正常高值血压痰湿壅盛证大鼠模型的建立及代谢组学分析

Establishment and Metabolomics Analysis of Rat Model of High-normal Blood Pressure with Excessive Phlegm-dampness Syndrome

目的:通过构建正常高值血压痰湿壅盛证大鼠模型,分析该模型大鼠的血清代谢物,阐释正常高值血压痰湿壅盛证大鼠模型的代谢特征。方法:采用纯化高脂饲料喂养及腹腔注射Nω-硝基-L-精氨酸(L-NNA,剂量7.625 mg·kg^(-1))的方式构建模型,正常组大鼠喂养普通生长繁殖饲料并腹腔注射等量生理盐水,通过观察两组大鼠一般情况,测量大鼠体质量和尾动脉血压,检测血清甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、血糖、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)含量对模型进行评价;运用液相色谱-质谱联用技术分析大鼠血清代谢物变化,采用流动相0.05%甲酸水溶液(A)-0.05%甲酸乙腈溶液(B)梯度洗脱(0~3 min,2%B;3~9 min,2%~40%B;9~18 min,40%~98%B),电喷雾离子源(ESI),正、负离子模式检测,采集范围m/z 80~1000;结合单变量和多变量统计分析筛选组间差异代谢物并进行代谢通路富集分析。结果:与正常组比较,模型组大鼠毛色较暗、懒动、便溏,血压稳定保持在160/88 mmHg左右(1 mmHg≈0.133 kPa),体质量、血糖、TG、TC、LDL-C、MDA含量明显升高(P<0.05,P<0.01),GSH-Px含量显著降低(P<0.01);正、负离子模式下共筛选出差异代谢物115个,其中45个上调,70个下调[变量投影重要性(VIP)值>1,P<0.05且差异倍数(FC)≥2],包括吲哚乙醛、5-羟色胺、溶血磷脂酰胆碱、磷脂酰胆碱、黄嘌呤等,主要通过甘油磷脂代谢、亚油酸代谢、色氨酸代谢、花生四烯酸代谢等途径调控血压。结论:采用纯化高脂饲料喂养结合腹腔注射L-NNA的方式可以成功构建正常高值血压痰湿壅盛证大鼠模型,吲哚乙醛、5-羟色胺、溶血磷脂酰胆碱、磷脂酰胆碱、黄嘌呤可作为该模型大鼠特征性血清差异代谢物。

Objective:To construct the rat model of high-normal blood pressure with excessive phlegmdampness syndrome and analyze the serum metabolites and explain the metabolic characteristics of the model rats.Method:The model rats of high-normal blood pressure with excessive phlegm-dampness syndrome were fed with high-fat diet and intraperitoneal injected with 7.625 mg·kg^(-1)of Nω-nitro-L-arginine(L-NNA),while rats in the normal group were fed chow diet and injected with the same amount of normal saline.The model was evaluated from the aspects of the general state,body weight,blood pressure and serum biochemical indexes,such as triglyceride(TG),total cholesterol(TC),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),blood glucose,malondialdehyde(MDA)and glutathione peroxidase(GSH-Px).Liquid chromatography-mass spectrometry was used to analyze the changes of metabolites in rats serum.The conditions were as follows:mobile phase of 0.05% formic acid aqueous solution(A)-0.05%formic acid acetonitrile solution(B)for gradient elution(0-3 min,2%B;3-9 min,2%-40%B;9-18 min,40%-98%B),electrospray ionization(ESI),positive and negative ion detection modes,acquisition range of m/z 80-1000.Univariate and multivariate statistical analysis were used to screen the differential metabolites between groups which were used to metabolic pathways enrichment analysis.Result:Compared with the normal group,the rats in the model group had the characteristics of darker fur,lazy movement and loose stools,the body weight,blood glucose and contents of TG,TC,LDL-C,MDA increased significantly(P<0.05,P<0.01)and GSH-Px level decreased significantly(P<0.01),the blood pressure remained stable at about 160/88 mmHg(1 mmHg≈0.133 kPa).A total of 115 differential metabolites were screened in the positive and negative ion modes,of which 45 were up-regulated and 70 were down-regulated[variable importance in the projection(VIP)value>1,P<0.05 and fold change(FC)≥2],including indoleacetaldehyde,5-hydroxytryptamine,lysophosphatidylcholine,phosphatidylcholine,xanthine and so on,which regulated blood pressure mainly through glycerophospholipid metabolism,linoleic acid metabolism,tryptophan metabolism and arachidonic acid metabolic pathways.Conclusion:Feeding with high-fat diet and intraperitoneal injection of L-NNA can successfully construct the rat model of high-normal blood pressure with excessive phlegm-dampness syndrome,and indoleacetaldehyde,5-hydroxytryptamine,lysophosphatidylcholine,phosphatidylcholine and xanthine can be used as characteristic serum differential metabolites of the rat model.