基于“多维谱效关系”的热毒宁注射液抗急性肺损伤药效物质基础研究

In this study, a strategy based on multidimensional spectrum-effect relationship was proposed, which not only applied several tools and approaches based on multidisciplinary, but also combined analyses in integrity and molecular level. The multidimensional chemical information of Reduing injection (RDN) was achieved using both RPLC-MS, HILIC-MS, and GC-MS analytical techniques, and the corresponding pharmacodynamic effects were obtained based on metabonomics and traditional in vivo approach. The global endogenous metabolic profile associated with acute lung injury was characterized and the intervention mechanisms of RDN were investigated. And the change of endogenous biomarkers related to syndrome was as one of pharmacodynamic activity evaluation. The multidimensional spectrum-effect relationship was analyzed and identified by multidimensional information processing technique. Then the effective component group of RDN would be tracked and clarified. Meanwhile, the chemical components of RDN and its corresponding potential targets which got from metabonomics analysis were virtually screening by the binding capacity between the components and targets using network pharmacology. The results of multidimensional spectrum-effect relationship study were validated by the method of network pharmacology. It will be a novel reference method for effective components study of TCM formula.

本项目以多维谱效关系为切入点，运用多学科研究手段和工具，进行以活性为中心的整体分析与分子水平分析相结合的中药复方药效物质基础研究。在化学物质组成与体内行为特征分析的基础上，获得基于GC-MS、RPLC-MS和HILIC-MS技术的多维化学成分指纹图谱；分析比较给药干预前后生物体内源性代谢物谱的变化，以反映病证本质特征的代谢物调控作为药效评价指标之一，同时测定传统药效评价指标，将两者整合为“综合药效评价指标”，之后将“多维化学成分指纹图谱”与“综合药效评价指标”进行关联，建立“多维谱效关系”，揭示热毒宁注射液发挥作用的药效成分群；通过代谢组学研究，以反映病症本质特征的代谢物调控为靶标，探索其发挥作用的靶点，进行分子模拟，计算预测各化学成分与靶点的结合能力，虚拟筛选药效成分，并对“多维谱效关系”结果进行验证，为中药复方的药效物质基础研究提供新的思路和方法依据。

本项目采用GC-MS、LC-MS和HLPC-UV技术，对热毒宁注射液进行化学物质组成分析，并对其主要成分绿原酸、栀子苷、新绿原酸、隐绿原酸、异绿原酸A、B、C进行了大鼠体内的药代动力学研究，获得了其体内过程特征信息，在此基础上，对给于低、中、高三个剂量组的大鼠血浆进行分析，获得了热毒宁注射液多维化学成分指纹图谱。采用UPLC-MS技术，分析比较给药干预前后大鼠血浆和肺组织的内源性代谢物谱的变化，找到了急性肺损伤的代谢组学特征。在血浆代谢组学研究中，发现并鉴定了8个潜在生物标志物。大鼠血浆与对照组相比，模型组血浆中LPC 18:2、LPC 20:4、LPC 16:0、LPC 18:0和肌酸的浓度降低，色氨酸、苯丙氨酸和不对称二甲基精氨酸的浓度升高，给药后，大鼠体内的相关生物标志物水平趋于正常。在组织代谢组学研究中，发现并鉴定了14个潜在生物标志物。与对照组相比，模型组肺组织中肌酸、肉毒碱、烟酰胺、次黄嘌呤、黄嘌呤、尿嘧啶和植物鞘氨醇的浓度降低，胆碱、甜菜碱、C16-二氢鞘氨醇、鞘氨醇、LPC 20:4、LPC16:0和LPC 18:0的浓度升高。给药后，相关生物标志物水平趋于正常。涉及到急性肺损伤大鼠体内氨基酸代谢、磷脂类代谢、鞘脂类代谢、核苷酸类代谢、能量代谢途径的失调，推测热毒宁注射液可能通过调节以上代谢途径起到治疗急性肺损伤的作用。以热毒宁注射液对代谢物调控作为药效评价指标，并与传统药效评价指标进行数据整合，获得综合药效指标。采用相关和回归分析，将多维化学成分指纹图谱与综合药效指标数据进行关联，建立“多维谱效关系”，找到了热毒宁注射液发挥抗急性肺损伤作用的药效成分为异绿原酸A、B、C、新绿原酸、栀子苷、京尼平酸和青蒿酮。通过分子模拟方法，对热毒宁注射液的化学成分及其代谢产物与相关靶标蛋白进行了分析，并构建了成分-靶标网络，推测热毒宁注射液的活性化学成分可能为异绿原酸A、B以及绿原酸的谷胱甘肽结合及水解产物。本项目系统探讨了热毒宁注射液的物质基础，并为其他中药复方的研究提供新的思路和方法依据。