目的 运用网络药理学方法推测肉苁蓉苯乙醇总苷在治疗不育症方面的潜在作用靶点和信号通路，并通过分子对接和动物体内实验来验证并进一步探讨其作用机制。方法 应用TCMSP等数据库及文献收集肉苁蓉苯乙醇总苷的活性成分及潜在作用靶点，通过基因数据库（Gene Cards）、人类孟德尔遗传数据库（OMIM）获取不育症疾病靶点；通过Venny 2.1软件获得交集靶点基因，结合STRING数据库绘制蛋白相互作用（PPI）网络，并通过Cytoscape 3.8.2软件筛选肉苁蓉苯乙醇总苷治疗不育症的核心作用靶点；运用DAVID数据库，分析交集基因的基因本体（GO）和京都基因与基因组百科全书（KEGG）富集情况。选取关键作用靶点与药物活性成分通过AutoDockTools 1.5.6软件进行分子对接验证；最后通过肉苁蓉苯乙醇总苷干预腺嘌呤诱导的不育症大鼠模型，通过反转录PCR（RT-PCR）法、蛋白质印迹（Western blotting）法验证大鼠睾丸组织中核心靶点和核心通路的表达。结果 肉苁蓉苯乙醇总苷共筛选出21个潜在活性成分，62个药物靶点，7 961个不育症靶点，药物与不育症交集靶点51个；核心活性成分主要为毛蕊花糖苷、松果菊苷、管花苷A等，核心靶点主要为肿瘤坏死因子（TNF）、基质金属蛋白酶9（MMP9）、哈维大鼠肉瘤病毒癌基因同源物（HRAS）、纤溶酶原基因（PLG）、热休克蛋白90α家族A类成员1（HSP90AA）等；GO功能富集主要包括蛋白水解、一碳代谢过程、细胞外基质分解等；KEGG富集通路主要包括雌激素信号通路、GnRH信号通路、氮代谢、代谢途径信号通路等；分子对接结果显示核心活性组分与不育症关键核心靶基因TNF、MMP9、HRAS、PLG、HSP90AA1的结合度高；体内实验结果表明，给予肉苁蓉苯乙醇总苷后，不育症大鼠睾丸组织ERα、HSP90 mRNA和蛋白表达升高，ERβ mRNA和蛋白表达降低（P＜0.05）。结论 肉苁蓉苯乙醇总苷治疗不育症是多靶点､多通路共同调控的结果，其作用机制可能与参与雌激素通路，调控核心靶点ERα、ERβ表达有关。

Objective To explore the potential targets and signaling pathways of phenylethanoid glycosides from Cistanche Herba in treatment of infertility by network pharmacology. Molecular docking and animal experiments were used to verify and further explore the mechanism of action. Methods The active components and potential targets of total phenylethanoid glycosides from Cistanche Herba were collected using TCMSP and other databases and literature, and infertility disease targets were obtained by Gene Cards and OMIM. The intersection target genes were obtained by Venny 2.1 software, the protein interaction network was plotted by STRING database, and the core target of the treatment of infertility was screened by Cytoscape 3.8.2 software. GO and KEGG enrichment of intersecting genes were analyzed using DAVID database. Key targets and active ingredients were selected for molecular docking verification by AutoDockTools 1.5.6 software. Finally, total phenylethanoid glycosides from Cistanche Herba were used to interfere with adenine-induced infertility rat models, and the expression of core targets and core pathways in testicular tissue of rats was verified by RT-PCR and Western blotting. Results A total of 21 potential active ingredients, 62 drug targets, 7 961 infertility targets, and 51 drug and infertility intersection targets were identified. The core active ingredients were mainly verminoside, echinoside, tuphonoside A, etc. The core targets were mainly TNF, MMP9, HRAS, PLG, HSP90AA1, etc. GO functional enrichment mainly includes proteolysis, carbon metabolism, extracellular matrix decomposition, etc. KEGG enrichment pathway mainly includes estrogen signaling pathway, GnRH signaling pathway, nitrogen metabolism, metabolic pathway signaling pathway, etc. Molecular docking results showed that the binding degree of the core active components to the key core target genes of infertility TNF, MMP9, HRAS, PLG, and HSP90AA1 was high. The results showed that the expression of ERα and HSP90 mRNA and protein increased, and the expression of ERβ mRNA and protein decreased after the administration of total phenylethanoid glycosides from Cistanche Herba（P < 0.05）. Conclusion The treatment of infertility by total phenylethanoid glycosides from Cistanche Herba is the result of multi-target and multi-pathway co-regulation, and the mechanism of action may be related to participation in estrogen pathway and regulation of core target ERα and ERβ expression.

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国家自然科学基金资助项目(82160772);新疆维吾尔自治区自然科学基金资助项目(2022D01C286,2022D01C260)