Toxicity Attenuation Mechanism on Processing Method for Aconiti Lateralis<italic style="font-style: italic"> </italic>Radix Praeparata in Guilingji Based on Urine Metabolomics

XIN Jiayun; CHEN Jia; XU Xike; QI Xingrui; YANG Meixin; LIN Tiantian; LEI Huibo; ZU Xianpeng

doi:10.13422/j.cnki.syfjx.20231168

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Toxicity Attenuation Mechanism on Processing Method for Aconiti Lateralis Radix Praeparata in Guilingji Based on Urine Metabolomics

更新时间：2024-01-15

- Toxicity Attenuation Mechanism on Processing Method for Aconiti Lateralis Radix Praeparata in Guilingji Based on Urine Metabolomics
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 30, Issue 4, Pages: 166-174(2024)
- 作者机构：
  
  1.海军军医大学药学院，基础医学院，上海 200433
  2.山东中医药大学药学院，济南 250355
  3.上海交通大学医学院附属第九人民医院，上海 200011
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20231168
  CLC： R22;R28;R969.1;O657
- Received：30 March 2023，
  
  Published Online：07 July 2023，
  
  Published：20 February 2024
- 稿件说明：
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辛佳芸,陈佳,徐希科等.基于尿液代谢组学探究龟龄集中附子炮制方法的减毒机制[J].中国实验方剂学杂志,2024,30(04):166-174.

XIN Jiayun,CHEN Jia,XU Xike,et al.Toxicity Attenuation Mechanism on Processing Method for Aconiti LateralisRadix Praeparata in Guilingji Based on Urine Metabolomics[J].Chinese Journal of Experimental Traditional Medical Formulae,2024,30(04):166-174.
辛佳芸,陈佳,徐希科等.基于尿液代谢组学探究龟龄集中附子炮制方法的减毒机制[J].中国实验方剂学杂志,2024,30(04):166-174. DOI： 10.13422/j.cnki.syfjx.20231168.

XIN Jiayun,CHEN Jia,XU Xike,et al.Toxicity Attenuation Mechanism on Processing Method for Aconiti LateralisRadix Praeparata in Guilingji Based on Urine Metabolomics[J].Chinese Journal of Experimental Traditional Medical Formulae,2024,30(04):166-174. DOI： 10.13422/j.cnki.syfjx.20231168.

摘要

目的

利用代谢组学方法，通过分析多条代谢通路，从氨基酸代谢、氧化应激和能量代谢等方面揭示附子炮制减毒的作用机理。

方法

将24只大鼠随机分为空白组、生品组、炮制品组（附子醋制后蜜制），每组8只，生品组和炮制品组每天分别灌胃0.64 g·kg

-1

的附子生品和附子炮制品，空白组每天灌胃等量生理盐水，连续给药7 d后，收集大鼠尿液、血清及心脏组织，利用苏木素-伊红（HE）染色法对心脏进行病理学检查，采用微板法和免疫抑制法分别对血清和心脏组织中的乳酸脱氢酶（LDH）及肌酸激酶同工酶（CK-MB）进行活性检测，比较附子炮制前后对大鼠心脏的影响；应用超高效液相色谱-四级杆-飞行时间质谱法（UPLC-Q-TOF-MS）进行尿液代谢组学分析，利用多元统计分析筛选与附子炮制减毒相关的差异代谢物，并进行通路富集分析，探究附子炮制减毒的作用机制。

结果

大鼠心脏组织HE染色结果显示，生品组大鼠的心脏组织可见明显的浆细胞、粒细胞等炎性细胞浸润的现象，表明生附子具有心脏损伤性；炮制品组大鼠的心脏组织HE染色结果与空白组无明显差异，表明经过炮制之后附子毒性明显降低。与空白组比较，生品组大鼠血清及心脏组织中LDH和CK-MB活性明显上升（

