFP line) are both recruited to the LPS resource in this inflammatory response. Then we take the example of lianggesan compound, which has been proved to have endotoxin properties by our team, to carry out the feasibility study of traditional Chinese medicines (monomer composition, active component, effective parts, water decoction, etc.) based on zebrafish model system, so as to establish a high-throughput and low-cost model for screening of anti-endotoxin composition from traditional Chinese medicine. This zebrafish model with a very promising applicable future and would be an entirely new model as screening system different from mammalian and cell model.

在现代医学对由内毒素触发全身炎症反应综合征的治疗仍然一筹莫展的今天，却不断有中药、复方或其所含成分被证明对受内毒素攻击的动物有保护作用，从中药寻找抗内毒素治疗措施呈现良好的前景，抗内毒素中药活性筛选成为关注的热点。本项目针对中药抗内毒素活性筛选面临的问题，发挥新型模式生物斑马鱼在抗炎免疫药理研究及药物筛选方面的特色与优势，采用荧光标记免疫细胞转基因斑马鱼（mpo-GFP转基因系），结合活体成像可视化示踪免疫细胞炎症行为等技术，构建LPS诱导的斑马鱼内毒素炎症模型。再以课题组先期研究证明具有抗内毒素作用的凉膈散及其组成药物为示范，开展基于斑马鱼内毒素炎症模型的中药复杂成分体系（单体成分、活性组分、有效部位、水煎剂等）抗内毒活性筛选适用性研究，建立一个高通量、低成本的中药抗内毒素活性斑马鱼活体筛选新方法，填补细胞模型等体外筛选体系和哺乳动物模型验证之间过渡模型的空白，具有良好的应用前景。

现代医学针对由内毒素触发的全身炎症反应综合征或脓毒症的治疗尚无特殊有效的措施，临床上仍以对症治疗加糖皮质激素为主，寻找拮抗内毒素治疗措施一直是基础医学与临床医学努力的方向。近十几年来，大量中药、复方或其所含成分被证明具有抗内毒素活性的作用，进一步筛选、分析这些方药的效应物质基础，追踪抗内毒素效应成分迫在眉睫。本项目从寻找快速、高效的中药抗内毒素筛选平台出发，采用模式动物斑马鱼，通过对LPS的染毒方式、剂量及鱼龄的探索，确定了采用3 dpf斑马鱼卵黄囊显微注射0.5 mg/mL LPS的方式，成功构建了斑马鱼内毒素炎症模型，并基于此模型，根据斑马鱼生存率、炎症细胞迁移、炎症因子表达等指标，成功开展了中药单体成分（绿原酸、连翘酯苷A、连翘苷、大黄素、黄芩苷、紫花牡荆素、甘草苷）、单味中药提取物（甘草、连翘的石油醚部位、乙酸乙酯部位、正丁醇部位、水部位）、中药复方及不同提取部位（凉膈散及其石油醚部位、乙酸乙酯部位、正丁醇部位、水部位）的抗内毒素活性评价工作，明确了上述方药及其所含成分的体内抗内毒素活性，还将应用范围成功扩展至筛选单体成分组合（连翘苷、大黄素、黄芩苷、甘草苷）的抗内毒素活性评价。以上研究说明本课题所建立的内毒素斑马鱼模型可为中药复杂体系及中药活性部位的研究提供快速、高效的筛选平台。