【作者】 刘虎；

【导师】 宋琦；

【作者基本信息】 河北大学 ， 中药学， 2024， 硕士

【摘要】 目的:复制慢性应激压力(Chronic Unpredictable Mild Stress,CUMS)模型,明确越鞠丸改善CUMS模型大鼠抑郁样行为的效应,挖掘血清、尿液差异代谢物及相关代谢通路,表征越鞠丸的血中移行成分,分析差异代谢物与血中移行成分的相关性,阐明越鞠丸抗抑郁的药效物质基础及作用机制。方法:复制CUMS模型,以体重、糖水偏好、旷场实验以及强迫游泳为评价方法,明确越鞠丸对CUMS模型大鼠抑郁样行为的改善作用;采用UPLC-Q/TOF-MS~E的代谢组学技术,以血清和尿液为研究对象,分析越鞠丸抗抑郁的代谢轮廓,通过多变量统计分析方法挖掘越鞠丸差异代谢物,并利用Metaboanalyst网站富集分析其影响代谢通路,揭示越鞠丸改善抑郁样行为的作用机制;利用UPLC-Q/TOF-MS~E技术鉴定越鞠丸体外化学成分,通过中药血清药物化学方法结合模式识别方法辨识对越鞠丸血中移行成分进行分析;运用灰色关联度分析和典型相关分析将血中移行成分与血清、尿液差异代谢物进行关联分析,以明确越鞠丸抗抑郁的药效物质基础。结果:1.体重、糖水偏好、旷场实验以及强迫游泳结果显示,造模各组与空白组相比体重显著下降,糖水偏好率显著降低,在旷场箱中的移动总距离和中央区域停留时间显著缩短,强迫游泳不动时间显著增加。越鞠丸给药后,大鼠体重显著增加,糖水偏好率显著升高,在旷场箱中的移动总距离和中央区域停留时间显著增加,强迫游泳不动时间显著下降。2.运用代谢组学技术,对空白组和模型组的血清和尿液进行代谢轮廓表征和多元统计分析,结果显示空白组与模型组的代谢轮廓明显分开。给药后,对代谢轮廓及差异代谢物分析,PCA得分图显示越鞠丸各剂量给药组均表现出远离模型组,接近空白组的方向。在已鉴定的14个血清差异代谢物中,高剂量给药组回调了13个,低剂量给药组回调了14个,高低剂量均回调13个;在已鉴定的19个尿液差异代谢物中,高剂量给药组回调了18个,低剂量给药组回调了19个,高低剂量均回调18个。这些血清差异代谢物主要涉及越鞠丸抗抑郁主要涉及半乳糖代谢、不饱和脂肪酸的生物合成等代谢通路;尿液差异代谢物主要涉及组氨酸代谢、磷酸戊糖途径等通路。3.利用UPLC-Q/TOF-MS~E技术鉴定出越鞠丸的120个体外化学成分,其中香附25个,苍术16个,栀子31个,川芎48个,神曲6个。通过中药血清药物化学方法对越鞠丸的血中移行成分进行分析,共表征了32个入血成分,分别为香附中的L-果糖、无色花青素、莎草二酮、罗森内酯、广藿香酮、对伞花烃、延胡索乙素,苍术中的色氨酸,栀子中的京尼平苷酸、藏红花酸O-β-D-吡喃糖苷、丹皮酚、戊基呋喃、异绿原酸B,川芎中的水杨酸、2-丙酰基呋喃、戊基呋喃、2-乙基己烯醛、14-甲基十六烷酸、川芎醌、亚油酸、丁酸、(1S,2S)-1-乙烯基-1-甲基-2-丙-1-烯-2-基环己烷、(3-甲基戊基)苯、1,3,8-对甲苯、1,4-二乙基-2-甲基苯、5,7-二乙基-5,6-二烯-3-炔、间甲乙苯、亚麻油酸、硬脂酸、芎萘呋内酯,神曲中的异绿原酸B。将上述成分与CUMS模型的血清和尿液差异代谢物进行相关性分析,解析越鞠丸体内显效成分与差异代谢物的关联度,发现13个成分与差异代谢物群极度相关,为越鞠丸抗抑郁的主要显效成分。研究结论:本研究从代谢网络层面揭示越鞠丸改善CUMS模型大鼠抑郁样行为的效应机制,主要调节了半乳糖代谢、不饱和脂肪酸的生物合成、组氨酸代谢、戊糖磷酸途径等通路。通过研究越鞠丸抗抑郁的血中移行成分与内源性代谢物的相关性,阐明13个成分是越鞠丸抗抑郁的药效物质基础,为越鞠丸的临床应用及深度开发提供依据。更多还原

