摘要
目的:以脾胃湿热证模型大鼠为研究对象,基于尿液代谢组学寻找潜在生物标志物及相关代谢通路,从内源性小分子代谢物层面探究脾胃湿热证候本质。方法:将16只SD大鼠随机分为正常组及模型组,正常组给予普通饲料喂养,模型组在此基础上每日增加200 g·L^(-1)蜂蜜水自由饮用,猪油与白酒隔日交替灌服,共喂养17 d,第10天起每日相同时间将大鼠置于温度30~34℃、相对湿度95%的环境中2 h,共7 d,建立脾胃湿热证大鼠模型,并通过观察一般体征,酶联免疫吸附测定法(ELISA)检测血浆胃动素(MTL)、胃泌素(GT)含量及胃肠组织病理学检查对模型进行评价;利用超高效液相色谱-四极杆-飞行时间质谱法(UPLC-Q-TOF-MS)进行尿液代谢组学分析,检测条件为ACQUITY^(TM)UPLC BEH C_(18)色谱柱(2.1 mm×100 mm,1.7μm),流动相0.1%甲酸水溶液(A)-0.1%甲酸乙腈溶液(B)梯度洗脱(0~3 min,1%~18%B;3~8 min,18%~40%B;8~10 min,40%~100%B),流速0.4 m L·min^(-1),电喷雾离子源(ESI),正、负离子模式检测,扫描范围m/z 50~1 000。利用单变量与多变量统计分析筛选组间差异离子,根据精确相对分子质量计算其元素组成,将碎片离子信息与人类代谢组数据库(HMDB)等数据库匹配以鉴定生物标志物。通过京都基因和基因组百科全书(KEGG)数据库获得代谢物的生物信息,采用Metabo Analyst 5.0软件分析其相关代谢通路。结果:与正常组比较,模型组肛温显著上升(P<0.01),血浆MTL和GT水平均明显下降(P<0.05,P<0.01),胃及结肠组织出现充血、出血及炎性浸润等病理性变化;尿液代谢组学研究发现L-组氨酸、柠檬酸、异柠檬酸等25个差异代谢物可作为脾胃湿热证的生物标志物,其中13个代谢物为内源性物质的Ⅱ相代谢产物(葡萄糖醛酸结合物、硫酸结合物和乙酰基结合物),涉及组氨酸代谢、三羧酸循环、乙醛酸和二羧酸代谢等代谢通路。结论:脾胃湿热证主要引起组氨酸代谢、三羧酸循环、乙醛酸和二羧酸代谢3条代谢通路的紊乱,以及内源性代谢物的活性/失活状态失衡,可能涉及机体免疫应答、物质及能量代谢、炎症反应和肠道菌群等方面,可为脾胃湿热证模型的建立和应用提供依据。
Objective:Taking the rat model of spleen-stomach damp-heat syndrome(SSDHS)as the research object,this study aimed to investigate the potential biomarkers of SSDHS and the related metabolic pathways based on urine metabolomics,and tried to reveal the essence of SSDHS at the level of endogenous small molecular metabolites.Method:Sixteen SD rats were randomly divided into normal and model groups.The normal group was fed normal chow and the model group was fed with 200 g·L^(-1) honey water daily,and lard and Chinese Baijiu alternately on alternate days for 17 days.The SSDHS model rats were exposed to external dampness-heat environment with temperature at 30-34℃,relative humidity of 95%for 2 h at the same time every day from the 10th day for 7 d.Then,the model was evaluated by observing the general conditions of the rats,measuring the contents of motilin(MTL)and gastrin(GT)in plasma by enzyme-linked immunosorbent assay(ELISA),and examining the histopathology of gastronitestinal tissues.In additon,the urine metabolomics analysis was performed by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS),and the detection conditions was as follows:ACQUITY™UPLC BEH C_(18) column(2.1 mm×100 mm,1.7μm),mobile phase of 0.1%formic acid aqueous solution(A)-0.1%formic acid acetonitrile solution(B)for gradient elution(0-3 min,1%-18%B;3-8 min,18%-40%B;8-10 min,40%-100%B),the flow rate of 0.4 mL·min-1,electrospray ionization(ESI)in positive and negative ion modes,scanning range of m/z 50-1000.The univariate and multivariate statistical analysis were constructed for screening inter-group differential ions,the element composition was calculated according to the precise relative molecular weight,and ion information was matched with databases such as Human Metabolome Database(HMDB)to identify biomarkers.Kyoto Encyclopedia of Genes and Genomes(KEGG)database was used to obtain the biological information of metabolites,and their associated metabolic pathways were analyzed by MetaboAnalyst 5.0.Result:Compared with the normal group,the rectal temperature of the model group increased significantly(P<0.01),the levels of plasma MTL and GT decreased significantly(P<0.05,P<0.01),and pathological changes such as bleeding,congestion and inflammatory infiltration in the gastric and colonic tissues.A total of 25 differential metabolites such as L-histidine,citric acid and isocitric acid were found to be the potential biomarker of SSDHS by urine metabolomics,13 of which were phaseⅡmetabolites of endogenous substances(glucuronic acid conjugates,sulfuric acid conjugates and acetyl conjugates),involving the metabolic pathways of histidine metabolism,tricarboxylic acid cycle,glyoxylate and dicarboxylate metabolism.Conclusion:SSDHS primarily causes disorders of histidine metabolism,tricarboxylic acid cycle,glyoxylate and dicarboxylate metabolism,as well as the imbalance of the activation/inactivation of endogenous metabolites,which may involve the immune response,material and energy metabolism,inflammatory response and intestinal flora,and may provide a basis for the establishment and application of SSDHS model.
作者
于静波
韩越
谢新
周梓洋
陈静梅
王韦轶
邱靖淞
王宇红
YU Jingbo;HAN Yue;XIE Xin;ZHOU Ziyang;CHEN Jingmei;WANG Weiyi;QIU Jingsong;WANG Yuhong(School of Pharmacy,Science&Technology Innovation Center/National Key Laboratory Cultivation Base of Chinese Medicinal Powder&Innovative Medicinal Jointly Established by Province and Ministry,Hunan Key Laboratory of Traditional Chinese Medicine Prevention&Treatment of Depressive Diseases,Hunan University of Chinese Medicine,Changsha 410208,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2023年第10期166-173,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(82204613)
湖南省自然科学基金项目(2020JJ5415,2021JJ40410)
湖南省教育厅项目(20C1413)
湖南中医药大学一流学科开放基金项目(2020ZYX06,2021ZYX25)
中药粉体与创新药物省部共建国家重点实验室培育基地开放基金项目(科函[2020]8号)。
