Analysis of Inhibitory Mechanism of Reyanning Mixture Combined with Linezolid Against MRSA and Its Biofilm Based on Metabonomics

Lu-lu ZHANG; Bo WEN; Cheng LYU; Wei-feng YANG; Ya-jun CHU; Mei BAO; Jian-feng YI; Li LI; Cheng-qiang DU; Yong TAN

doi:10.13422/j.cnki.syfjx.20210853

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Analysis of Inhibitory Mechanism of Reyanning Mixture Combined with Linezolid Against MRSA and Its Biofilm Based on Metabonomics

Drug Metabolism | 更新时间：2021-06-25

- Analysis of Inhibitory Mechanism of Reyanning Mixture Combined with Linezolid Against MRSA and Its Biofilm Based on Metabonomics
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 27, Issue 13, Pages: 89-96(2021)
- 作者机构：
  
  1.中国中医科学院中医临床基础医学研究所，北京 100700
  2.宜春学院化学与生物工程学院，江西宜春 336000
  3.中国中医科学院医学实验中心，北京 100700
  4.清华德人西安幸福制药有限公司，西安 710043
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20210853
  CLC： R24;R378.1+1;R28;R969.3
- Received：20 October 2020，
  
  Published Online：07 February 2021，
  
  Published：05 July 2021
- 稿件说明：
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张璐璐,温博,吕诚等.基于代谢组学分析热炎宁合剂联合利奈唑胺对耐甲氧西林金黄色葡萄球菌及其生物膜的抑制机制[J].中国实验方剂学杂志,2021,27(13):89-96.

ZHANG Lu-lu,WEN Bo,LYU Cheng,et al.Analysis of Inhibitory Mechanism of Reyanning Mixture Combined with Linezolid Against MRSA and Its Biofilm Based on Metabonomics[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(13):89-96.
张璐璐,温博,吕诚等.基于代谢组学分析热炎宁合剂联合利奈唑胺对耐甲氧西林金黄色葡萄球菌及其生物膜的抑制机制[J].中国实验方剂学杂志,2021,27(13):89-96. DOI： 10.13422/j.cnki.syfjx.20210853.

ZHANG Lu-lu,WEN Bo,LYU Cheng,et al.Analysis of Inhibitory Mechanism of Reyanning Mixture Combined with Linezolid Against MRSA and Its Biofilm Based on Metabonomics[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(13):89-96. DOI： 10.13422/j.cnki.syfjx.20210853.

摘要

目的

探讨热炎宁合剂（RYN）联合利奈唑胺（LNZ）对耐甲氧西林金黄色葡萄球菌（MRSA）及其生物膜的抑制作用。

方法

采用微量稀释法测定RYN和LNZ对MRSA的最低抑菌浓度（MIC）；微孔板法检测在生物膜生长过程中4个时间点（0，6，12，24 h）的MRSA给药前后活菌量的变化；扫描电镜观察24 h时MRSA形态学变化；细胞代谢组学技术检测在4个时间点时两药联合干预MRSA的内源性小分子终端代谢物的变化。

结果

RYN和LNZ的MIC分别为1/2原液和4 mg·L

-1

。单用LNZ（2 mg·L

-1

）0 h活菌量抑制效果优于1/16 RYN单用，6，12，24 h则单用1/16 RYN优于单用LNZ。RYN联合LNZ在4个时间点的抑制作用均优于单用组。两药联用对24 h时MRSA生物膜形态学结构的破坏优于单用组。环磷酸腺苷（cAMP），二磷酸腺苷（ADP）-

