基于代谢组学的“附子理中汤”治疗脾虚证的药效物质基础与作用机理研究

In this project, metabolomics-based strategy was adopted to design experimental programme. Ultra Performance Liquid Chromatography (UPLC/Q-TOF-MS) was applied to detect Fuzi Lizhong decoction (FLD) samples and biological samples of Fuzi Lizhong decoction treated splenic asthenia rats (FLDTSAR) as a key technology. Further, pharmacodynamic chemome were filtered out by means of Partial Least Squares Discriminant Analysis (PLS-DA) and chemical structures of pharmacodynamic components were identified based on accurate molecular weight, MS2 spectrum and retention time. Pharmacodynamic material basic of FLD treating splenasthenic syndrome could be illustrated on the basis of the above results. UPLC/Q-TOF-MS was also used to detect biological samples of healthy rats, splenic asthenia rats and FLDTSAR. Further, in vivo targeted chemome of FLD were screened out by means of PLS-DA. Moreover, expression of critical genes regulated metabolic pathways which was significantly related to the above targeted components was quantified by real-time reverse transcriptional quantitative polymerase chain reaction (RT-qPCR) technology. On the basis of analysis of variance (ANOVA) results of critical genes expression, efficacy of FLD could be evaluated and mechanism of action of FLD treating splenasthenic syndrome could be clarified at the molecular level. Results of this research could provide reference index for clinical diagnosis of splenasthenic syndrome and efficacy evaluation of FLD, and supply a scientific basis for drug developing of treating splenic asthenia.

本项目基于代谢组学的研究策略设计实验方案，以超高效液相色谱-质谱联用法（UPLC/Q-TOF-MS）为关键技术，一方面对附子理中汤样品、附子理中汤治疗的脾虚大鼠生物样品进行检测，借助于偏最小二乘判别分析（PLS-DA）法筛选附子理中汤的药效物质组，并进行结构鉴定，以阐明附子理中汤治疗脾虚证的药效物质基础；另一方面对正常大鼠、脾虚大鼠、以及附子理中汤治疗的脾虚大鼠生物样品进行检测，借助于PLS-DA法筛选附子理中汤药效作用的靶标物质组，并以与这些靶标物质显著相关的代谢通路为切入点，采用实时定量逆转录聚合酶链反应（RT-qPCR）技术，定量分析调控相关代谢通路的关键基因表达水平，通过关键基因表达的方差分析结果评价附子理中汤的疗效，从分子水平上揭示附子理中汤治疗脾虚证的作用机理。本研究可以为脾虚证的临床诊断，以及附子理中汤的疗效评价提供参考指标，为开发治疗脾虚证的新药提供科学依据。

本项目基于附子理中汤对脾虚证疗效确切，但药效物质基础与作用机理不明确的问题开展研究，拟筛选出附子理中汤治疗脾虚证的药效成分、靶标生物标志物、靶标基因及功能。. 一方面，以超高效液相色谱-质谱联用技术作为大鼠体内内、外源性代谢物的检测手段。通过对附子理中汤化学成分、附子理中汤治疗的脾虚大鼠血清样品中外源性代谢物进行分析，共鉴定出22种附子理中汤血中移行成分，其中20种为原型成分的代谢产物；通过对空白对照组、脾虚模型组、附子理中汤治疗组大鼠血清、尿液样品中内源性代谢物进行分析，借助于偏最小二乘判别分析（PLS-DA）法，筛选脾虚证以及附子理中汤疗效作用的生物标志物，结果在血清、尿液中分别鉴定出15、12个脾虚证相关生物标志物，其中血清中6个、尿液中10个为附子理中汤疗效作用的靶标标志物。. 另一方面，以转录组测序技术作为大鼠体内基因的检测手段，对空白对照组、脾虚模型组、附子理中汤治疗组大鼠脾组织中基因表达水平进行分析，借助于DEGseq筛选脾虚证相关基因以及附子理中汤疗效作用的靶标基因，结果共筛选出脾虚证相关基因328个，其中100个被初步确定为附子理中汤疗效作用的靶标基因。进一步借助clusterProfiler进行基因功能富集分析，并选择10个脾虚证相关基因，进行实时荧光定量聚合酶链反应定量分析，阐述附子理中汤干预的基因位点及功能。. 以上研究结果表明，附子理中汤在宏观上通过干预体内脂肪酸降解、甘油磷脂代谢、三羧酸循环、苯甲酸代谢、α-亚油酸代谢途径，在微观上通过干预细胞蛋白质代谢过程、核糖体结构、能量产生和转化功能相关基因的表达发挥健脾功效。本研究结果可为脾虚证的临床诊断及附子理中汤对脾虚证的疗效评价提供参考指标，为开发治疗脾虚证的新药提供前期实验数据。