应用微阵列技术分析五子衍宗丸干预少弱精症小鼠睾丸组织的长链非编码RNA表达  

Preliminary study on the expression of long non-coding RNA in testis of oligoasthenospermia treated with Wuzi Yanzong Pill

作　　者：邹迪新 鹿伟[2] 孟雪丹 张钟秀 王汉 段宇 代一航 路广琦 张岳阳[4] 林瑞超[3] ZOU Dixin;LU Wei;MENG Xuedan;ZHANG Zhongxiu;WANG Han;DUAN Yu;DAI Yihang;LU Guangqi;ZHANG Yueyang;LIN Ruichao(State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs,Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]中国中医科学院中药研究所道地药材品质保障与资源持续利用全国重点实验室,北京100700 [2]内蒙古医科大学附属医院中医科 [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 [4]中国中医科学院望京医院男科

出　　处：《环球中医药》2024年第10期1937-1947,共11页Global Traditional Chinese Medicine

基　　金：国家自然科学基金(82304718、81760837);北京市自然科学基金(7232303);中国中医科学院科技创新工程项目(CI2023E002、CI2021A02208、CI2021A02210);中央级公益性科研院所基本科研业务费专项(ZXKT22009)。

摘　　要：目的研究五子衍宗丸干预少弱精症相关的睾丸长链非编码RNA(long noncoding RNA,lncRNA),初步探讨该方调控lncRNA在其干预少弱精症中的潜在作用机制。方法采用白消安法建立少弱精症动物模型,使用五子衍宗丸干预14天后,应用小动物精子自动检测仪测定附睾精子数量和活力及与雌鼠交配受孕率评估生育力。分别抽提正常组、模型组、药物组各6例睾丸组织的RNA,应用转录组测序技术分析并筛选五子衍宗丸调控的差异表达lncRNA,实时荧光定量(quantitative real-time PCR,qRT-PCR)技术对差异表达lncRNA数据验证。通过生物信息学对获得的药物调控lncRNA结合前期报道该方调控mRNAs的实测值,进行关联分析,建立该方调控lncRNA靶向mRNAs的共表达网络,筛选网络lncRNA靶向的核心mRNAs,对核心mRNAs进行KEGG信号通路分析,构建该方调控lncRNA靶向信号通路网络,分析五子衍宗丸调控lncRNA干预少弱精症的信号通路。结果五子衍宗丸可显著提高少弱精症精子质量、雌鼠受孕率,并明显调控296条睾丸lncRNA,其中上调215条,下调81条,qRT-PCR验证lncRNA测序结果可靠。通过lncRNA-mRNAs的关联分析筛选到药物调控的核心lncRNA(102条)和mRNAs(81条),该方调控lncRNA靶向信号通路网络显示,在102条lncRNA中,有80条lncRNA靶向2条及以上信号通路,信号通路中有40条对应2条及以上lncRNA,这些lncRNA靶向的信号通路涉及到睾丸细胞的生长、分化、代谢、凋亡、信号传导等整个细胞周期过程,并且主要富集在范科尼贫血通路、细胞周期、剪接体、叉头状转录因子通路、p53通路、同源重组等通路上。结论本研究提供了五子衍宗丸干预少弱精症的睾丸lncRNA表达谱,这些lncRNA参与了药物修复睾丸生精损伤,提示其可能在药效中起到了重要作用,是五子衍宗丸潜在的干预靶点。Objective To study the intervention of Wuzi Yanzong Pill on long non coding RNA(lncRNA)in the testis related to oligoasthenozoospermia,and to preliminarily explore the potential mechanism of drug regulation of lncRNA in its intervention of oligoasthenozoospermia.Methods Establishment of oligoasthenospermia mouse model by intraperitoneal injection of busulfan,after 14 days of intervention with Wuzi Yanzong Pill,the number and vitality of epididymal sperm in small animals were measured using an automatic sperm detector,and the fertility was evaluated by mating with female mice,RNA was extracted from testicular tissues of 6 cases in the normal,model and drug groups,and transcriptomic sequencing technology was used to analyze and screen differentially expressed lncRNA regulated by Wuzi Yanzong Pill.Real-time fluorescent quantitative polymerase chain reaction(Real time PCR)was used to verify the differentially expression of lncRNA data.Bioinformatics was used to correlate the obtained drug-regulated lncRNA with the previously reported drug-regulated mRNAs,establish a coexpression network for drug regulated lncRNA targeting mRNAs,and screen the core mRNAs targeted by lncRNA in the network,which was performed by KEGG analysis,construct a drug regulated lncRNA targeted signaling pathway network and analyze the signaling pathway of Wuzi Yanzong Pill in regulating lncRNA intervention in oligoasthenozoospermia.Results Wuzi Yanzong Pill significantly improved the quality of sperm in oligoasthenozoospermia,the fertility rate of mating with female mice with oligoasthenozoospermia,obviously regulated 297 testicular lncRNA,of which 215 were up-regulated and 81 were down-regulated.The method of Real time PCR was used to confirmed the reliability of lncRNA sequencing results.The association analysis of drug regulated lncRNA mRNAs identified the core lncRNA(102)and mRNAs(81)regulated by drugs,lncRNA targeting signaling pathway association analysis showed that among 102 lncRNA,80 lncRNA targeted two or more signaling pathways,and 40

关 键 词：五子衍宗丸 长链非编码RNA 少弱精症 精子发生 

分 类 号：R285.5[医药卫生—中药学]