基于网络药理学及实验验证的强精片治疗解脲支原体感染男性不育的机制研究

Mechanism of Qiangjing(强精)Tablets in the Treatment of Male Infertility with Ureaplasma Urealyticum Infection Based on Network Pharmacology and Experimental Verification

目的:通过网络药理学探讨强精片治疗解脲支原体感染男性不育症的潜在作用机制,并对解脲支原体模型雄性大鼠的相关作用靶点进行实验验证。方法:通过TCMSP收集强精片的活性成分和潜在靶点,使用Genecard数据库筛选解脲支原体感染男性不育症的疾病靶点;将疾病靶点与药物预测靶点取交集,借助Cytoscape3.7.2软件构建交集靶点网络,利用Bioconductor平台和R语言进行GO和KEGG富集分析。采用解脲支原体感染雄性大鼠模型,检查精子质量、血清T含量,采用HE染色、电镜观察睾丸结构,Elisa检测血清IL-17、IL-1β、TNF-ɑ含量,免疫组化检测睾丸组织TRAF6、NF-κB P65蛋白表达,探讨强精片治疗解脲支原体感染男性不育症的潜在机制。结果:共获得槲皮素、山柰酚、木犀草素、淫羊藿苷、五味子苷、人参皂苷Re等109个强精片活性成分及286个可用于后续分析的靶点,249个解脲支原体感染男性不育症靶点,映射得出43个药物-疾病共同靶点。GO富集结果表明,强精片治疗解脲支原体男性不育症的可能机制主要是对细菌来源分子及氧化应激的细胞因子及其受体结合反应。KEGG通路分析提示主要与TNF信号通路、IL-17信号通路、NK-κB信号通路、感染信号通路等信号通路相关,JUN、IL6、MAPK14、IL1B、IL4、MAPK1等是其关键作用蛋白。动物实验验证表明强精片可以改善解脲支原体感染雄性大鼠精液质量,提高血清T含量,改善睾丸组织结构,降低大鼠血清IL-17、IL-1β、TNF-α含量,降低睾丸组织TRAF6、NF-κB P65蛋白表达,抑制IL-17通路,发挥抗炎及免疫作用。结论:网络药理学显示强精片可以通过多通路多靶点对解脲支原体感染男性不育症的免疫及炎症相关机制进行调节,实验验证表明强精片可以抑制IL-17通路,改善解脲支原体感染大鼠睾丸组织结构及生精功能,发挥治疗作用。

Objective:To explore the potential mechanism of Qiangjing(强精)Tablets in the treatment of male infertility with UU(Ureaplasma Urealyticum)infection through network pharmacology,and to verify the relevant targets of UU-infected male rats by experiments.Methods:The active components and potential targets of Qiangjing Tablets were collected by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),and the targets of male infertility with UU infection were screened by GeneCards.Then the disease targets and drug prediction targets were intersected to construct an intersection target network by Cytoscape 3.7.2.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were performed using Bioconductor and R language.UU-infected male rat model was established to examine sperm quality and serum T content.HE staining and electron microscopy were used to observe testicular structure.Enzyme-linked immunosorbent assay(ELISA)was performed to detect serum contents of interleukin 17(IL-17),IL-1β,and tumor necrosis factorɑ(TNF-ɑ),and immunohistochemistry was employed to detect the expressions of TNF receptor associated factor 6(TRAF6)and nuclear factor kappa B(NF-κB)p65 in testicular tissue.Results:A total of 109 active components of Qiangjing Tablets such as quercetin,kaempferol,luteolin,icariin,schizandriside,and ginsenoside Re.were screened,and 286 targets that could be used for subsequent analysis were obtained.A total of 249 targets of male infertility with UU infection were obtained,and 43 common targets were mapped.The GO enrichment analysis showed that the possible mechanism of Qiangjing Tablets in the treatment of male infertility with UU infection was mainly the binding response to bacteria-derived molecules and oxidative stress-related cytokines and their receptors.The KEGG pathway analysis revealed that the TNF,IL-17,NF-κB and infection signaling pathways were mainly involved,with JUN,IL6,mitogen-activated protein kinase 14(MAPK14),IL1 B,IL4,and MAPK1 as the key proteins.Animal experiments indicated that Qiangjing Tablets enhanced the semen quality of UU-infected male rats,increased serum T content,improved testicular structure,reduced serum contents of IL-17,IL-1βand TNF-ɑand the protein expressions of TRAF6 and NF-κB p65 in testicular tissue,and inhibited IL-17 pathway,thus exerting the anti-inflammatory and immune effects.Conclusion:Network pharmacology demonstrated that Qiangjing Tablets could modulate the immune system and inflammation-related mechanisms of male infertility with UU infection through multiple pathways and targets.Experimental validation suggested that Qiangjing Tablets could inhibit IL-17 pathway and improve testicular structure and spermatogenic function of UU-infected rats,thereby producing the therapeutic effects.