基于UPLC-Q-TOF-MS的左归丸干预早发性卵巢功能不全大鼠的血清代谢组学研究

Serum Metabolomics Studies on Zuogui Pills in the Treatment of Premature Ovarian Insufficiency Rats Based on UPLC-Q-TOF-MS

目的基于超高效液相色谱-四级杆-飞行时间质谱(UPLC-Q-TOF-MS)技术探寻左归丸干预早发性卵巢功能不全(POI)大鼠的代谢物靶点,以阐明左归丸治疗POI的作用机制。方法以环磷酰胺诱导建立POI大鼠模型,以左归丸(1.851 g·kg^(-1))灌胃给药干预,每日1次,连续60 d。采用卵巢组织病理学观察(HE染色法)、各级卵泡计数,联合酶联免疫吸附试验(ELISA)检测血清卵泡刺激素(FSH)和雌二醇(E_(2))水平,评价POI大鼠模型及左归丸的干预作用。采用UPLC-Q-TOF-MS技术进行血清非靶向代谢物检测,运用代谢组学方法分析POI大鼠血清中潜在的生物标志物,以探寻左归丸作用的代谢物靶点及代谢通路。结果左归丸干预能明显增加POI大鼠卵巢组织中的生长卵泡数目(P<0.05),降低闭锁卵泡数目(P<0.05)及血清FSH水平(P<0.05)。共鉴定出POI大鼠血清中潜在的生物标志物93种;左归丸作用的差异性代谢物共20种,左归丸能显著回调α-羧乙基羟色曼、2-羟基-6-十五烷基苯甲酸、11(R)-羟二十烷四烯酸和D-果糖的水平;左归丸作用的差异性代谢物主要富集在鞘脂代谢、花生四烯酸代谢、亚油酸代谢等代谢通路。结论左归丸可能通过影响多种物质代谢,从抗氧化、抗炎等多个方面改善POI大鼠的卵巢功能。

Objection To investigate the metabolite targets of Zuogui Pills in rats with premature ovarian insufficiency(POI)based on ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF-MS)technique and to elucidate the mechanism of action of Zuogui Pills in the treatment of POI.Methods A rat model of POI was established by cyclophosphamide induction,and Zuogui Pills(1.851 g·kg^(-1))was administered by gavage once daily for 60 continuous days.Serum follicle stimulating hormone(FSH)and estradiol(E_(2))levels were measured by histopathological observation of ovaries(HE staining)and follicle counting at all levels,combined with enzyme-linked immunosorbent assay(ELISA),and the POI model and the intervention effect of Zuogui Pills in rats was evaluated.Serum non-targeted metabolites were detected by UPLC-Q-TOF-MS,and the potential biomarkers in the serum of POI rats were analyzed by metabolomics to explore the metabolite targets and metabolic pathways of the effects of Zuogui Pills.Results Zuogui Pills intervention significantly increased the number of growing follicles(P<0.05)and decreased the number of atretic follicles(P<0.05)and serum FSH level(P<0.05)in the ovarian tissues of POI rats.A total of 93 potential biomarkers were identified in the serum of POI rats and 20 metabolite targets were identified;Zuogui Pills significantly modulated the levels ofα-carboxyethylhydroxychroman,2-hydroxy-6-pentadecylbenzoic acid,11(R)-hydroxyeicosatetraenoic acid and Dfructose;The metabolite targets of Zuogui Pills are mainly enriched in sphingolipid metabolism,arachidonic acid metabolism and linoleic acid metabolism.Conclusion Zuogui Pills may improve ovarian function in POI rats in several ways,including antioxidant and anti-inflammatory,by affecting the metabolism of various substances.