以lipid A 为靶点的黄连解毒汤抗内毒素的物质基础及作用机制研究

The biological activity of the toxin center lipid A as the target was considered and biosensor technology was used in order to study the material basis and mechanism on antiendotoxin of Huanglian Jiedu Decoction. And the difficult problem of molecular mechanism on prevention and treatment of sepsis was solved. Combining the traditional and modern separation and analysis methods, and high performance liquid chromatography spectra of Huanglian Jiedu Decoction was established. With the help of negative and double negative and single herb spectra, chemical compositions and compatibility of antiendotoxic were discussed. The lipid A was coated on the biosensor hydrophobic sample pool and its biological hydrophilic end was exposed. And that was used as the target of active components screening and detection and tracking and screening of compounds with high affinity to the lipid A were carried out. The activity of compounds on antiendotoxin were examed in vitro and in vivo. With the help of the above researching of Huanglian Jiedu Decoction, the material basis and mechanism were discussed. Lipid A was used as the target and biological sensor technology was used for the fist time in the researching on material basis and mechanism on antiendotoxin of Huanglianjiedu Decoction, and it is a new breakthrough in the studying of molecular mechanism of action on the prevention and treatment of sepsis. Therefore, the item was important scientific significance. At the same time, a technical platform for the researching of material basis and mechanism on antiendotoxin of Huanglian Jiedu Decoction was provided and the foundation for the effective prevention and treatment of sepsis was found. The item was good prospects for studying.

以内毒素的生物活性中心类脂A为靶点，采用生物传感器技术，探讨黄连解毒汤抗内毒素的物质基础和作用机制，解决防治脓毒症的分子机制的难题。将传统和现代的分离分析方法相结合，建立黄连解毒汤特征高效液相图谱，并结合其阴性，双阴性，单味药的图谱分析讨论黄连解毒汤抗内毒素的化学成分和配伍问题。将类脂A包被于生物传感器疏水样品池并使其生物学作用的亲水端外露，以此作为活性组分筛选、跟踪和检测的靶点，筛选出与类脂A亲和力较高的化合物。并对该化合物进行体外、体内抗内毒素的活性研究，同时结合上述研究讨论黄连解毒汤的物质基础和作用机制。首次提出以类脂A为靶点，利用生物传感器技术研究黄连解毒汤抗内毒素的物质基础和作用机制，对防治脓毒症的分子作用机制研究具有重要的科学意义；同时为黄连解毒汤的抗内毒素的物质基础和作用机制研究提供一个技术平台，为脓毒症的有效防制和治疗奠定基础，具有良好的研究前景。

黄连解毒汤（HLJDT）由黄连、黄柏、黄芩、桅子四味药组成，为清热解毒的代表方。本研究主要探讨HLJDT的抗内毒素的药效物质基础和作用机制。在HLJDT制备过程中有沉淀产生，结合HLJDT全方和拆方的高效液相色谱和文献研究发现沉淀主要有黄芩中黄酮类成分、黄连和黄柏中的生物碱类成分以及栀子中的有机酸类成分组成的；上清液中主要含有生物碱、黄酮和环烯醚萜类成分。MTT实验中小檗碱和巴马汀呈现较弱的降低细胞活力作用，与文献报道小檗碱和巴马汀均具有较低的毒性相一致；栀子苷和黄芩苷对细胞活力无影响。表明HLJDT四味药并用，可以降低黄连解毒汤中低毒的生物碱类成分，使其达到减毒增效的作用。. 基于生物传感器技术对HLJDT的有效部位群进行筛选，并分别从高亲和力的有效部位群沉淀水洗、上清醇洗和沉淀醇洗中分离得到小檗碱、巴马汀、药根碱、黄芩苷、黄芩素、汉黄芩素、栀子苷和绿原酸。应用生物传感器技术对单体化合物进行活性筛选得到高亲和力的小檗碱、巴马汀、黄芩苷和栀子苷，并进行体内和体外活性研究。实验结果表明小檗碱和巴马汀中均呈现较好的中和LPS的活性，抑制LPS诱导炎症因子的释放，脏器的保护作用；黄芩苷、栀子苷具有一定Lipid A结合力和中和LPS活性，但是能显著降低炎症因子水平，均具有脏器保护作用。表明HLJDT的多成分起到抗内毒素的作用。结合体内体外活性研究结果及文献报道小檗碱、巴马汀、黄芩苷和栀子苷可能通过中和LPS 和作用TLR4-NF-κB信号通路抑制炎症因子的释放，进一步说明HLJDT通过多靶点起到抗内毒素、治疗脓毒症的作用。. 文献报道生物碱类成分具有抗菌、抗病毒、抗炎、降血糖等作用；黄酮类成分具有抗炎、抗氧化、抗菌、抗病毒等作用；环烯醚萜类成分主要有保肝、抗炎、抗肿瘤、降血糖等活性。但有些成分具有一定毒副作用，如黄芩苷可致过敏反应，小檗碱剂量过大会引起恶心、呕吐、腹泻等胃肠道反应和肾脏炎症，栀子苷具有肝毒性等，但四药合用后并未有不良反应。说明HLJDT配伍后，起到降毒解毒、增强疗效的作用，证明HLJDT配伍的科学性以及中药复方多成分、多靶点治疗的物质基础。黄连解毒汤治疗脓毒症充分发挥了多成分、多途径、多靶点和“菌(细菌)、毒(内毒素)、炎(炎症介质)”并治的特色和优势，为防治毒热证脓毒症提供有效手段和方法。