应用靶向亲和-色谱-质谱联用技术对梅花鹿茸促进睾酮合成关键酶mRNA表达的研究

Sika Pilose Antler is a famous TCM with Strengthen Yang action. The effect of improving spermatogenic and strengthening Yang of Sika Pilose Antler is closely related totestosterone level of human because testosterone is an key factor to enhance human strength , libido , immune function. The preliminary research of our workgroup discovered that the total proteins, peptides, oligopeptides and alkaloids of Sika Pilose Antler could stimulated testosterone secretion in rat Leydig cells. And the actions of total oligopeptides and alkaloids were better than ones of total proteins and peptides. So we proposed the following hypothesis that the effect of improving spermatogenic and strengthening Yang of Sika Pilose Antler on serum gonadal hormone levels were due to the total oligopeptides and alkaloids acted on different key testosterone synthesis enzymes compositively. In this study we would study on Sika Pilose Antler improving mRNA expression of key steroidogenic enzymes of rats by using target molecule affinity coupled with highperformance liquid chromatography and tandem mass spectrometry. Furthermore targeted isolation and structure elucidation were done with modern chromatography and spectroscopy and the activity and mRNA expression of key steroidogenic enzymes of rats of every compound was measured. The results of the project shows that mechanism of improving spermatogenic and strengthening Yang of SikaPilose Antler come from their actions in multi-target and multi-component pattern on different key testosterone synthesis enzymes compositively. It not only has important theoretical significance to seek new methods for studying on TCM with Strengthen Yang action but also provides a scientific basis to create more economic value and social benefits.

鹿茸是传统壮阳中药，睾酮是增强人体力量、性欲、免疫等功能的重要影响因素，故鹿茸的壮阳生精功效与机体睾酮水平密切相关。项目组前期证实梅花鹿茸总蛋白、多肽、寡肽和生物碱均有促进大鼠睾丸间质细胞分泌睾酮的作用，而寡肽和生物碱效果明显强于总蛋白和多肽。因此提出如下假说：梅花鹿茸针对性腺轴的壮阳生精功效主要源于寡肽类和生物碱类成分对不同睾酮合成关键酶的综合作用。本项目通过梅花鹿茸寡肽和生物碱成分与睾丸间质细胞及睾酮合成关键酶共同孵育，结合色谱-质谱联用技术对共孵育后能与细胞或关键酶靶向亲和的化学成分进行分析，快速确定较强活性的化学成分在其指纹图谱中的归属和活性差异。进而考察分得单体活性成分对睾酮分泌及合成关键酶mRNA的表达，确定其促睾酮分泌的分子机制，最终阐明梅花鹿茸的壮阳生精的药效物质基础。该项目探索了壮阳中药药效物质基础研究新方法，而且为鹿茸合理应用和创造更多的经济价值和社会效益提供科学依据。

本项目阐明了梅花鹿茸针对性腺轴壮阳生精的主要药效物质是寡肽类成分，其分子机制为寡肽对睾酮合成关键酶P450scc mRNA表达的正向影响而发挥作用。基于靶细胞亲和-色谱-质谱联用技术对梅花鹿茸增强免疫、壮阳生精活性成分分析，结果表明有4个生物碱和2个寡肽能够分别与脾细胞和睾丸间质细胞亲和。应用高效液相色谱法，对11批不同产地的梅花鹿茸和8批马鹿茸的水提取物所得指纹图谱进行了主成分分析（PCA），研究结果为鹿茸药材的品种鉴别与质量控制提供了可靠的依据。应用液质联用技术分析了梅花鹿茸中的水溶性成分，鉴定了其中8个生物碱和6个寡肽；同时还应用HPLC法同时测定了梅花鹿茸中含量相对较高的6个生物碱类成分，方法简便、准确、重复性好，可用于鹿茸的质量控制。有针对性的分离、纯化了11个核苷和碱基以及2个寡肽并鉴定了它们的结构，其中9个化合物为首次从梅花鹿茸中分离得到。在体外活性研究，发现总生物碱、尿嘧啶、腺苷具有明显的促进小鼠脾细胞增殖作用的作用；发现总寡肽和1个单体寡肽具有明显的促进大鼠睾丸间质细胞增殖和睾酮分泌作用，即表明它们是梅花鹿茸针对性腺轴壮阳生精和免疫增强的主要药效物质。通过对鹿茸总寡肽治疗肾阳虚大鼠的考察，发现其是过多途径、多靶点来实现，包括对下丘脑-垂体-靶腺轴上与肾阳虚相关的激素类考察指标的显著影响及对睾酮合成关键酶P450scc mRNA表达的正向影响。该项目探索了壮阳中药药效物质基础研究新方法，而且为鹿茸合理应用和创造更多的经济价值和社会效益提供科学依据。