“糖谱法”结合“受体垂钓技术”研究竹荪多糖的优势免疫活性部位及其作用机制

Polysaccharides are considered the major immunomodulatory components in Dictyophora indusiata, and the immunomodulatory activity of D. indusiata polysaccharides is closely correlated to their dominant active fragments. Nevertheless, it is a great challenge and a bottleneck for the investigation of dominant immunomodulatory fragments and quality evaluation of D. indusiata polysaccharides due to their complex and diverse chemical structures, respectively. The current methods for the investigation of dominant active fragments of polysaccharides have several drawbacks, such as poor repeatability and stability, low selectivity, tedious steps, and time consuming. Thus, in this project, we would like to develop a stable, specific, efficient, and convenience approach for the investigation of dominant immunomodulatory fragments of polysaccharides. Briefly, the chemical structures and immunomodulatory activities of D. indusiata polysaccharides before/after enzymatic digestions are compared, and the quality evaluation system of D. indusiata polysaccharides based on their specific chemical structures are established by saccharide mapping; Meanwhile, the potentially dominant immunomodulatory fragments of D. indusiata polysaccharides are efficiently and specifically selected by receptor fishing technology. And the detailed chemical structures of dominant immunomodulatory fragments are elucidated based on their multiple MS data and glycan database; Furthermore, the immunomodulatory activities of the potential immunomodulatory fragments are confirmed both in vitro and in vivo, and their possible immunomodulatory mechanism are revealed according to the inhibition of pattern-recognition receptors on the surface of macrophages. Results may provide a key approach for the investigation of structure-bioactivity relationships and quality evaluation of polysaccharides with immunomodulatory activity from edible mushrooms, and an important theoretical basis for exploring the theory of active domains in polysaccharides with immunomodulatory activity from edible mushrooms.

多糖作为竹荪的主要免疫活性成分，其免疫活性与竹荪多糖的优势活性部位相关。然而，由于竹荪多糖结构的复杂性和多样性，其优势免疫活性部位的研究，一直是竹荪多糖免疫构效关系研究的难题，也是其质量检控研究的瓶颈。现有的优势活性部位研究方法，重复性差、选择性低、步骤繁琐、工作量大。因此，本项目拟构建一种稳定、特异、高效、简便快捷的多糖优势活性部位研究方法。即采用糖谱法，比较糖苷酶定向切割前、后，竹荪免疫活性多糖的化学特征与活性差异，建立竹荪活性多糖的糖谱检控体系；并结合巨噬细胞受体垂钓技术，特异、高效地筛选竹荪多糖的潜在优势免疫活性部位，采用多元质谱和聚糖结构MS数据库解析其结构特征；进一步验证其免疫活性，并结合多糖识别受体阻断实验，明确优势免疫活性部位的作用机制。研究结果有望为食用菌免疫活性多糖的构效关系及其质量检控等共性问题研究提供关键技术，为探索食用菌免疫活性多糖的活性中心理论提供重要理论依据。

多糖作为竹荪的主要免疫活性成分，其免疫活性与竹荪多糖的优势活性部位相关。然而，由于竹荪多糖结构的复杂性和多样性，其优势免疫活性部位的研究，一直是竹荪多糖免疫构效关系研究的难题，也是竹荪活性多糖质量检控研究的瓶颈。因此，本项目拟构建一种稳定、特异、高效、简便快捷的竹荪多糖优势活性部位研究方法。. 通过本项目实施，构建了竹荪免疫活性多糖优势活性部位研究的新方法，并明确了竹荪免疫活性多糖优势活性部位的作用机制。首先，采用超声辅助靶向提取竹荪免疫增强活性多糖，并解析其精细结构特征和确认其免疫增强活性；随后，采用糖苷酶或超声辅助非金属芬顿体系选择性降解竹荪免疫增强活性多糖，制备竹荪免疫活性多糖的潜在优势免疫活性部位，并采用巨噬细胞受体垂钓技术分析优势免疫活性部位的免疫活性潜力，进一步采用基于多维色谱/光谱的糖谱技术解析优势免疫活性部位的结构特征和构建其多维特征糖谱；最后，进一步采用体内外实验确认了优势活性部位的免疫增强活性及其潜在作用机制。本项目所提出的活性中心理论和构建的优势活性部位研究方法为研究食用菌多糖的构效关系及其质量检控提供了科学的理论基础和技术支撑。. 本项目公开发表与项目相关的SCI收录论文11篇、正在撰写与项目相关的SCI收录论文2篇；已联合培养硕士研究生5名，在读硕士研究生1名；指导1名硕士研究生获“2021届四川省优秀大学毕业生”荣誉称号；指导1名硕士研究生获四川农业大学“优秀学术成果一等奖”；指导2名硕士研究生获“2020年硕士研究生国家奖学金”。