0.05），炮制品组大鼠血清及心脏组织中LDH和CK-MB活性明显下降（

0.05），提示炮制后附子的心脏损伤性降低。尿液代谢组学共筛选了108个附子生品明显影响的内源性代谢物，其中附子炮制品明显回调了其中51个差异代谢物，通路富集结果显示，与生附子毒性相关的通路主要包括色氨酸代谢，精氨酸和脯氨酸代谢，苯丙氨酸代谢，氨酰tRNA生物合成，丙氨酸、天冬氨酸和谷氨酸代谢等，炮制品附子减毒相关的体内代谢通路主要包括氨酰tRNA生物合成代谢，色氨酸代谢，苯丙氨酸、酪氨酸和色氨酸的生物合成，苯丙氨酸代谢，咖啡因代谢等。

结论

龟龄集中所载附子醋制后蜜制的炮制工艺可明显减弱作用生附子的心脏损伤性，减毒机制主要涉及氨基酸代谢、氧化应激和能量代谢等方面，为附子炮制减毒机制及临床安全应用相关研究提供实验依据。

Abstract

Objective

Metabolomics was used to reveal the mechanism of Aconiti Lateralis

Radix Praeparata（ALRP）

in attenuating toxicity by processing from the aspects of amino acid metabolism， oxidative stress and energy metabolism by analyzing multiple metabolic pathways.

Method

Twenty-four rats were randomly divided into control group， raw group and processed group， 8 rats in each group. The raw and processed group were given with 0.64 g·kg

-1

of raw ALRP and processed ALRP respectively every day， the control group was given with an equal amount of normal saline once a day. After continuous administration for 7 days， the urine， serum and heart tissue of rats were collected. Pathological examination of the heart was carried out using hematoxylin-eosin（HE） staining， and the activities of lactate dehydrogenase（LDH） and creatine kinase-MB（CK-MB） in serum and cardiac tissues were detected by microplate assay and immunoinhibition assay. The effects of ALRP on rat heart before and after processing were compared and analyzed. Ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS） was used to perform urine metabolomics analysis， and multivariate statistical analysis was used to screen for differential metabolites related to ALRP

in attenuating toxicity by processing， and pathway enrichment analysis was carried out to explore the processing mechanism.

Result

HE staining showed that no obvious pathological changes were observed in the heart tissue of the control group， while obvious infiltration of inflammatory cells such as plasma cells and granulocytes was observed in the heart tissue of the raw group， indicating that the raw ALRP had strong cardiotoxicity. There was no significant difference in HE staining of heart tissue between the processed group and the control group， indicating that the toxicity of ALRP was significantly reduced after processing. Compared with the control group， the activities of LDH and CK-MB were significantly increased in serum and heart tissue of the raw group， and those were significantly decreased in serum and heart tissue of the processed group， suggesting that the myocardial toxicity of processed ALRP was reduced. A total of 108 endogenous differential metabolites associated with the raw ALRP were screened using multivariate statistical analysis in positive and negative modes， of which 51 differential metabolites were back-regulated by the processed ALRP. Biological analysis of the key regulatory pathways and associated network changes showed that the pathways related to toxicity of ALRP mainly included tryptophan metabolism， arginine and proline metabolism， phenylalanine metabolism， aminoacyl-tRNA biosynthesis， alanine， aspartate and glutamate metabolism， etc. The metabolic pathways related to the attenuation of processed ALRP mainly included aminoacyl-tRNA biosynthesis， tryptophan metabolism， phenylalanine， tyrosine and tryptophan biosynthesis， phenylalanine metabolism and caffeine metabolism.

Conclusion

The processing technology of ALRP in Guilingji can significantly attenuate the cardiotoxicity of raw products， the mechanism mainly involves amino acid metabolism， oxidative stress and energy metabolism， which can provide experimental bases for the research related to the mechanism of toxicity reduction of ALRP by processing and its clinical safety applications.

关键词

Keywords

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Toxicity Attenuation Mechanism on Processing Method for Aconiti Lateralis Radix Praeparata in Guilingji Based on Urine Metabolomics

DOI：10.13422/j.cnki.syfjx.20231168

摘要

Abstract

关键词

Keywords

references