基于代谢组学的研究方法探讨毒性剂量对乙酰氨基酚对小鼠血浆氨基酸代谢的影响  

Effect of Over-dose APAP on Mice Amino Acid Metabolism in Plasma Based on Metabonomics

作　　者：杨虹 彭芳 刘刚 时京珍 郝小燕[2] 钱海兵 Yang Hong;Peng Fang;Liu Gang;Shi Jingzhen;Hao Xiaoyan;Qian Haibing(Guizhou university of chinese medicine,Guiyang 550002;Guizhou medical university,Guiyang 550002)

机构地区：[1]贵州中医药大学,贵阳550002 [2]贵州医科大学,贵阳550002

出　　处：《中药药理与临床》2020年第2期162-167,共6页Pharmacology and Clinics of Chinese Materia Medica

基　　金：国家自然科学基金资助项目(编号:81460640、81860700);贵州省普通高等学校特色重点实验室[黔教合KY字(2017)006];贵州省国内一流建设学科(中药学)[GNYL(2017)008号];贵州省科技合作计划项目[黔科合LH字(2016)7122号];黔科合[SY(2010)3016];贵州中医药大学校内项目(编号:3040-040170106)。

摘　　要：目的:基于代谢组学的研究方法,考察毒性剂量对乙酰氨基酚造模1 h后(ALT尚未升高,GSH已明显降低),小鼠血浆氨基酸相关代谢物的改变情况,为临床诊断寻找潜在生物标志物。方法:30只雄性balb/c小鼠,随机分为两组,正常对照组(10只)、对乙酰氨基酚肝损伤组(20只)。腹腔注射对乙酰氨基酚300 mg/kg建立急性肝损伤模型,造模后将对乙酰氨基酚组小鼠进一步随机分为对乙酰氨基酚1 h组、对乙酰氨基酚16 h组。正常对照组、对乙酰氨基酚1 h组于造模后1 h取血、肝组织,对乙酰氨基酚16 h组于造模后16 h取血及肝组织。检测血浆谷丙转氨酶(ALT)活力,测定肝组织还原型谷胱甘肽(GSH)水平。超高效液相色谱串联三重四级杆飞行时间质谱法(UPLC-Triple-TOF-MS)检测小鼠造模后1 h血浆代谢产物进行代谢组学分析,正交偏最小二乘法(OPLS-DA)区分组间整体代谢轮廓差异,寻找与对乙酰氨基酚代谢相关的差异代谢物,以及氨基酸相关差异代谢物。差异代谢物进行KEGG通路注释分析,并将差异代谢物与血浆对乙酰氨基酚水平进行相关性分析。结果:与正常对照组比较,对乙酰氨基酚组给药1 h后血浆ALT水平未见明显改变,肝组织GSH含量明显降低,给药16 h后血浆ALT水平明显升高(P<0.05),肝组织GSH含量与正常对照组未见明显差异。经OPLS-DA分析,结果显示对乙酰氨基酚及其代谢产物对乙酰氨基酚硫酸结合物水平与正常组比较明显升高(P<0.05),相关差异代谢物包括D-色氨酸、吲哚乳酸、吲哚-3-丙酸、5’-甲硫腺苷,涉及色氨酸代谢通路及半胱氨酸及蛋氨酸代谢通路,其中D-色氨酸、吲哚乳酸、吲哚-3-丙酸与对乙酰氨基酚水平显著相关(P<0.01)。结论:对乙酰氨基酚染毒1 h后,发现与氨基酸代谢相关的4个差异代谢物,其中3个差异代谢物与色氨酸代谢相关。Objective: To investigate the amino acid metabolism induced by over-dose APAP(toxic dose, 1 h) in mice in plasma metabonomics study, in order to find its clinical potential biomarker. Methods: 30 balb/c male mice were randomly divided into the control group(10) and APAP group(20). Mice in APAP group were intraperitoneally injected with APAP 300 mg/kg to establish acute liver injury model. After modeling, mice were divided into APAP 1 h and 16 h groups. Mice livers and plasma in APAP 1 h group and control group were collected 1 h after injection, livers and plasma in APAP 16 h group were collected 16 h after injection. The ALT activity in plasma and GSH level in liver tissues were detected. The plasma metabolites after 1 h were detected by UPLC-Triple-TOF-MS. The integral differences of metabolic profiling were described by OPLS-DA. The differential metabolites related to APAP metabolic process and amino acid metabolism were searched and identified. Differential metabolites were investigated by KEGG pathway analysis, and its relativity analysis was performed with APAP level in plasma. Results: Compared with the control group, no significant change was observed on ALT level 1 h after modeling, GSH level of liver tissues were significantly decreased. 16 h after modeling,ALT level in mice serum were significantly increased,while no significant change was observed on GSH level. OPLS-DA result showed, compared with the control group, levels of APAP and APAP-sulfuric acid complex were significantly increased in APAP group. D-tryptophan, Indolelactic acid,3-Indolepropionic acid and 5’-methylthioadenosine were identified as differential metabolites. The levels of D-tryptophan, Indolelactic acid and 3-Indolepropionic acid were significant correlated with APAP level.Conclusion: 1 h after over-dose APAP administration,4 related differential metabolites were found in mice plasma,in which 3 differential metabolites were related with tryptophan metabolism.

关 键 词：对乙酰氨基酚 药物性肝损伤 色氨酸代谢 生物标志物 

分 类 号：R965[医药卫生—药理学]