基于转录组学探讨穿心莲内酯对铜绿假单胞菌的抗菌增效机制  

Study on the mechanism of antibacterial potentiation of Pseudomonas aeruginosa by Andrographolide based on transcriptomics

作　　者：陈思敏[1,2] 周婧 赵元静 王辉 徐浩然 谌立巍 CHEN Simin;ZHOU Jing;ZHAO Yuanjing;WANG Hui;XU Haoran;SHEN Liwei(School of Pharmacy,Chengdu University of Traditional Chinese Medicine,Chengdu 611137,Sichuan;State Key Laboratory of Southwestern Chinese Medicine Resources,Chengdu 611137,Sichuan;School of Health and Rehabilitation,Chengdu University of Traditional Chinese Medicine,Chengdu 611137,Sichuan)

机构地区：[1]成都中医药大学药学院/现代中药产业学院,成都610037 [2]成都中医药大学西南特色中药资源国家重点实验室,成都611137 [3]成都中医药大学养生康复学院,成都611137

出　　处：《中药与临床》2024年第5期44-52,共9页Pharmacy and Clinics of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(82374056);国家自然科学基金青年基金(81503325);成都市科技局重点研发支撑计划-技术创新研发项目(2022-YF05-01876-SN)。

摘　　要：目的:基于前期研究结果,探索穿心莲内酯(Andrographolide,Andr)对铜绿假单胞菌(Pseudomonas aeruginosa,PA)的抗菌增效作用机制。方法:利用illunima二代测序平台,比较PA标准菌株ATCC27853在有无Andr干预下的细菌转录组改变,采用基因组比对、差异鉴定、通路和表型分析确定核心靶标;考察在不同浓度穿心莲内酯(100、50、25μg·mL^(-1))干预下ATCC27853绿脓毒素(Pyocyanin,PYO)的分泌量;结合Andr可能的作用机制,RT-q PCR检测其对PA代谢过程中关键基因phz M、phz H、phz S和pvd I表达的影响;尾静脉注射临床分离耐药PA(PA2019-32)构建小鼠急性细菌感染模型,分别给予左氧氟沙星(阳性对照组)、左氧氟沙星联合Andr(联合用药组),观察记录各组小鼠的存活情况,连续观察5 d,绘制生存曲线,测评Andr联合抗菌药物的体内抗感染保护作用。结果:转录组学共组装对比出289个显著差异基因,其中145个基因显著上调,144个基因显著下调。差异基因的GO富集分析显示,差异基因主要涉及细胞代谢过程、细胞组成及“铁离子结合”;KEGG分析差异基因在42条通路中高度富集,主要涉及各类抗菌药物抗性通路,能量代谢相关通路以及毒力因子相关通路,其中色氨酸代谢、乙醛酸和二羧酸代谢、泛醌和其他萜类-泛醌、辅助因子的生物合成、β-内酰胺抗药性、离子抗菌肽(CAMP)抗药性显著下调(pvalue<0.01~0.05)。RT-q PCR检测结果显示,Andr对pvd I无影响,但显著降低phz H和phz S的表达,同时phz M的表达升高,影响铜绿假单胞菌PYO的生物合成过程。验证实验证实Andr降低了PYO的生成,并且体内感染模型实验表明Andr与左氧氟沙星联合使用可显著提高感染小鼠的存活率。结论:通过干扰PYO的合成,调控Fe转运,增强CAMPs活性,可能是Andr最终实现“减毒”和“增效”的重要原因。Objective:Based on the results of previous studies,the antibacterial synergistic effect of Andrographolide(Andr)on Pseudomonas aeruginosa(PA)was explored.Methods:Illumina next-generation sequencing technology was employed to analyze transcriptomic changes in the PA standard strain ATCC27853 in the presence and absence of andrographolide.Core targets were identified through genomic alignment,differential expression analysis,pathway analysis,and phenotypic evaluation.Additionally,we investigated the impact of andrographolide on the expression of key genes involved in pyocyanin metabolism—specifically phzM,phzH,phzS,and pvdI—using RT-qPCR.The production of pyocyanin by ATCC27853 was assessed at various concentrations of Andr(100,50,25μg·mL^(-1)).An acute bacterial infection model was established in mice through tail vein injection of clinically isolated,drug-resistant PA.Mice were divided into a positive control group(levofloxacin)and a combination therapy group(levofloxacin plus Andr).Survival rates were monitored over a five-day period,and survival curves were generated to assess the in vivo anti-infective efficacy of Andr in conjunction with antimicrobial agents.Results:Transcriptomic analysis revealed 289 significantly differentially expressed genes,consisting of 145 upregulated and 144 downregulated genes.Gene Ontology(GO)enrichment analysis indicated that these differentially expressed genes were primarily associated with cellular metabolic processes,cellular composition,and"iron ion binding."Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis demonstrated significant enrichment of these genes across 42 pathways,predominantly related to antimicrobial resistance,energy metabolism,and virulence factors.Notable downregulation was observed in pathways linked to tryptophan metabolism,glyoxylate and dicarboxylate metabolism,ubiquinone and other terpenoid-ubiquinone biosynthesis,cofactor biosynthesis,β-lactam resistance,and cationic antimicrobial peptide(CAMP)resistance(p-value<0.01-0.05).RT-qPCR results ind

关 键 词：转录组 穿心莲内酯 铜绿假单胞菌 绿脓毒素 抗菌增效 

分 类 号：R285.5[医药卫生—中药学]