冠心病心血瘀阻证大鼠心肌代谢组学研究

Myocardial Metabolomics in Rats with Coronary Heart Disease and Blood Stasis Obstruction Syndrome

目的运用代谢组学的方法探讨冠心病(CHD)心血瘀阻证的潜在生物学基础。方法20只SD大鼠随机分为正常组8只和心血瘀阻证组12只。心血瘀阻证组采用高脂饲料喂养结合维生素D3灌胃、异丙肾上腺素皮下注射制备冠心病心血瘀阻证模型。观察两组大鼠心脏组织病理学以及心肌细胞线粒体超微结构变化;运用液相色谱-质谱联用(LC-MS)技术对大鼠心肌组织进行代谢组学研究,结合主成分分析(PCA)和正交偏最小二乘法分析(OPLS-DA)筛选组间差异代谢物并进行相关代谢通路的分析,并利用受试者工作特征曲线(ROC)对潜在生物标志物用于冠心病心血瘀阻证诊断的准确性进行评价。结果正常组大鼠心肌细胞排列整齐,结构清晰,线粒体排列规则,结构完整;心血瘀阻证组大鼠心肌细胞肥大水肿,肌纤维肿胀,可见炎症细胞浸润,线粒体大小不一,数量减少,排列紊乱,部分线粒体已溶解破裂,膜结构消失。两组大鼠的内源性代谢物存在显著差异,得到19种组间差异代谢物。与正常组比较,心血瘀阻证组中磷酸三苯酯、肌苷、TrHA、牛磺酸、泛酸、丙基硫氧嘧啶表达水平上调;LysoPC[18∶2(9Z,12Z)]、PE[18∶1(9Z)/0∶0]、PE[0∶0/18∶2(9Z,12Z)]、PC[18∶2(2E,4E)/0∶0]、PC[18∶2(9Z,12Z)/0∶0]、PC[18∶1(9Z)/0∶0]、PG(16∶0/0∶0)[U]、L-异亮氨酸、2-羟基肉桂酸、DG[15∶1(9Z)/18∶1(9Z)/0∶0][iso2]、黄嘌呤、甲胺、L-苯丙氨酸表达水平下调。涉及苯丙氨酸、酪氨酸和色氨酸的生物合成,氨酰-tRNA生物合成,缬氨酸、亮氨酸和异亮氨酸的生物合成,牛磺酸和亚牛磺酸代谢等多条代谢通路。ROC评价结果显示,L-苯丙氨酸、L-异亮氨酸、牛磺酸这3种差异代谢物对冠心病心血瘀阻证预后判断具有较高的预测价值。结论冠心病心血瘀阻证大鼠心肌代谢产物主要涉及氨基酸代谢、脂类代谢和嘌呤代谢等多个层面,其中L-苯丙氨酸、L-异亮氨酸、牛磺酸这3种代谢物有望作为标志物单独或者联合用于冠心病心血瘀阻证的早期诊断。

Objective To explore the potential biological basis of coronary heart disease(CHD)with heartblood stasis obstruction syndrome by using metabolomics.Methods Twenty SD rats were randomly divided into normal group(8 rats)and the heart-blood stasis obstruction syndrome group(12 rats).The model of CHD with heartblood stasis obstruction syndrome was prepared by high-fat diet combined with vitamin D3 and subcutaneous injection of isoproterenol.The changes of cardiac histomorphology and mitochondrial ultrastructure of cardiomyocytes were observed.The myocardial tissue samples of rats were collected for metabolomics research using liquid chromatographymass spectrometry(LC-MS).The differential metabolites between the groups were screened through principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA),and the related metabolic pathways were analyzed.Results The cardiomyocytes in the normal group were arranged orderly and the structure was clear,with regularly arranged and complete mitochondria.Cardiac myocyte hypertrophy,muscle fibers swelling,and inflammatory cell infiltration were observed in the heart blood stasis obstruction syndrome group,with decreased and disorderly arranged mitochondria in different sizes,as well as certain dissolved and ruptured mitochondria and disappeared membrane structure.The endogenous metabolites of rats in both groups were significantly different,and 19 differential metabolites were detected.The rats in the heart blood stasis obstruction syndrome group had increased expression levels of triphenyl phosphate,inosine,TrHA,taurine,pantothenic acid and propylthiouracil,as well as decreased expression of LysoPC[18:2(9 Z,12 Z)],PE[18:1(9 Z)/0:0],PE[0:0/18:2(9 Z,12 Z)],PC[18:2(2 E,4 E)/0:0],PC[18:2(9 Z,12 Z)/0:0],PC[18:1(9 Z)/0:0],PG(16:0/0:0)[U],L-isoleucine,2-hydroxycinnamic acid,DG[15:1(9 Z)/18:1(9 Z)/0:0][iso2],xanthine,methylamine,and L-phenylalanine Acid than those in the normal group.Multiple metabolic pathways were involved such as phenylalanine,tyrosine and tryptophan biosynthesis,aminoacyl tRNA biosynthesis,valine,leucine and isoleucine biosynthesis,taurine and taurine metabolism.The ROC results showed that the three differential metabolites including L-phenylalanine,L-isoleucine and taurine had a high predictive value for prognosis of CHD with heart blood stasis obstruction syndrome.Conclusion The myocardial metabolites of CHD rats with heart blood stasis obstruction syndrome mainly involve amino acid metabolism,lipid metabolism and purine metabolism.Three metabolites including L-phenylalanine,L-isoleucine and taurine,alone or in combination,are expected to be used as markers for the early diagnosis of CHD with heart and blood stasis obstruction syndrome.