色谱 ›› 2023, Vol. 41 ›› Issue (1): 47-57.DOI: 10.3724/SP.J.1123.2022.04017

• 研究论文 • 上一篇    下一篇

松花粉干预卵巢早衰大鼠肝脏的广泛靶向代谢组学分析

曲涛2, 陈阳1, 杨长军2, 刘其耸2, 陈惠2, 何志勇1, 王召君1, 陈洁1, 曾茂茂1,*()   

  1. 1.江南大学, 食品科学与技术国家重点实验室, 江苏 无锡 214122
    2.烟台新时代健康产业有限公司, 山东 烟台 264006
  • 收稿日期:2022-04-21 出版日期:2023-01-08 发布日期:2023-01-12
  • 通讯作者: 曾茂茂
  • 基金资助:
    国家食品科学与工程一流学科建设项目(JUFSTR20180201)

Pseudotargeted metabolomics analysis of pine pollen intervention in the liver of premature ovarian failure rats

QU Tao2, CHEN Yang1, YANG Changjun2, LIU Qisong2, CHEN Hui2, HE Zhiyong1, WANG Zhaojun1, CHEN Jie1, ZENG Maomao1,*()   

  1. 1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China
    2. New Era Health Industry (Group) Co., Ltd., Yantai 264006, China
  • Received:2022-04-21 Online:2023-01-08 Published:2023-01-12
  • Contact: ZENG Maomao
  • Supported by:
    National First-class Discipline Program of Food Science and Technology(JUFSTR20180201)

摘要:

卵巢早衰是妇科领域的常见病,中医认为卵巢早衰与肝肾的正常与否息息相关,通过药食两用物质对肝脏代谢的调理是治疗卵巢早衰的一种重要手段。该研究基于超高效液相色谱-三重四极杆质谱(UHPLC-MS/MS)建立的广泛靶向代谢组学技术,探讨破壁松花粉对环磷酰胺诱导的卵巢早衰模型大鼠肝脏代谢的影响,旨在通过测定对照组、模型组、雌激素阳性对照组及施以不同剂量的松花粉干预组的SD大鼠肝脏组织中代谢物的含量变化,结合主成分分析(PCA)、正交偏最小二乘判别分析(OPLS-DA)等多元统计方法揭示松花粉干预卵巢早衰大鼠肝脏代谢的作用机制。通过正、负离子模式共检测出687种肝脏代谢物,PCA与OPLS-DA显示环磷酰胺诱导的模型组能够与对照组、阳性对照组、松花粉干预组等组别之间的代谢物较好的分离。通过单变量分析中的t检验(p<0.05)、变异倍数(FC>2或<0.5)与多变量分析中变量投影重要性(VIP值)>1相结合对差异代谢物进行筛选。与对照组相比,模型组SD大鼠肝脏中的32个生物标志物含量显著升高,28个生物标志物含量显著降低,主要涉及α-亚麻酸代谢、维生素B6代谢、嘌呤代谢、赖氨酸降解和糖酵解/糖异生途径;松花粉的干预可以使代谢物回调,以雌激素组为参考,松花粉的有效干预主要涉及维生素B6的代谢途径。研究表明,花粉提取物可以调节伴随卵巢早衰的肝脏代谢紊乱,促进代谢向正常水平回归。

关键词: 超高效液相色谱-三重四极杆质谱, 广泛靶向代谢组学, 维生素, 松花粉, 卵巢早衰, 肝脏

Abstract:

Premature ovarian failure (POF) is a prevalent gynecological disease. In traditional Chinese medicine, it is believed that POF is directly related to abnormal function of the liver and kidneys. As such, regulation of the liver metabolism through the use of medicinal and edible substances is important for the treatment of POF. Pine pollen, a traditional Chinese medicinal and edible pollen variety, contains various active substances, such as sex hormones and phytohormones, which have been used to inhibit inflammation, regulate the immune system, and protect reproductive tissues. Using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS), this study examined the influence of pine pollen on the liver metabolome of cyclophosphamide-induced POF model Sprague Dawley (SD) rats. The variations in the metabolites present in the liver tissue of control SD rats, model SD rats, and SD rats treated with various doses of pine pollen or estrogen were analyzed using principal component analysis (PCA) in combination with orthogonal partial least squares discriminant analysis (OPLS-DA) and other multivariate statistical methods to reveal the mechanism of pine pollen intervention in the livers of POF SD rats.

An animal model experiment was conducted using six groups of ten-week-old rats. Cyclophosphamide was administered intraperitoneally to the model group and four intervention groups at a dosage of 60 mg/kg for 1 d followed by a dosage of 10 mg/kg for 14 d. Within the following four weeks, each of the four intervention groups received the intragastric administration of 0.1, 0.5, or 1.5 g/kg bodyweight (BW) of pine pollen, or 0.075 g/kg BW of conjugated estrogens (positive control). Equal quantities of normal saline were administered to the control and cyclophosphamide-treated model groups. Subsequently, the rat livers were subject to pseudotargeted metabolomics, and a total of 687 liver metabolites were discovered using both positive and negative ions. The metabolites differing in content were screened using the t-test (p<0.05) and the fold change (FC>2 or <0.5) in univariate analysis, and the variable importance in projection (VIP>1) in multivariate analysis. It was found that in comparison with the control group, the contents of 32 metabolites significantly increased, while those of 28 metabolites significantly decreased in the model group. The majority of these metabolites were involved α-linolenic acid metabolism, vitamin B6 metabolism, and purine metabolism, along with the lysine degradation and glycolysis/gluconeogenesis metabolic pathways. Compared with the cyclophosphamide-induced model group, the estrogen group exhibited increased levels of 47 metabolites and decreased levels of 29 metabolites, wherein 34 metabolites were restored to the levels found in the control group. These metabolites mainly involved the vitamin B6, lysine, glycolysis/gluconeogenesis, arginine and proline, and cysteine and methionine metabolic pathways. In the low/medium/high-dose pine pollen groups, the contents of 34/32/34 metabolites increased, the contents of 30/37/24 metabolites decreased, and the contents of 47/38/34 metabolites were restored to the levels found in the control group, respectively. These metabolites were mainly involved in vitamin B6 metabolism, purine metabolism, and the glycolysis/gluconeogenesis metabolic pathway. These results therefore indicate that the restoring effect of pine pollen is equivalent or superior to that of conjugated estrogen. Additionally, based on the known metabolic pathways, it appears that when estrogen interferes with the liver metabolism, the key metabolic pathways that become affected are the arginine and proline metabolism and cysteine and methionine metabolism pathways. In contrast, pine pollen intervention affected existing metabolic pathways that were known to be disordered by cyclophosphamide. The use of pine pollen may therefore restore the levels of many metabolites. It should be noted that 23 overlaps exist between the estrogen-restored metabolites and the pine pollen-restored metabolites, including a variety of acylcarnitines, such as ACar 10∶0. As a result, pine pollen extract may be able to normalize the liver metabolic abnormalities induced by POF. This study therefore establishes a theoretical reference for the development of functional applications for pine pollen and for the treatment of POF.

Key words: ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS), pseudotargeted metabolomics, vitamins, pine pollen, premature ovarian failure, liver

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