【Abstract】 Objective:The Chronic Unpredictable Mild Stress(Chronic Unpredictable Mild Stress,CUMS)model was replicated to reveal the effect of Yueju Wan on improving depression-like behavior,to mine differential metabolites in serum and urine and related metabolic pathways,to characterize effective ingredients of Yueju Wans in vivo,to analyze the correlation between differential metabolites and ingredients,and to clarify the pharmacodynamic substances and mechanism of Yueju Wan in anti-depression.Method:The CUMS model was evaluated by body weight,sugar water preference,open field test,and forced swimming,to clarify the improvement effect of Yueju Wan on depression-like behavior in CUMS model rats.Based on UPLC-Q/TOF-MS~Eserum and urine metabolomics,we analyzed the metabolic profile of the antidepressant effect of Yueju Wan,mined differential metabolites by multivariate statistical analysis methods,and performed pathway analysis using the online website Metabo Analyst,revealing the mechanism of Yueju Wan in improving depression-like behavior.UPLC-Q/TOF-MS~Eanalysis revealed the ingredients of Yueju Wan in vitro,and effective ingredients of Yueju Wan in vivo were analyzed by the serum pharmacochemistry of TCM combined with pattern recognition method.Grey correlation analysis and Canonical correlation analysis were used to analyze the correlation between the effective ingredients in vivo and differential metabolites in serum and urine,so as to clarify the pharmacodynamic substances of the antidepressant effect of Yueju Wan.Result:1.The results of weight,sugar preference,open field experiments,and forced swimming showed that the model group had a significant decrease in weight and sugar preference rate compared to the control group.The total distance of movement in the open field box and the duration of stay in the central area were significantly shortened,and the duration of forced swimming immobility was significantly increased.After administration of Yueju Wan,the weight of rats significantly increased,the preference for sugar water significantly increased,the total distance of movement in the open field box and the duration of stay in the central area significantly increased,and the duration of forced swimming immobility significantly decreased.2.Metabolomics data were used to characterize the metabolic profiles and multivariate statistical analysis of serum and urine of the control group and model group.The results showed that the metabolic profiles of the control group and model group were significantly separated.After administration,the PCA score showed that all dosage groups of Yueju Wan showed a direction away from the model group and close to the control group.Among the 14differential serum metabolites,13 were reversed in the high-dose administration group,14were reversed in the low-dose administration group,and 13 were reversed in both high and low doses.Among the 19 differential urine metabolites identified,18 were reversed in the high-dose administration group,19 were reversed in the low-dose administration group,and18 were reversed in both high and low doses.These differential serum metabolites were mainly involved in galactose metabolism,biosynthesis of unsaturated fatty acids and etal.The differential urine metabolites mainly involved pathways such as histidine metabolism,pentose phosphate pathway,and etal.3.There are 120 chemical components of Yueju Wans were identified in vitro by UPLC-Q/TOF-MS~E.Among them,there are 25 in Cyperus Rotundus,16 in Rhizoma Atractylodis,31 in Gardenia,48 in Ligusticum Wallichii,and 6 in Medicated Leaven.The ingredients of Yueju Wan in vivo were analyzed by the serum pharmacochemistry of TCM combined with pattern recognition method,and a total of 30 ingredients were characterized,including L-Fructose、Leucocyanidin、Cyperadione、Rosenonolactone、Patchoulenone、p-Cymene、Tetrahydropalmatine in Cyperus Rotundus,Tryptophanin Rhizoma Atractylodis,Geniposidic acid、Picrocrocinic acid O-beta-D-glucopyrinoside、Paeonol、Pentylfuran、Isochlorogenic acid B in Gardenia,Salicylaldehyde、2-Propionylfuran、Pentylfuran、2-Ethyl-hexenal、14-Methylhexadecanoic acid、Senkyunone、Linoleic acid、Butyric acid、(1S,2S)-1-Ethenyl-1-methyl-2-prop-1-en-2-ylcyclohexane、(3-Methylpentyl)benzene、1,3,8-p-Menthatriene、1,4-Diethyl-2-methyl-benzene、5,7-Diethyl-5,6-decadien-3-yne、m-Ethyltoluene、cis-9,cis-12-Linoleic acid、Stearic acid、Wallichilide in Ligusticum Wallichii and Isochlorogenic acid B in Medicated Leaven.Through the correlation between the above ingredients and the differential metabolites in serum and urine,13 ingredients were found to be extremely correlated with the biomarker,which were the main effective ingredients of the antidepressant effect of Yueju Wan.Conclusion:This study reveals the mechanism of Yueju Wan in improving depression-like behavior of CUMS model rats at the metabolic network level,mainly regulating pathways such as galactose metabolism,unsaturated fatty acid biosynthesis,histidine metabolism,and pentose phosphate pathway.By studying the correlation between the ingredients in vivo and differential metabolites of Yueju Wan in antidepressants,it is elucidated that 13 ingredients are the pharmacodynamic substances of the antidepressant effect of Yueju Wan,which provides a basis for its clinical application and in-depth development.更多还原