五味扶正益精汤治疗男性不育症精子DNA损伤患者的蛋白质组学分析及初步实验验证  

Proteomic Analysis and Preliminary Experimental Verification of Wuwei Fuzheng Yijing Decoction in Treatment of Male Infertility Patients with Sperm DNA Damage

作　　者：马鹏 张琦[2] 王祖龙[2] MA Peng;ZHANG Qi;WANG Zulong(The First Clinical Medical College of Henan University of Chinese Medicine,Zhengzhou Henan China 450000;Department of Andrology,the First Affiliated Hospital to Henan University of Chinese Medicine,Zhengzhou Henan China 450000)

机构地区：[1]河南中医药大学第一临床医学院,河南郑州450000 [2]河南中医药大学第一附属医院男科,河南郑州450000

出　　处：《中医学报》2025年第5期911-918,共8页Acta Chinese Medicine

基　　金：国家自然科学基金面上项目(82174377);河南省自然科学基金项目(242300420431)。

摘　　要：目的:分析五味扶正益精汤治疗男性不育症精子DNA损伤患者的蛋白质组学特征,并基于鞘脂代谢通路进行实验验证。方法:20例男性不育症精子DNA损伤患者随机分为试验组与验证组各10例,均接受五味扶正益精汤治疗12周。另将10例健康体检者作为对照组。采用流式细胞术检测患者精子DNA碎片指数(DNA fragmentation index,DFI)。采用DIA蛋白质组学技术检测精浆蛋白质谱,筛选差异表达蛋白。采用DAVID数据库对差异表达蛋白进行京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析。采用ELISA法检测精浆鞘磷脂磷酸二酯酶1(sphingomyelin phosphodiesterase 1,SMPD1)、鞘氨醇-1-磷酸(sphingosine-1-phosphate,S1P)、神经酰胺(ceramide,Cer)含量。采用RT-qPCR法检测精浆SMPD1、S1P、Cer基因表达水平。结果:与治疗前比较,试验组和验证组治疗后患者精子DFI水平降低(P<0.001)。DIA蛋白质组学鉴定出2753种定量蛋白质。与对照组比较,试验组治疗前精浆中有542个差异表达蛋白(308个蛋白上调,234个蛋白下调);与治疗前比较,试验组治疗后精浆中有505个差异表达蛋白(267个蛋白上调,238个蛋白下调);两者的交集蛋白235个。KEGG分析显示,差异表达蛋白和交集蛋白集中富集于鞘脂代谢通路。ELISA检测显示,与对照组比较,验证组治疗前精浆中SMPD1、Cer含量升高,S1P含量降低(P<0.01);与治疗前比较,验证组治疗后精浆中SMPD1、Cer含量降低,S1P含量升高(P<0.01)。RT-qPCR检测显示,与对照组比较,验证组治疗前精浆SMPD1 mRNA、Cer mRNA水平升高,S1P mRNA水平降低(P<0.01);与治疗前比较,验证组治疗后精浆SMPD1 mRNA、Cer mRNA水平降低,S1P mRNA水平升高(P<0.01)。结论:五味扶正益精汤可改善男性不育症患者的精子DNA损伤,其机制可能与调节鞘脂代谢通路有关。Objective:To analyze the proteomic characteristics of Wuwei Fuzheng Yijing Decoction in patients with sperm DNA damage in male infertility,and to carry out experimental verification based on sphingolipid metabolism pathway.Methods:A total of 20 patients with sperm DNA damage in male infertility were randomly divided into the experimental group and the verification group,with 10 cases in each group,and all of them were treated with Wuwei Fuzheng Yijing Decoction for 12 weeks.In addition,10 healthy subjects were used as the control group.Flow cytometry was used to detect the DNA fragmentation index(DFI)of the patient′s sperm.DIA proteomics technology was used to detect seminal plasma protein profile,and differentially expressed proteins were screened.The Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis of differentially expressed proteins was performed using the DAVID database.The contents of sphingomyelin phosphodiesterase 1(SMPD1),sphingosine-1-phosphate(S1P)and ceramide(Cer)were detected by ELISA.RT-qPCR was used to detect the expression levels of SMPD1,S1P and Cer genes in seminal plasma.Results:Compared with before treatment,the DFI level of sperm in the experimental group and the validation group decreased after treatment(P<0.001).DIA proteomics identified 2753 quantitative proteins.Compared with the control group,there were 542 differentially expressed proteins in the seminal plasma in the experimental group before treatment(308 proteins were up-regulated and 234 proteins were down-regulated).Compared with before treatment,there were 505 differentially expressed proteins in the seminal plasma in the experimental group after treatment(267 proteins were up-regulated and 238 proteins were down-regulated).There were 235 intersecting proteins between the two.KEGG analysis showed that the differentially expressed proteins and intersecting proteins were concentrated and enriched in the sphingolipid metabolism pathway.ELISA showed that compared with the control group,the contents of SMPD1 and Cer i

关 键 词：五味扶正益精汤 男性不育症 精子DNA损伤 DNA碎片指数(DFI) 蛋白质组学 鞘脂代谢 

分 类 号：R285.5[医药卫生—中药学]