基于二维代谢调控网络的黄连解毒汤治疗脓毒症的功效物质基础研究

Huanglian Jiedu Decoction (HLJDD) is a representative Traditional Chinese Medicine formula with efficacy of “Clearing away Heat and Toxins”. Using techniques of metabolomics, gut metagenomes and network biology, with CLP induced sepsis as the model, this project investigate the disposal of drugs by the body and the function of drugs on the body at the same time, and the diversity and structural discrepancy of the gut flora, thereby constructing two-dimensional metabolic regulation networks between the body (including gut flora) and the formula, which combined with pharmacodynamics and pharmacology evaluation, could give an overall characterization of the efficacy of HLJDD in the treatment of sepsis. This method, when combined with the spectrum-activity relationships obtained from the treatment of sepsis by HLJDDs with different combination ratios of the component herbs, could weigh the importance of principles to the efficacy. Based on the correlation networks of differential metabolites, by tracing to its upstream enzymes and genes, the metabolite-enzyme (target) networks were constructed, which were further integrated with the drug-protein (target) network and protein-disease network, established the drug-target-disease network. In our early stage research, cholinergic anti-inflammatory pathway (CAP) was demonstrated to be a good exhibition of the integral advantage of the whole formula. According to the roles and relationships of biomolecules, specific nodes of the integrated network that were related to CAP were selected for further network validation and formula optimization, thereby clarified the material base for the efficacy of HLJDD in the treatment of sepsis and the underlying mechanisms.

黄连解毒汤是清热解毒代表方，本课题以符合其病证特点的严重威胁人类健康的脓毒症为模型，借助代谢组学、肠道菌群宏基因组学和网络生物学技术同时考察生理或病理状态下机体对药物的处置和药物对机体的作用，以及肠道菌群多样性及其结构差异，以构建机体（肠道菌群）-复方药物两个维度的代谢调控网络。结合药效评价，整体表征黄连解毒汤治疗脓毒症的功效；通过对不同组方比例的黄连解毒汤治疗脓毒症谱效关系分析，确定所含成分对药效的权重。由差异代谢物相关网络进一步回溯其上游酶和基因，建立代谢-酶(靶点)网络，并与药物-蛋白(靶点)网络，蛋白-疾病网络融合建立药物-靶点-疾病网络。依据生物分子在网络中的地位和相互关系，选取与前期研究发现的能体现黄连解毒汤整体性优势的胆碱能抗炎通路相关的特定节点进行网络的验证和优化，从而在多个层次说明黄连解毒汤治疗脓毒症的体内功效物质基础及作用机制。

本项目以符合黄连解毒汤清热解毒功效适应症的严重威胁人类健康的脓毒症为病症模型，同时考虑机体-药物的双向作用，基于代谢组学、宏基因组测序结合分子生物学等技术，从多个层面说明黄连解毒汤治疗脓毒症的体内功效物质基础及作用机制。.CLP脓毒症造模后12小时内和12小时后大鼠分别处于过度炎症和免疫抑制的状态。黄连解毒汤给药可以剂量依赖性降低大鼠促炎细胞因子和抑制免疫细胞凋亡，黄连解毒汤可以调节短链脂肪酸产生菌属的丰度提高其含量，缓解脓毒症造成的肠道菌群结构紊乱和代谢失调，既表现出抗炎活性，也具有抗免疫抑制功能。黄连为君药，其特征性生物碱成分可能是黄连解毒汤主要的影响肠道菌群的物质。结果表明，黄连水提物可以有效改善脓毒症大鼠血小板指标和凝血障碍。与黄连解毒汤类似，黄连水提物也可以剂量依赖性降低促炎细胞因子和免疫细胞凋亡说明黄连可以抗炎以及抗免疫抑制。黄连作用于肠道菌群后，改善了肠道菌群组成，纠正紊乱的氨基酸、能量代谢等相关代谢物，进而发挥抗炎、保护肠道屏障、维护肠道缺氧微环境和抗免疫抑制功能。基于上述结果，我们进一步探究黄连解毒汤中重要的生物碱小檗碱对脓毒症造成的肝损伤的保护机制。结果发现小檗碱可以有效调节脓毒症造成的肝细胞代谢紊乱、减轻炎症反应和氧化损伤进而抑制肝细胞凋亡。进一步研究发现小檗碱处理能促进PXR及其调节基因的表达同时抑制NF-κB的激活，抑制炎症反应，表明小檗碱是通过PXR-NF-κB的串扰缓解脓毒症导致的肝损伤，为小檗碱的抗炎机制提供新思路。根据黄连解毒汤四味药在复方中的地位将整方拆方分为四个去除某一味药的子方，通过对比研究拆方与原方治疗脓毒症的作用疗效，分析了其君臣佐使药配伍意义及物质基础，构建了机体（肠道菌群）-复方药物两个维度的代谢调控网络，探讨了该方君、臣、佐、使”配伍治疗脓毒症的配伍内涵。.本项目揭示了黄连解毒汤的成分变化与其治疗脓毒症药效之间的内在关系，从而阐明了其组方的合理性和其清热解毒功效的科学内涵。本项目以黄连解毒汤治疗脓毒症为示例，建立了一个整合中医药学、化学、系统生物学体系，适用于研究外部干预系统(中药复方)与生物应答系统(人体复杂系统包括肠道菌群)之间的相互作用。