717解毒合剂抗蝮蛇毒物质基础及对蛇伤NF-κB和MAPK信号通路干预机制研究

Snakebite is the clinical common disease harmful to human health. Traditional Chinese medicine to cure snakebite has unique advantages, Researching Chinese herbal compound has the role of resistance to snake venom is of great significance. This topic select 717 jiedu decoction can treat viper bited patients fastly and effectively in my section office as the research object, based on component tracking method and the whole party research strategy, comprehensively evaluate the resistance of snake venom activity of the decoction and its disassembled prescriptions through animal experiments, set up 717 jiedu decoction of in vitro and in vivo fingerprint with HPLC technology chromatographic separation technology and NMR spectrum analysis technology, Combined with traditional Chinese pharmacy,pharmacology and TCM theory,evaluate the efficacy of 717 jiedu decoction resisting to viper poison ,and clarify its material foundation;Simultaneously the study choose activated the NF-Kappa B and MAPK two signal pathways as the breakthrough point ,adopt ELISA, RT-PCR, flow cytometry instrument, Western blot method ,etc. From the molecules, cells and the overall level,discuss the mechanism of action of 717jiedu decoction resisting to snake venom activity ,ascertain the aging correlation and dose dependence of Resistance of snake venom activity, reveal the new targets of common regulation effects on compatibility of medicines,so as to provide theoretical basis for the clinical application, explore the new direction and new ideas of traditional Chinese medicine compound with integrity, efficiency and systematic characteristics treat snakebite.

毒蛇咬伤是临床常见的危害人类健康的疾病。中医药治疗蛇伤具有独特优势，研究具有抗蛇毒作用的中药复方对蛇伤治疗具有重要意义。本课题以我科临床治疗蝮蛇咬伤快速、有效的717解毒合剂为研究对象，基于成分追踪法与整方研究策略，动物实验综合评价该合剂及拆方的抗蛇毒活性，采用HPLC等色谱分离技术和NMR等波谱分析技术建立717解毒合剂的体外和体内指纹图谱，结合中药化学、药理学及中医药理论，评价717解毒合剂抗蛇毒药效，明确其物质基础；同时以NF-κB和MAPK两条信号通路为切入点，采用ELISA、RT-PCR、流式细胞仪、Western blot 等方法，从分子、细胞和整体水平探讨717解毒合剂抗蛇毒活性分子机制，阐明其抗蛇毒活性的时效相关性与剂量依赖性,揭示其配伍共同调控作用新的靶点，从而为其临床应用提供理论依据，探索出以整体性、高效性和系统性为特点的中药复方治疗毒蛇咬伤的新方向与新思路。

毒蛇咬伤是临床常见的危害人类健康的疾病。中医药治疗蛇伤具有独特优势，研究具有抗蛇伤作用的中药复方对蛇伤治疗具有重要意义。蝮蛇是我国分布最广，数量最多，也是我省伤人最多的蛇种。本课题检测了蝮蛇毒全蛋白成分，分析了蝮蛇毒早期创面蛋白表达差异，并以大鼠、斑马鱼为对象，评价蝮蛇伤致毒作用；重点研究临床治疗蝮蛇咬伤快速、有效的717解毒合剂抗蝮蛇毒物质基础；同时以NF-κB和MAPK信号通路为切入点，探讨其抗蝮蛇毒分子机制。结果显示：蝮蛇毒含蛋白成分224种，其中Metalloproteinase-disintegrin-like、Phospholipase A2、L-amino-acid oxidase等类蛋白为主要的高丰度蛋白。蝮蛇蛇伤表达上调斑点160个，表达下调的斑点141个，并鉴定出其中四种表达上调的蛋白；在屏蔽环境下，蝮蛇毒主要导致心、肝、肾脏毒性，生化指标具有可逆性，但病理损伤存在时间较长，提示抗感染治疗的重要性。实验证实717解毒合剂抗蝮蛇毒成分集中在乙酸乙酯部位，结合PAMPA和MDCK药物吸收模型，通过LC-MS/MS技术检测，并比对经文献检索建立717解毒合剂成分库，两种吸收模型一共鉴定出的147种化合物，其中以β-蜕皮激素、香叶木素、苜蓿素、欧前胡素（或其同分异构体）、邻苯二甲酸二( 2-乙基己基) 酯、山奈酚以及木犀草素类、芹菜素类、槲皮素类等物质含量相对较多而两种吸收模型相同的54种化合物，两部分都含有较多的同分异构体，这为该合剂抗蝮蛇毒物质基础指明了方向同时，也给实验带来大量的未知性，该复方的物质基础的揭示还有待进一步深入研究。蝮蛇毒通过促进IκBα、STAT3及p38蛋白的磷酸化，从而激活NF-κB、STAT3、MAPK等信号通路，促进蛇伤后炎症及并发症的发生发展，717解毒合剂能多途径，多靶点抑制TNF-α、IL-6、CRP等因子表达，阻断重要蛋白的磷酸化，间接抑制NF-κB、STAT3、MAPK信号通路激活，从而减少这些信号通路下游因子的表达，以降低蛇伤后SIRS、MODS、DIC的发生率，有效改善预后。本课题为科学阐释中医药抗蝮蛇伤物质基础及分子机制提供了大量的数据，并为进一步研究中药复方防治各类蛇伤的临床实践提供新的理论基础。