黑果枸杞叶多糖免疫调节活性构效关系及作用机制研究

Lycium ruthenicum, a famous traditional Tibetan medicine, has been used for treatment of heartdisease, irregular menstruation and menopause. Modern pharmacological studies have shown that Lycium ruthenicum has the effects of immunodulatory, hypoglycemic and anti-fatigue, polysaccharide was the main functional factor. In our previous study, five arabinogalactans with highly branched were obtained from the leaves of Lycium ruthenicum，corresponding research about chemical structure and biological activity indicated that it had a clear correlation between structure and antioxidant and immunoregulation activities, therefor, the relationship between fine structure and biological activity and mechanism should be further studied. In present study, an immunologically active homogeneous arabinogalactan LRLP3 will be selectively degraded by enzymatic digestion, acetolysis and mild acid hydrolysis to research the corresponding relationship between structure of LRLP3 and various fragment fractions and immunological activity using the methods of immunosuppressive mice model established by subcutaneous cyclophosphamide and mouse macrophage RAW264.7 cells, and then to elucidate the structure-activity relationship of LRLP3 of immune function. Receptors on Macrophage membrane and downstream molecules of signaling pathway will be examined by the methods of receptor inhibitor treatment, ELISA, RT-PCR and Western bolt to investigating the potential mechanisms involved in the effects of LRLP3. This study will be of great significance to the comprehensive utilization of Lycium ruthenicum polysaccharides, and will provided a basis for further enriching and improving structure-activity relationship of polysaccharides from traditional Chinese medicine.

黑果枸杞是我国民族医药常用药材，具有免疫调节、降血糖和抗疲劳功效，多糖是其主要药效成分之一。本项目前期从黑果枸杞叶片中分离纯化到五个具有多分支阿拉伯半乳聚糖组分，化学结构和生物活性对应研究表明具有免疫调节和抗氧化构-效相关性，该多糖精细结构与活性关系及作用机制尚待深入研究。本项目拟以免疫调节活性最强的黑果枸杞叶多糖均一性组分LRLP3为研究对象，在重复单元结构明确的基础上，运用酶法和化学法进行可控降解，结合环磷酰胺小鼠免疫抑制模型和小鼠巨噬细胞RAW264.7模型，研究LRLP3及降解后各种结构片段与免疫调节活性之间的对应关系，阐明LRLP3免疫调节作用的构效关系；采用受体抑制剂处理，多维联用检测等手段，研究LRLP3免疫调节活性相应的膜受体与信号通路，揭示LRLP3免疫调节活性的分子机制。本项目研究将对黑果枸杞多糖资源开发利用有重要意义，将为进一步丰富和完善中药多糖构效关系理论奠定基础。

黑果枸杞是我国民族医药常用药材，具有免疫调节、降血糖和抗疲劳功效，多糖是其主要药效成分之一。本项目前期从黑果枸杞叶片中分离纯化到五个具有多分支阿拉伯半乳聚糖组分，化学结构和生物活性对应研究表明具有免疫调节和抗氧化构-效相关性，该多糖精细结构与活性关系及作用机制尚待深入研究。本项目以免疫调节活性最强的黑果枸杞叶多糖均一性组分LRLP3为研究对象，在重复单元结构明确的基础上，运用酶法和化学法进行了可控降解，得到4个不同结构片段（LRLP3-T、LRLP3-B、LRLP3-AF、Oligo-S），并对降解产物进行了结构表征；采用环磷酰胺小鼠免疫抑制模型和小鼠巨噬细胞RAW264.7模型，研究了LRLP3及降解后各种结构片段与免疫调节活性之间的对应关系，结果发现，LRLP3在不同程度失去支链阿拉伯糖后，其免疫调节活性降低甚至消失；阿拉伯糖支链和（1→3）Galp半乳糖主链对于小鼠的非特异性免疫同等重要；而低分子寡糖链Oligo-S对淋巴细胞、巨噬细胞激活有明显作用。采用受体抑制剂处理，ELISA、PT-PCR、Western Blot等多维联用检测手段，对LRLP3免疫调节活性相应的膜受体与信号通路进行了研究，结果发现，LRLP3可被巨噬细胞表面膜受体TLR4和TLR2识别，通过MyD88依赖途径活化TRAF6和NF-κB，激活巨噬细胞，发挥免疫调节活性。本研究进一步丰富和完善了中药多糖构效关系研究方法和理论基础，同时对黑果枸杞多糖资源开发利用有重要意义。在项目的支持下，已发表SCI论文3篇，中文核心期刊1篇，提交会议论文2篇；另有2篇SCI论文正在审稿中；申请发明专利1项；培养已毕业硕士研究生1人，在读硕士研究生2人；获陕西省高等学校科学技术奖一等奖1项；项目负责人入选“陕西省2019年度青年科技新星”。