-核糖和2-甲基丁酰辅酶A（2M-CoA）是与被膜形成相关的代谢物；LNZ对这3个代谢物无治疗作用，RYN在12，24 h分别对2M-CoA和ADP-

-核糖产生影响；两药联用在24 h对三者均有治疗作用。

-色氨酸、苯丙酮酸、胞苷和癸二酸是LNZ的药效代谢标志，相关的生物通路有苯丙氨酸、酪氨酸和色氨酸的生物合成，苯丙氨酸代谢。

-组氨酸，尿酸，

-赖氨酸等4个代谢物是RYN的药效标志，相关的生物通路有苯丙氨酸代谢和氨酰转移核糖核酸的生物合成。

-色氨酸，

-赖氨酸，鞘氨醇-1-磷酸等9个代谢物与两药联用的药效相关，相关的生物通路有氨酰转移核糖核酸的生物合成，苯丙氨酸、酪氨酸和色氨酸的生物合成，新生霉素生物合成和酪氨酸代谢。

结论

RYN联合LNZ干预MRSA在其生物膜生长的各时间点均有抑菌增效作用；抑制被膜的机制与cAMP代谢相关；两药联合增效作用则与氨酰转移核糖核酸的生物合成，苯丙氨酸、酪氨酸和色氨酸的生物合成相关。RYN与LNZ联用可作为临床治疗MRSA感染的潜在有效方案。

Abstract

Objective

To investigate the inhibitory effects and mechanism of Reyanning mixture （RYN） combined with linezolid （LNZ） against methicillin-resistant

Staphylococcus aureus

（MRSA） and its biofilm.

Method

The minimum inhibitory concentrations （MICs） of RYN and LNZ against MRSA were determined by microdilution assay. The microplate method was used to detect the changes in viable count before and after MRSA administration at four time points （0， 6， 12， 24 h） in the process of biofilm growth. The morphological changes of MRSA after 24 h were observed by scanning electron microscope. Metabonomic technique was applied to analyze the changes in terminal metabolites of endogenous small molecules from MRSA treated by the two drugs at four time points.

Result

The MICs of RYN and LNZ were 1/2 of the stock solution concentration and 4 mg·L

-1

， respectively. The inhibitory effect of LNZ （2 mg·L

-1

） against viable bacteria at 0 h was better than that of 1/16 RYN. At 6， 12， 24 h， 1/16 RYN was superior to LNZ in inhibiting MRSA. The inhibitory effects of RYN combined with LNZ were better than those of RYN or LNZ alone at the four time points. RYN combined with LNZ caused more severe damages to the morphological structure of MRSA biofilm at 24 h than RYN or LNZ alone. Cyclic adenosine monophosphate （cAMP）， adenosine diphosphate （ADP）-

-ribose and 2-methylbutanoyl-coenzyme A （2M-CoA）， as the metabolites related to biofilm formation， were immune to LNZ， but 2M-CoA and ADP-

-ribose were influenced by RYN at 12 h and 24 h. The combined use of RYN and LNZ interfered with the three metabolites at 24 h.

-tryptophan， phenylpyruvic acid， cytidine and sebacic acid were the pharmacometabolic markers of LNZ， and the related biological pathways were phenylalanine， tyrosine and tryptophan biosynthesis and phenylalanine metabolism. Four metabolites such as

-histidine， uric acid， and

-lysine were the pharmacometabolic markers of RYN， with phenylalanine metabolism and aminoacyl-transfer ribonucleic acid （tRNA） biosynthesis confirmed as the related biological pathways. Nine metabolites such as

-tryptophan，

-lysine， and sphingosine-1-phosphate were responsible for the efficacy of RYN combined with LNZ. The related biological pathways involved aminoacyl-tRNA biosynthesis， phenylalanine， tyrosine and tryptophan biosynthesis， novobiocin biosynthesis， and tyrosine metabolism.

Conclusion

RYN combined with LNZ better exerts the inhibitory effects against MRSA at each time point of its biofilm formation， which is attributed to cAMP metabolism. The synergistic effect resulted from aminoacyl-tRNA biosynthesis and phenylalanine， tyrosine and tryptophan biosynthesis. RYN combined with LNZ can serve as a potentially effective solution to MRSA infection.

关键词

Keywords

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