目的 運用網(wǎng)絡藥理學方法推測肉蓯蓉苯乙醇總苷在治療不育癥方面的潛在作用靶點和信號通路，并通過分子對接和動物體內(nèi)實驗來驗證并進一步探討其作用機制。方法 應用TCMSP等數(shù)據(jù)庫及文獻收集肉蓯蓉苯乙醇總苷的活性成分及潛在作用靶點，通過基因數(shù)據(jù)庫（Gene Cards）、人類孟德爾遺傳數(shù)據(jù)庫（OMIM）獲取不育癥疾病靶點；通過Venny 2.1軟件獲得交集靶點基因，結合STRING數(shù)據(jù)庫繪制蛋白相互作用（PPI）網(wǎng)絡，并通過Cytoscape 3.8.2軟件篩選肉蓯蓉苯乙醇總苷治療不育癥的核心作用靶點；運用DAVID數(shù)據(jù)庫，分析交集基因的基因本體（GO）和京都基因與基因組百科全書（KEGG）富集情況。選取關鍵作用靶點與藥物活性成分通過AutoDockTools 1.5.6軟件進行分子對接驗證；最后通過肉蓯蓉苯乙醇總苷干預腺嘌呤誘導的不育癥大鼠模型，通過反轉錄PCR（RT-PCR）法、蛋白質(zhì)印跡（Western blotting）法驗證大鼠睪丸組織中核心靶點和核心通路的表達。結果 肉蓯蓉苯乙醇總苷共篩選出21個潛在活性成分，62個藥物靶點，7 961個不育癥靶點，藥物與不育癥交集靶點51個；核心活性成分主要為毛蕊花糖苷、松果菊苷、管花苷A等，核心靶點主要為腫瘤壞死因子（TNF）、基質(zhì)金屬蛋白酶9（MMP9）、哈維大鼠肉瘤病毒癌基因同源物（HRAS）、纖溶酶原基因（PLG）、熱休克蛋白90α家族A類成員1（HSP90AA）等；GO功能富集主要包括蛋白水解、一碳代謝過程、細胞外基質(zhì)分解等；KEGG富集通路主要包括雌激素信號通路、GnRH信號通路、氮代謝、代謝途徑信號通路等；分子對接結果顯示核心活性組分與不育癥關鍵核心靶基因TNF、MMP9、HRAS、PLG、HSP90AA1的結合度高；體內(nèi)實驗結果表明，給予肉蓯蓉苯乙醇總苷后，不育癥大鼠睪丸組織ERα、HSP90 mRNA和蛋白表達升高，ERβ mRNA和蛋白表達降低（P＜0.05）。結論 肉蓯蓉苯乙醇總苷治療不育癥是多靶點､多通路共同調(diào)控的結果，其作用機制可能與參與雌激素通路，調(diào)控核心靶點ERα、ERβ表達有關。

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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國家自然科學基金資助項目(82160772);新疆維吾爾自治區(qū)自然科學基金資助項目(2022D01C286,2022D01C260)