基于微透析-代谢组学-因果辨识数理模型的丹参-降香抗脑缺血再灌注损伤效应物质研究

Searching effective drugs to cure cerebral ischemia/reperfusion injury has already to be problem and urgent task in clinical study. Radix Salvia Miltiorrhizae-Dalbergia odorifera has obvious therapeutic effects on this disease, nevertheless, the core effective substance of this herb-pair need more attention and urgent research work. In previous study，metabolism of Radix Salvia Miltiorrhizae-Dalbergia odorifera in plasma of animal model with blood stasis have been investigated, effective substances with remarkable causal relationship such as IDHP, 4-methylbenzamide and so on was found in plasma as specific metabolites cause by compatibility of the two herbs. Meanwhile, IDHP was also found in cerebrospinal fluid of the hippocampus of healthy rats in pretest study. Based on this, this project plan to utilize the combination technique of MD-SPE-HPLC\MS，supervise metabolic profiling of Radix Salvia Miltiorrhizae-Dalbergia odorifera in brain dynamically, investigate cerebrospinal fluid metabolomics at different time periods and position related to the disease. Considering the interaction of drugs and the interaction of between drug and body, the mathematical model modern statistical technique will be employed in processing and analyzing data from metabolomics research, and then the mathematical model of causal relation of ingredients, metabolites of drugs and endogenous substance will be established. Furthermore, the causal identification method of microdialysis, metabolomics and mathematical model will be built. Therefore, this study will acquire the core effective substance of Radix Salvia Miltiorrhizae-Dalbergia odorifera metabolizing in brain from the point of causal relation, and verify the effective substance by pharmacological research, thus interpretating the content of compatibility relation of the herb-pair mentioned above, providing the reference for investigating effective substances of herb-pair.

探寻防治脑缺血再灌注损伤的有效药物已成为临床研究难点和前沿任务。丹参-降香在此方面具有显著疗效，然而其配伍核心效应物质亟待深入研究。项目组前期在丹参-降香血瘀动物血浆代谢研究时，发现了IDHP、4-methylbenzamide等配伍引起的强因果相关效应物质，在正常大鼠海马CA3区初步发现了代谢产物IDHP。据此，本研究拟运用MD-SPE-HPLC\MS-NMR技术，实时、动态、全面监测药物脑内代谢谱，展开不同时间段、疾病相关不同部位脑脊液代谢组学研究；同时采用课题组建立的非平稳随机数理模型，对各组代谢组学数据整理剖析，综合药物之间、药物与机体之相互作用，构建上述药对配伍前后药物物质群、药物代谢物群及内源性物质群之因果关系数理模型，形成微透析-代谢组学-数理模型因果辨识方法，从因果相关角度辨识该药对配伍增效之核心效应物质，辅以药理学确证，更全面诠释其配伍内涵，为中药药对效应物质研究提供借鉴

本项目针对脑缺血再灌注损伤相关疾病的危重性及药物的匮乏，基于微透析、代谢组学等优势联合项目组独有因果数理模型技术，对丹参-降香药对抗脑缺血损伤效应物质进行研究。项目执行中（1）对制备的香丹、丹参及降香注射液进行了成分分析及质量研究，发现注射液符合澄明度、溶血性等检查要求且主要成分含量均符合质控标准；同时完成了注射液中丹参素、原儿茶醛、迷迭香酸、丹酚酸及丹参酮ⅡA等14种成分含量测定；鉴定出香丹注射液53个水溶性和9个挥发性成分。（2）发现丹参-降香配伍后显著降低MCAO大鼠脑含水量、减少脑梗死面积、改善大鼠神经行为学障碍，显著升高脑内LDH、CAT和SOD含量，降低LD、MDA与炎症因子IL-1β、TNF-α和IL-8水平，与抑制酸中毒、抗自由基损伤和抗炎作用有关，且效果明显优于单用组。完成了上述药对抗脑缺血尿样、血样及脑代谢组学研究，发现香丹注射液可回调MCAO大鼠体内发生紊乱的36种差异性物质且效果优于单用组，涉及氨基酸代谢、脂肪酸β氧化、脂类代谢及柠檬酸循环等代谢通路。（3）发现上述药对在正常、脑缺血大鼠体内的39个药物成分及79个代谢物，在心、脑等首次发现咖啡酸异丙酯（KYZ）并初步确定其作用与迷迭香酸、丹酚酸A、丹参素异丙酯、KYZ等相关。（4）基于上述结果，运用微透析、固相萃取及质谱技术展开了丹参-降香在脑缺血再灌注损伤大鼠脑内代谢研究，发现香丹给药后，皮层区以改善神经递质紊乱为主，海马区除了上述现象外，发现了丹参素、迷迭香酸、丹酚酸A及代谢物丹参素异丙酯和KYZ，结合因果数理模型辨识技术，推测丹参素异丙酯、IDHP等透过血脑屏障的药物成分及特征代谢物是其发挥抗脑缺血作用效应物质。（5）对丹参-降香特有代谢产物KYZ进行合成及抗脑缺血再灌注损伤药效学验证，发现其可显著提高大鼠运动能力、减轻脑组织损伤等。. 项目实施期间，共发表SCI论文6篇、中文2篇，拟投稿SCI论文2篇，授权发明专利1项、申请国际发明专利1项，获陕西高等学校科学技术奖一、二等奖各1项，顺利完成了原项目计划书中预期目标。