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

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),FSH下降但差异无统计学意义;附睾中精子数量和活力显著提高(P<0.01);五子衍宗丸可明显调控半去势小鼠睾丸的159条mRNA,其中32条上调,127条下调,经Real-time PCR验证转录组检测数据可靠。GO和KEGG分析显示,五子衍宗丸调控的转录组功能涉及到睾丸内间质细胞的性激素生成,生精细胞的更新、分化、代谢、凋亡、信号传导等精子发育的整个细胞周期过程,并和生精干细胞功能、内质网蛋白处理及代谢程序等信号通路的生物学行为密切相关。结论:五子衍宗丸可有效改善半去势雄性小鼠造成的生精功能低下,其机制可能与五子衍宗丸干预睾丸转录调节网络,调控睾丸间质细胞性激素合成、生精干细胞功能、精子发生的整个细胞周期过程,以及内质网蛋白处理及代谢程序相关基因转录的表达有关。

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 development and other morphological abnormalities,and serum T significantly decreased,LH significantly increased(P<0.01),and FSH elevated but no statistically significant difference,the count and vitality of epididymal sperm significantly decreased(P<0.01).There were 882 differentially expressed mRNAs in the testicular tissues,of which 565 were up-regulated and 317 were down-regulated.Cluster analysis showed that these differentially expressed mRNA could effectively distinguish between the sham-operated group and the model group.Compared with the model group,the damage to testicular tissue in the Wuzi Yanzongwan group was reduced,the structure of the seminiferous tubules was intact,vacuolization was reduced,and the number of spermatogenic cells at all levels was significantly increased and arranged tightly. The serum T significantly increased, LH significantly decreased(P<0.01), and FSH decreased but the difference was not statistically significant. The count and vitality of sperm in the epididymis were significantly increased(P<0.01). Moreover, Wuzi Yanzongwan could regulate 159 mRNA levels in the testes of semi-castrated mice, of which 32 were up-regulated and 127 were down-regulated, and the data of the transcriptome assay was verified to be reliable by Real-time PCR. GO and KEGG analysis showed that the transcriptome functions regulated by Wuzi Yanzongwan were involved in the whole cell cycle process of sperm development such as sex hormone production of interstitial cells in testis, renewal, differentiation, metabolism,apoptosis and signal transduction of spermatogenic cells, and were closely related to the biological behaviors of signaling pathways such as spermatogenic stem cell function, endoplasmic reticulum protein processing and metabolic program. Conclusion:Wuzi Yanzongwan can effectively improve the low spermatogenic function of semi-castrated male mice, and its mechanism may be related to the regulation of testicular transcriptional regulatory network, the synthesis of sex hormones in testicular interstitial cells, the function of spermatogenic stem cells, the whole cell cycle process of spermatogenesis, as well as the expression of endoplasmic reticulum protein processing and metabolic program related genes transcription.