Analysis of Intervention of Erxiantang in Ovariectomized Rats by LC-MS Serum Metabolomics

YANG Ying; HU Jing; LIU Haixia; LIU Yanjun; SONG Changheng; MA Yujie; ZHANG Zhiguo; CHEN Yanjing

doi:10.13422/j.cnki.syfjx.20231161

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Analysis of Intervention of Erxiantang in Ovariectomized Rats by LC-MS Serum Metabolomics

更新时间：2023-09-21

- Analysis of Intervention of Erxiantang in Ovariectomized Rats by LC-MS Serum Metabolomics
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 20, Pages: 118-125(2023)
- 作者机构：
  
  中国中医科学院中医基础理论研究所，北京100700
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20231161
  CLC： R22;R28;R969.1;O657
- Published：20 October 2023，
  
  Published Online：07 June 2023，
  
  Received：06 April 2023，
- 稿件说明：
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杨莹,胡晶,刘海霞等.二仙汤对去卵巢大鼠干预作用的LC-MS血清代谢组学分析[J].中国实验方剂学杂志,2023,29(20):118-125.

YANG Ying,HU Jing,LIU Haixia,et al.Analysis of Intervention of Erxiantang in Ovariectomized Rats by LC-MS Serum Metabolomics[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(20):118-125.
杨莹,胡晶,刘海霞等.二仙汤对去卵巢大鼠干预作用的LC-MS血清代谢组学分析[J].中国实验方剂学杂志,2023,29(20):118-125. DOI： 10.13422/j.cnki.syfjx.20231161.

YANG Ying,HU Jing,LIU Haixia,et al.Analysis of Intervention of Erxiantang in Ovariectomized Rats by LC-MS Serum Metabolomics[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(20):118-125. DOI： 10.13422/j.cnki.syfjx.20231161.

摘要

目的

基于液相色谱-质谱法（LC-MS）探讨去卵巢大鼠血清内源性代谢物的改变及二仙汤的干预作用。

方法

将24只健康雌性SD大鼠随机分为假手术组、模型组和二仙汤组，每组8只，模型组和二仙汤组切除双侧卵巢组织，假手术组切除双侧腹腔内小块脂肪组织，术后2周开始灌胃给药。二仙汤组给予7.5 g·kg

-1

二仙汤进行灌胃，假手术组和模型组灌胃等体积蒸馏水。给药结束后，腹主动脉取血，采用LC-MS对大鼠血清进行非靶向代谢组学研究，结合正交偏最小二乘法判别分析（OPLS-DA）筛选差异代谢物，基于基因组百科全书（KEGG）数据库进行代谢通路分析并采用酶联免疫吸附测定法（ELISA）验证代谢通路关键酶的水平。

结果

代谢组学结果显示，模型组与假手术组间82个差异代谢物包括甘油磷脂、脂肪酰基、类固醇及其衍生物等14类成分，其中差异最显著的为甘油磷脂类。二仙汤可回调82个差异代谢物中的65个，其中11个具有统计学意义，主要为甘油磷脂中的磷脂酰胆碱（PC）和溶血磷脂酰胆碱（LysoPC），其次为类固醇和类固醇衍生物中的皮质酮和11-脱氧皮质醇；代谢通路分析表明模型组甘油磷脂代谢、类固醇激素生物合成等通路发生变化，并在二仙汤给药后得以回调。ELISA结果显示，与假手术组比较，模型组大鼠血清中甘油磷脂代谢物PC、LysoPC生成的关键代谢酶磷酸胆碱胞苷酰转移酶（CCT）、分泌型磷脂酶A2（sPLA2）及溶血磷脂酰胆碱酰基转移酶（LPCAT）水平均明显降低（

0.05，

0.01），胆碱磷酸转移酶1（CPT1）水平有所下降但差异无统计学意义；与模型组比较，二仙汤组CCT、sPLA2、CPT1水平均有显著升高（

0.01）。此外，与假手术组比较，模型组总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）水平显著升高（

