基于血浆代谢组学的新生化颗粒对急性血瘀大鼠作用机制研究

目的 研究新生化颗粒对急性血瘀大鼠血浆中内源性代谢物的影响，探讨新生化颗粒治疗急性血瘀证的可能机制。方法 将50只SD大鼠随机分为对照组、模型组、复方丹参滴丸（阳性药，0.10 g·kg^-1）组和新生化颗粒低、高剂量（4.86、9.72 g·kg^-1）组，给药体积10 mL·kg^-1，对照组和模型组大鼠ig给予等体积0.5%羧甲基纤维素钠（CMC-Na），每天早晚各ig给药1次，共7次。第5次给药后，除对照组外，采用sc盐酸肾上腺素和冰水浴制备大鼠急性血瘀模型。通过测定全血黏度（WBV）、血浆黏度（PV）、活化部分凝血活酶时间（APTT）、凝血酶原时间（PT）、纤维蛋白原（FIB）和凝血酶时间（TT），观察不同剂量的新生化颗粒对急性血瘀大鼠血液流变学和凝血功能的影响；采用超高效液相色谱-四极杆飞行时间质谱联用（UHPLCQTOF-MS）法检测各组大鼠血浆中的内源性代谢物，通过多变量统计分析筛选潜在生物标志物，结合质谱信息、数据库检索和标准品比对鉴定潜在生物标志物，并将鉴定到的生物标志物导入MetaboAnalyst 5.0数据库推测其可能的代谢通路。结果 与模型组比较，新生化颗粒显著降低急性血瘀大鼠WBV、PV和FIB，显著延长APTT、PT和TT（P＜0.05、0.01）。代谢组学结果显示，从急性血瘀大鼠血浆中共鉴定出21个差异代谢物（准确鉴定7个），与对照组比较，模型组大鼠血浆中乳酸、肉碱和肌酐等10个内源性代谢物显著上调，苹果酸、琥珀酸和色胺等11个内源性代谢物显著下调（P＜0.05、0.01）；除了硫酸吲哚酚和脱氧胞苷外，新生化颗粒对其他19个生物标志物均有显著回调作用（P＜0.05、0.01）。这些标志物主要涉及苯丙氨酸、酪氨酸和色氨酸生物合成、苯丙氨酸代谢、亚油酸代谢、三羧酸循环和色氨酸代谢。结论 新生化颗粒对急性血瘀大鼠体内紊乱代谢物有较好的回调作用，其作用机制主要与调节苯丙氨酸、酪氨酸和色氨酸生物合成，苯丙氨酸代谢，亚油酸代谢，三羧酸循环和色氨酸代谢通路有关。

Metabolomics analysis of Xinshenghua Granules for activating blood circulation on acute blood stasis rats

Objective To explore the effects of Xinshenghua Granules on endogenous metabolites in rats with acute blood stasis syndrome, and to investigate the possible mechanism of Xinshenghua Granules for promoting blood circulation on acute blood stasis rats. Methods Tatolly 50 SD rats were randomly divided into control group, model group, compound Danshen Dripping Pills (positive drug, 0.10 g·kg^-1) group and Xinshenghua Granules low-dose and high-dose groups (4.86 and 9.72 g·kg^-1). The volume of administration was 10 mL·kg^-1. Intragastrically, once in the morning and evening, for seven times in total. After the fifth administration, except for the control group, the acute blood stasis model of rats were induced by ice water bath and subcutaneous injection of adrenaline. To evaluate the effects of promoting blood circulation, whole blood viscosity (WBV), plasma viscosity (PV), activated partial thrombin time (APTT), prothrombin time (PT), fibrinogen (FIB) and thrombin time (TT) in different groups were determined. UHPLC-QTOF-MS was applied to investigate the plasma metabolomics changes of acute blood stasis rats, and multivariate statistical analysis was used to screen the potential biomarkers, then the potential biomarkers were identified via database querying and comparison of reference standards, and finally the related metabolic pathways were discovered via MetaboAnalyst 5.0 software. Results Xinshenghua Granules obviously decreased WBV, PV, and FIB, while promoted the APTT, PT and TT (P < 0.05, 0.01). Totally 21 potential biomarkers were identified from blood stasis rats. Compared with control group, the levels of 10 metabolites (such as lactic acid, carnitine and creatinine) increased in model group; while the levels of 11 metabolites (such as malic acid, succinic acid and tryptamine) inclined in model group (P < 0.05, 0.01). Except for indoxyl-sulfate and deoxycytidine, the disturbed levels of other 19 metabolites could be reversed to normal-like levels after the drug treatment of Xinshenghua Granules (P < 0.05, 0.01). The mainly metabolic pathways were involved with phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, linoleic acid metabolism, TCA cycle and tryptophan metabolism. Conclusion Xinshenghua Granules could exert the effects of promoting blood circulation on blood stasis rats by regulating the disturbed endogenous metabolites and the related metabolic pathways.