基于脂质代谢调控和质谱成像技术的栀子大黄汤抗肝损伤作用机制研究

In view of the unique advantages of traditional Chinese medicine (TCM) in the treatment of liver injury, it is significant to research on liver protecting TCM prescriptions for the treatment of liver diseases. In this project, the classic prescription Zhi-Zi-Da-Huang decoction (ZZDHD), which was originally reported in the Synopsis of the Golden Chamber, will be studied based on the lipid metabolism regulation and imaging mass spectrometry (IMS). Firstly, the serial metabolic profile spectrums of active constituents of ZZDHD and endogenous lipids in rat blood, bile and liver will be established respectively with analysis techniques such as UHPLC-FT-ICR MS. And then, the impact of ZZDHD on lipid metabolism regulation will be discussed by characterization of lipid biomarkers closely related with the action mechanism based on lipidomics analytic strategy. Furthermore, a brand new method based on IMS will be firstly investigated for visual analysis of active constituents of ZZDHD and endogenous lipids in rat liver tissue sections to explore the correlation between them. In a word, embarking on concept of the wholism for traditional Chinese medicine theories, the action mechanism of ZZDHD on hepatoprotective effect will be elucidated by means of the combinations of chemical and pharmacological, the active constituents in vivo and endogenous lipids study, lipidomics data and pathological physiology along with molecular biology information. And also a new way with innovativeness, efficiency and systematicness aiming at TCM will be explored by this research.

中医药治疗肝损伤具有独特优势，研究具有保肝作用的传统复方对肝病治疗具有重要意义。本课题以《金匮要略》之经方栀子大黄汤为研究对象，拟基于脂质代谢调控和质谱成像技术研究栀子大黄汤抗肝损伤作用机制。利用UHPLC-FT-ICR MS等分析手段建立栀子大黄汤体内效应成分和内源性脂质在大鼠“血-胆汁-肝组织”中的系列代谢轮廓谱；应用脂质组学分析策略，表征与作用机理密切相关的生物标志物，探讨栀子大黄汤对脂质代谢调控的影响；同时利用质谱成像技术可视化地开展栀子大黄汤效应成分和内源性脂质在大鼠肝组织的原位成像研究，并探索二者的联系。本研究将从中医学整体观出发，通过药理与化学相结合、体内效应成分与内源性脂质研究相结合、脂质组学数据与病理生理及分子生物学信息相结合的方法，阐释栀子大黄汤抗肝损伤作用机制，探索以创新性、高效性和系统性为特点的中药研究新思路。

本项目选择具有防治肝损伤功效的传统经方栀子大黄汤为研究对象，在前期研究的基础上，复制了α-萘异硫氰酸酯（ANIT）诱导的胆汁淤积性肝损伤大鼠模型；采用LC-FT-ICR MS技术，分别建立了栀子大黄汤体外化学成分谱和其在大鼠血清、胆汁、肝组织中效应成分的代谢轮廓谱，并对健康大鼠和模型大鼠体内效应成分进行了对比；在栀子大黄汤中共检测并鉴定出91种化学成分，进一步完善了栀子大黄汤的化学物质信息；在健康大鼠和模型大鼠血清中均检测出23种原形成分和19种代谢物，通过对健康和模型两组大鼠血清中效应成分的表达水平进行统计分析，发现了7种效应成分存在显著性差异；在健康大鼠胆汁中共鉴定出30种原形成分和14种代谢物，在模型大鼠胆汁中共检测到26种原形成分和13种代谢物；在健康大鼠肝组织中共鉴定出19种原形成分和9种代谢物，在模型大鼠肝组织中共检测到19种原形成分和7种代谢物。基于RPLC-FT-ICR MS与HILIC-FT-ICR MS技术，分别建立了大鼠血清、胆汁和肝组织中内源性脂质的代谢轮廓谱，利用脂质组学分析策略识别并表征了与栀子大黄汤调控胆汁淤积性肝损伤大鼠脂质代谢相关的潜在生物标志物；在大鼠血清中共发现31个、在大鼠胆汁中共发现9个、在大鼠肝组织中共发现6个与栀子大黄汤抗大鼠胆汁淤积性肝损伤相关的潜在脂质生物标志物；结果表明，栀子大黄汤对大鼠胆汁淤积性肝损伤具有保护作用，其作用机制可能与调节大鼠体内甘油磷脂代谢、鞘脂代谢、甘油酯代谢和糖基磷脂酰肌醇(GPI)-锚生物合成途径有关。基于MALDI-FT-ICR MS技术探索开展了大鼠肝组织质谱成像研究，在大鼠肝组织中获得了4种栀子大黄汤相关成分和28种内源性脂质成分的可视化信息。综上，本研究从中医学整体观出发，以“中药体内效应成分-内源性生物标志物-作用机制”为主线，利用高分辨质谱和质谱成像分析技术，综合表征了栀子大黄汤的体内效应成分，阐释了栀子大黄汤对胆汁淤积性肝损伤大鼠脂质代谢网络调控的影响和抗肝损伤作用机制，为传统经方栀子大黄汤的临床合理应用与进一步开发奠定了基础。