清热化痰通腑方干预卒中相关性肺炎大鼠的血清代谢组学研究

Metabolomics Study on the Mechanism of Qingre Huatan Tongfu Decoction on Stroke-associated Pneumonia in Rats

目的基于超高效液相色谱-四级杆-飞行时间串联质谱(UPLC-Q-TOF/MS)技术探讨清热化痰通腑方(HTF)对卒中相关性肺炎(SAP)大鼠血清代谢组的影响,旨在寻找血清中的内源性差异代谢物及其相关代谢通路,以阐释HTF治疗SAP的作用机制。方法将雄性SD大鼠随机分为假手术组、模型组和治疗组,每组7只。采用改良线栓法结合气管注射铜绿假单胞菌法复制SAP模型。造模同时给予大鼠HTF药液38 g·kg^(-1)·d^(-1)灌肠,给药体积为38 m L·kg^(-1),每天1次,连续7 d。应用UPLC-Q-TOF/MS技术进行血清样品的分离和数据采集,通过主成分分析(PCA)及正交偏最小二乘判别分析(OPLS-DA)法分析HTF对内源性差异代谢物的影响,并通过Metabo Analyst 3.0数据库分析其代谢通路。结果在正、负离子模式下共筛选鉴定出22种内源性差异代谢物。与模型组比较,HTF治疗组大鼠血清中的N-乙酰谷氨酸、顺乌头酸、雌激素、癸二酸、维生素E、酪氨酸、脱氧胞苷、莽草酸及胞苷含量显著升高(P<0.01),3-甲氧基-L-酪氨酸、白三烯B4、苯乙酰甘氨酸、磷酸丝氨酸、前列腺素E2、前列腺素F2α、血栓素B2、11-脱氧皮质酮、19-氧睾酮、前列腺素E3、前列腺素F1α、油酸以及棕榈酸含量显著降低(P<0.01)。22个潜在生物标志物主要涉及15条异常代谢通路,其中与苯丙氨酸、酪氨酸和色氨酸生物合成,花生四烯酸代谢,柠檬酸循环(TCA循环),脂肪酸生物合成,类固醇激素生物合成,嘧啶代谢以及甘氨酸、丝氨酸和苏氨酸代谢等7条代谢通路密切相关。结论HTF对SAP大鼠的治疗作用可能与其改善血清中内源性代谢物的水平,调节花生四烯酸代谢、脂质代谢以及能量代谢等有关。

Objective Using the metabolomics technology based on UPLC-Q-TOF/MS,the effects of Qingre Huatan Tongfu decoction(HTF) on stroke-associated pneumonia(SAP) rats was explored,and the relevant potential biomarkers and metabolic pathways of serum were screened to explain the mechanism of HTF treatment of SAP.MethodsMale SD rats were randomly divided into Sham-operated group(J),model group(M)and treatment group(Z),7 rats in each group.Except the group J,SAP models were established in the other two groups by modified suture embolization combined with tracheal injection.Group M was treated with HTF 38 g·kg^(-1)·d^(-1),dosing volume was 38 m L·kg^(-1),while the other two groups were treated with normal saline for 7 days.UPLC-Q-TOF/MS technology was applied to analyze the serum of rats in each group,multivariate statistical methods such as Principal Component Analysis(PCA)and Orthogonal Partial Least Squares Discriminant Analysis(OPLS-DA)were used to screen the relevant potential metabolites,and metabolic pathways were analyzed by Metabo Analyst 3.0.Results Twenty-two potential differential endogenous metabolites were identified in the positive and negative ion modes.Compared with the M group,HTF can significantly increase the concentrations of estrogen,N-acetylglutamate acid,sebacic acid,cytidine,deoxycytidine,cisaconitic acid,vitamin E,tyrosine and shikimic acid(P<0.01),and while significantly decreased the biomakers,concentrations of Phosphoserine,3-methoxy-L-tyrosine,thromboxane B,leukotriene B4,prostaglandin E2,prostaglandin F1α,phenylacetylglycine,prostaglandin F2α,11-deoxycorticosterone,19-oxytestosterone,The concentration of prostaglandin E3,palmitic acid and oleic acid(P<0.01).The relevant potential biomarkers totally involved in 15 metabolic pathways,and among them are arachidonic acid metabolism,glycine,serine and threonine metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,citric acid cycle(TCA cycle),steroid hormone biosynthesis,pyrimidine metabolism and fatty acids,a total of7 metabolic pathways are closely related.ConclusionThe therapeutic effect of HTF on SAP rats may be related to improving the levels of endogenous metabolites in serum,regulating arachidonic acid metabolism,fatty acids and energy metabolism,and then restoring the metabolism to be normal in vivo.