基于蛋白质组学和代谢组学分析初步探讨苯并(k)荧蒽致小鼠生殖损伤的作用机制  

Mechanism of benzo(k)fluoranthene induced reproductive damage in mice based on proteomics and metabolomics analysis

作　　者：李雅文 王丹丹 王芙蓉 周妮娅 王大朋[1] 曹佳 LI Yawen;WANG Dandan;WANG Furong;ZHOU Niya;WANG Dapeng;CAO Jia(School of Public Health,Key Laboratory of Environmental Pollution Monitoring and Disease Control of Ministry of Education,Guizhou Medical University,Guiyang,Guizhou Province,561113;Institute of Toxicology,Faculty of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]贵州医科大学公共卫生与健康学院,环境污染与疾病监控教育部重点实验室,贵阳561113 [2]陆军军医大学(第三军医大学)军事预防医学系毒理学研究所,重庆400038

出　　处：《陆军军医大学学报》2024年第13期1523-1534,共12页Journal of Army Medical University

基　　金：国家自然科学基金重点项目(82130097);重庆市科卫联合医学科研项目(2021MSXM123)。

摘　　要：目的通过蛋白质组学和代谢组学探究苯并(k)荧蒽[Benzo(k)fluoranthene,BkF]对雄性小鼠生殖损伤潜在的作用机制。方法选取20只健康清洁级6周龄雄性昆明小鼠[体质量(18±2)g],按完全随机分组分为对照组、低剂量BkF组(7.5 mg/kg)、中剂量BkF组(15 mg/kg)、高剂量BkF组(30 mg/kg),每组5只。对照组灌胃给予玉米油,低、中、高剂量BkF组分别给予7.5、15.0、30.0 mg/(kg/d)剂量的BkF,按照10 mL/kg的体积,经口连续灌胃35 d生精周期。每周5 d进行灌胃,连续5周。建模完成后,轻断食10 h进行取材。采用计算机辅助精子分析(computer-assisted sperm analysis,CASA)软件检测分析精液参数;HE染色分析睾丸组织病理结构;采用生物信息学技术分析差异蛋白通路;采用火山图分析高剂量BkF组和对照组差异表达前10的蛋白质;液相色谱-串联质谱法(LC-MS/MS)非靶向代谢组学技术,筛选出差异代谢物;通过KEGG及KEGG信号通路注释分析和GO富集分析差异代谢物。结果与对照组比较,BkF处理的小鼠精子数量和活力有下降趋势,差异具有统计学意义(P<0.05)。病理切片结果显示,与对照组比较,BkF处理的小鼠生精小管管腔扩张,生精细胞排列紊乱。蛋白质组学检测睾丸组织蛋白水平,发现Spata46、Rab5b等蛋白水平降低,Zscan21、Aifm2等蛋白水平升高(P<0.01)。蛋白质组学京都基因和基因组百科全书(Kyoto Encyclopeclia of Genes and Genomes,KEGG)富集分析显示,其主要涉及吞噬体、蛋白质输出、核糖体等通路。基因本体(Gene Ontology,GO)富集分析显示其主要涉及雄性减数分裂Ⅰ、组蛋白乙酰化、p53信号通路的调控、细胞周期的正向调节、细胞死亡的正向调节等信号通路。代谢组学KEGG显示发现氨基糖和核苷酸糖代谢与其他代谢途径联系最为密切。结论蛋白质组学和代谢组学分析结果表明BkF暴露与精子生成、细胞凋亡和周期、DNA损伤、氨基糖和核苷酸糖代�Objective To explore the potential mechanism of Benzo(K)fluoranthene(BkF)on male reproductive injury in mice by proteomics and metabolomics.Methods Twenty healthy and clean male Kunming mice(6 weeks old,18±2 g)were randomly divided into control group(corn oil group),low-,medium-and high-dose BkF groups(7.5,15.0 and 30.0 mg/kg),with 5 mice in each group.The corresponding agents were gavaged at a dose of 10 mL/kg,5 d per week,for 35 consecutive days.After modeling,the rats were fasted for 10 h,and then sperm samples and testicular tissues were harvested.Computer assisted sperm analysis(CASA)was used to detect and analyze semen parameters.HE staining was employed to observe the histopathological structure of the testicular tissue.Bioinformatics analysis was applied to analyze the differential protein pathways.Volcano plot were conducted to analyze the top 10 differentially expressed proteins(DEPs)in the control and high-dose BkF group.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)untargeted metabolomics techniques were utilized to screen out differential metabolites.KEGG signaling pathway and KEGG annotation analyses and GO enrichment analysis were used to analyze the differential metabolites.Results Compared with the control group,the sperm number and motility of BkF-treated mice showed a decreased trend,with statistical differences(P<0.05).Pathological observation showed that BkF treatment resulted in dilated seminal tubules and badly-arranged spermatogenic cells when compared with the control group.Proteomics analysis found that the protein levels of Spata46 and Rab5b were decreased,while those of Zscan21 and Aifm2 were increased(P<0.01).Proteomic KEGG enrichment analysis showed that it was mainly involved in phagosome,protein export,ribosome and other pathways.GO enrichment analysis indicated that it was mainly involved in male meiosis I,histone acetylation,regulation of p53 signaling pathway,positive regulation of cell cycle,positive regulation of cell death and other signaling pathways.Metabonomics

关 键 词：苯并(k)荧蒽 雄性生殖损伤 精子生成 蛋白质组学分析 代谢组学分析 

分 类 号：R322.64[医药卫生—人体解剖和组织胚胎学] R394-33[医药卫生—基础医学] R994.6