0.01），高密度脂蛋白胆固醇（HDL-C）水平明显降低（

0.05）；与模型组比较，二仙汤组TC、TG、LDL-C水平显著降低，HDL-C水平显著升高（

0.01）。

结论

去卵巢大鼠内源性代谢物及相关代谢通路发生改变，二仙汤可回调部分差异代谢物及相关通路，如调节代谢酶CCT、sPLA2、CPT1，从而升高PC、LysoPC的水平以调控甘油磷脂代谢，进而改善去卵巢大鼠脂代谢紊乱等病理变化。

Abstract

Objective

To investigate the changes of endogenous metabolites in serum of ovariectomized rats and the effect of Erxiantang on them based on liquid chromatography-mass spectrometry（LC-MS）.

Method

Twenty-four healthy female SD rats were randomly divided into sham-operated group， model group and Erxiantang group（7.5 g·kg

-1

）， with 8 rats in each group. Bilateral ovarian tissues were excised in the model and Erxiantang groups， and small pieces of adipose tissues were excised in the abdominal cavity of the sham-operated group bilaterally， and gastric administration was started 2 weeks after surgery， and equal volumes of distilled water were gavaged in the sham-operated and model groups. After 12 weeks of administration， blood was collected from abdominal aorta， and non-targeted metabonomics was performed on rat serum by LC-MS， and orthogonal partial least squares-discriminant analysis（OPLS-DA） was used to screen differential metabolites. Metabolic pathway analysis was performed based on Kyoto Encyclopedia of Genes and Genomes（KEGG）， and the levels of key enzymes of metabolic pathways were verified by enzyme-linked immunosorbent assay（ELISA）.

Result

The results of metabonomics showed that 82 differential metabolites between the model group and the sham-operated group were glycerophospholipids， fatty acyls， steroids and steroid derivatives， of which the most significant difference was glycerophospholipids. At the same time， Erxiantang could call back 65 out of 82 differential metabolites， of which 11 were statistically significant， mainly phosphatidylcholine（PC） and lysophosphatidylcholine（LysoPC） in glycerophospholipids， followed by corticosterone and 11-deoxycortisol in steroids and steroid derivatives. Metabolic pathway analysis showed that the pathways of glycerophospholipid metabolism and steroid hormone biosynthesis in model group were changed， and were recovered after the administration of Erxiantang. ELISA results showed that compared with the sham-operated group， serum levels of cholinephosphate cytidylytransferase（CCT）， secretory phospholipase A2（sPLA2） and lysophosphatidylcholine acyltransferase（LPCAT）， which were the key metabolic enzymes of glycerophospholipid metabolite PC and LysoPC， were significantly decreased in the model group（

0.05，

0.01）， and choline phosphotransferase 1（CPT1） levels decreased but the difference was not statistically significant， compared with the model group， the levels of CCT， sPLA2 and CPT1 were significantly increased in Erxiantang group（

0.01）. In addition， compared with the sham-operated group， the levels of cholesterol（TC）， triglyceride（TG） and low density lipoprotein cholesterol（LDL-C） were significantly increased in the model group（

0.01）， the high density lipoprotein cholesterol（HDL-C） level was decreased（

0.05）， compared with the model group， the levels of TC， TG and LDL-C were significantly decreased and the level of HDL-C was significantly increased in Erxiantang group（

0.01）.

Conclusion

Endogenous metabolites and related metabolic pathways in ovariectomized rats were altered， and Erxiantang can reverse some of the different metabolites and related pathways， such as regulating glycerophospholipid metabolism by regulating metabolic enzymes CCT， sPLA2 and CPT1 to increase the levels of PC and LysoPC， and then improve the pathological changes such as lipid metabolism disorder in ovariectomized rats.

关键词

二仙汤液相色谱-质谱法（LC-MS）代谢组学脂代谢甘油磷脂去卵巢大鼠内源性代谢物

Keywords

Erxiantangliquid chromatography-mass spectrometry（LC-MS）metabolomicslipid metabolismglycerophospholipidsovariectomized ratsendogenous metabolites

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