基于核磁共振氢谱代谢组学结合分子对接技术筛选补肾壮骨汤促睾酮合成活性成分  

Screening of Active Ingredients of Bushen Zhuanggu Decoction for Promoting Testosterone Synthesis Based on ^(1)H NMR Metabolomics Combined with Molecular Docking Technology

作　　者：吕经纬 李春楠 高晓晨 张楠茜 张凯月 赫玉芳 张辉 孙佳明 LYU Jing-Wei;LI Chun-Nan;GAO Xiao-Chen;ZHANG Nan-Xi;ZHANG Kai-Yue;HE Yu-Fang;ZHANG Hui;SUN Jia-Ming(Jilin Ginseng Academy,Changchun University of Chinese Medicine,Changchun 130117,China;School of Health Management,Changchun University of Chinese Medicine,Changchun 130117,China)

机构地区：[1]长春中医药大学吉林省人参科学研究院,长春130117 [2]长春中医药大学健康管理学院,长春130117

出　　处：《分析化学》2021年第5期779-788,I0001-I0006,共16页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.82074127);吉林省科技发展计划项目(No.20200404070YY)资助。

摘　　要：基于核磁共振氢谱(^(1)H NMR)代谢组学结合分子对接技术筛选补肾壮骨汤促睾酮合成的活性成分。应用酶联免疫吸附分析(ELISA)法分析补肾壮骨汤及其膜分离组分对过氧化氢诱导的小鼠睾丸间质细胞(Mouse leydig cells,TM3细胞)上清液睾酮含量的影响,应用超高效液相色谱-四极杆飞行时间质谱(RRLC-Q-TOF-MS)技术快速分析其升睾活性组分中的化学成分组成。利用^(1)H NMR代谢组学技术揭示过氧化氢诱导TM3细胞在给予升睾活性组分后的差异代谢物,进而通过京都基因与基因组百科全书(KEGG)数据库分析差异代谢物的代谢靶点,并与睾酮合成相关靶点交集,得到升睾活性组分促睾酮合成的效应靶点。最后,确定能够影响效应靶点表达的关键蛋白,应用分子对接技术从活性组分中筛选出与此关键蛋白结合较好的化学成分。在补肾壮骨汤升睾活性组分中解析了27种化学成分的可能结构;细胞代谢组学分析共鉴定出牛磺酸、谷氨酸、乙酸等33种差异代谢物,得到促睾酮合成的效应靶点为睾丸激素17-β-脱氢酶(Testosterone 17-β-dehydrogenase,17β-HSD),KEGG分析表明,抑制关键蛋白α蛋白激酶1(α-kinase 1,ALPK1)磷酸化影响17β-HSD表达能够促进睾酮合成。分子对接结果表明,樱桃苷、原儿茶酸等14个化学成分与ALPK1结合较好,可作为补肾壮骨汤促睾酮合成潜在活性成分,为此复方的药效物质基础研究提供了参考。Bushen Zhuanggu Decoctions(BSZGD)is an effective prescription in the treatment of osteoporosis(OP)caused by testosterone deficiency,but the active ingredients for promoting testosterone synthesis are still unclear.The present study aims to reveal the active compounds of BSZGD for promoting testosterone synthesis by using^(1)H NMR-based metabolomics and molecular docking methods.After 24 h of administration,BSZGD and its membrane fractions could effectively improve the content of testosterone in the supernatant of Mouse Leydig cells(TM3 cells)induced by hydrogen peroxide.On the basis of medicinal materials,reference materials,literature reports and mass spectrometry data,a total of 27 kinds of chemical composites in the active fractions of BSZGD were identified by ultra-high performance liquid chromatography-quadrupole time-of-flight(RRLC-Q-TOF-MS)technique.^(1)H NMR metabolomics technology was used to reveal the differential metabolites of hydrogen peroxide-induced TM3 cells after the active fraction was given.A total of 41 metabolites were identified by^(1)H NMR-based metabolomics,of which 33 differential metabolites were found.Testosterone 17-β-dehydrogenase(17β-HSD)as the common metabolic targets of different metabolites and testosterone synthesis was identified using MetScape analysis.KEGG analysis showed that competitive inhibitors of the key protein kinase1(ALPK1)could activate 17β-HSD and promote the synthesis of testosterone.Fourteen compounds were identified as potential active ingredients in BZGD for promoting testosterone synthesis by molecular docking technique.These results provided further evidence of BSZGD for promoting testosterone synthesis and were beneficial for its clinical application.The potential target and active components found in this study may provide valuable information for further studying the material basis of BSZGD.In addition,this work provided new insights for revealing the active ingredients of complex herbal preparations.

关 键 词：补肾壮骨汤 睾酮 活性成分 细胞代谢组学 分子对接 

分 类 号：R284.1[医药卫生—中药学]