经典名方五子衍宗丸干预半去势雄性小鼠生精功能的转录组学分析  

Transcriptomic Analysis of Wuzi Yanzongwan on Testicular Spermatogenic Function in Semi-castrated Male Mice

作　　者：邹迪新 张岳阳[2] 孟雪丹 鹿伟[4] 吕爽 曾凡俊 陈坤 刘畅 张钟秀 段宇 代一航 王昭懿[3] 王智民[1] 林瑞超[3] ZOU Dixin;ZHANG Yueyang;MENG Xuedan;LU Wei;LYU Shuang;ZENG Fanjun;CHEN Kun;LIU Chang;ZHANG Zhongxiu;DUAN Yu;DAI Yihang;WANG Zhaoyi;WANG Zhimin;LIN Ruichao(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Wangjing Hospital of China Academy of Chinese Medical Sciences,Beijing 100102,China;Chinese Medicine Quality Evaluation Center,Beijing Key Laboratory,School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100102,China;The Affiliated Hospital of Inner Mongolia Medical University,Hohhot 010050,China;The First Hospital of Hebei Medical University,Shijiazhuang 050031,China;General Administration of Customs(Beijing)International Travel Health Care Center,Beijing 100013,China;Beijing Center for Vaccine Control,Beijing Institute for Drug Control,Beijing 102206,China;School of Sport Science,Beijing Sport University,Beijing 100084,China)

机构地区：[1]中国中医科学院中药研究所,北京100700 [2]中国中医科学院望京医院,北京100102 [3]北京中医药大学中药学院,中药品质评价中心/北京市重点实验室,北京100102 [4]内蒙古医科大学附属医院,呼和浩特010050 [5]河北医科大学第一医院,石家庄050031 [6]海关总署(北京)国际旅行卫生保健中心,北京100013 [7]北京市药品检验研究院北京市疫苗检验中心,北京102206 [8]北京体育大学运动人体科学学院,北京100084

出　　处：《中国实验方剂学杂志》2024年第1期61-69,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：中国中医科学院科技创新工程项目(CI2021A02210,CI2021A02208);北京市自然科学基金项目(7232303);国家自然科学基金项目(82304718,81760837);中央级公益性科研院所基本科研业务费专项(ZXKT22009)。

摘　　要：目的:筛选五子衍宗丸干预半去势雄性小鼠生精功能相关的转录组,探讨其在干预生精功能低下进展中的潜在作用机制。方法:Balb/c小鼠按体质量随机分为假手术组、模型组、丙酸睾丸素组、五子衍宗丸组,每组12只,模型组和给药组摘取右侧睾丸和附睾构建半去势生精功能低下模型,假手术组仅剪开毛皮和右侧阴囊并立即消毒缝合,饲养1周后,五子衍宗丸组每天灌胃五子衍宗丸混悬液1.56 g·kg^(-1),丙酸睾丸素组肌肉注射丙酸睾丸素0.2 mg·kg^(-1),每周2次,假手术组和模型组每天灌胃等体积生理盐水,连续给药3周。干预结束后,苏木素-伊红(HE)染色观察睾丸组织病理;酶联免疫吸附测定法(ELISA)检测血清睾酮(T)、促黄体生成素(LH)和卵泡刺激素(FSH)含量;小动物精子自动检测仪测定附睾精子数量和活力;利用转录组学芯片技术检测各组睾丸组织mRNA表达水平,筛选五子衍宗丸调控相关基因转录组,并从中任选3条mRNA进行实时荧光定量聚合酶链式反应(Real-time PCR)验证转录组数据。验证合格的转录组数据通过基因本体(GO)注释分析及京都基因和基因组百科全书(KEGG)信号通路富集分析,解析五子衍宗丸调控模型动物生精功能相关的生物条目及信号通路。结果:与假手术组比较,模型组小鼠睾丸组织出现生精损伤,生精小管腔收缩、空泡化、各级生精细胞减少、间隙变宽,生精细胞发育阻滞等形态异常;血清T显著降低,LH显著升高(P<0.01),FSH升高但差异无统计学意义;附睾精子数量、活力均显著降低(P<0.01);睾丸组织中有882条差异表达mRNA,其中565条上调,317条下调,聚类分析表明,这些差异表达的mRNA可以很好地区分假手术组与模型组。与模型组比较,五子衍宗丸组小鼠睾丸组织损伤减轻,生精小管腔结构完整、空泡化减少、各级生精细胞明显增加、排列紧密;血清中T显著升高,LH显著下降(P<0.01)Objective:To screen out the transcriptomes related to the intervention of Wuzi Yanzongwan on the spermatogenic function of semi-castrated male mice,and to explore its potential mechanism in the intervention of the progress of low spermatogenic function.Method:Balb/c mice were randomly divided into sham-operated group,model group,testosterone propionate group(0.2 mg·kg^(-1)·d^(-1),intramuscular injection)and Wuzi Yanzongwan group(1.56 g·kg^(-1)·d^(-1),intragastric administration)according to body weight,with 12 mice in each group.The right testicle and epididymis were extracted from the model group and the drug administration group to construct the semi-castrated model of low spermatogenic function,while the fur and the right scrotum of the sham-operated group were only cut and immediately sterilized and sutured.At the end of the intervention,hematoxylin-eosin(HE)staining was used to observe the histopathology of testis,enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of serum testosterone(T),luteinizing hormone(LH)and follicle stimulating hormone(FSH).The sperm count and motility of epididymis were measured by automatic sperm detector of small animal.Transcriptomic microarray technology was used to detect the mRNA expression level of testicular tissue in each group,the transcriptome of genes related to the regulation of Wuzi Yanzongwan was screened,and three mRNAs were selected for Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)to verify the transcriptome data.Through the annotation analysis of Gene Ontology(GO)and the signaling pathway analysis of Kyoto Encyclopedia of Genes and Genomes(KEGG),the related functions of drugs regulating transcriptome were analyzed.Result:Compared with the sham-operated group,the testicular tissue of mice in the model group showed spermatogenic injury,contraction and vacuolization of the seminiferous tubules,reduction of spermatogenic cells at all levels,widening of the interstitial space,obstruction of spermatogonial cell develo

关 键 词：经典名方 五子衍宗丸 半去势 生精功能 转录组学 男性不育 实时荧光定量聚合酶链式反应(Real-time PCR) 

分 类 号：R22[医药卫生—中医基础理论] Q786[医药卫生—中医学] R28[生物学—分子生物学] R69