柴胡中基于P-selectin靶点治疗急性肺损伤的新型糖类先导化合物的发现与作用机制研究

Bupleurum chinense DC. belongs to the family of Umbelliferae, which is abundantly distributed in the northeast region of China, locally known as "Bei-Chai-Hu". B. chinense is a traditional Chinese herbal medicine (TCM) and has been used in the treatment of influenza, fever, malaria for thousands years. Though B. chinense has been reported to possess extensive pharmacological functions, however, the research on Bupleurum chinense polysaccharide is relatively weak. As important active substances of traditional Chinese medicine, the discovery of new lead compounds from polysaccharides has practical significance for the rational exploitation and utilization of traditional Chinese medicine resources. The incidence rate and mortality rate of acute lung injury (ALI) were higher in recent years, which have become a hotspot in scientific research. ALI is characterized by excessive inflammatory reaction, under inflammatory chemokines stimulation, the abundant neutrophils (PMN) in pulmonary circulation are quickly and continuously recruited into the lung tissue, excessive infiltration and activation, leading to excessive or uncontrolled systemic inflammatory response, eventually resulting in severe lung and vascular injury. As a new therapeutic target, P-selectin is an important adhesion molecule involved in early leukocyte recruitment during inflammatory response. Therefore, the development of P-selectin antagonists from TCM has been considered as a promising therapeutic strategy for inflammatory diseases, such as ALI. .Our previous study found that a polysaccharide fraction isolated from Bupleurum chinense, BCSP-2, could effectively antagonize the binding of P-selectin to its physiological ligands, block P-selectin-mediated leukocyte adhesion and the infiltration of leukocytes into inflammatory tissue, and inhibit the development of inflammation. Also, it suggested the possible presence of glycosidic bond-specific fragments in BCPS-2 can antagonize physiological function of P-selectin with high affinity. Based on the previous investigation, the present project will try to degrade BCPS-2 by controllable degradation method and purify the functional oligosaccharide fragment with high affinity to P-selectin by affinity chromatography; then resolve the chemical structure of the oligosaccharide fragment by combination of classical chemical and instrumental analysis methods; and systematically evaluate the mechanism and structural basis for prevention and treatment of ALI using in vitro and in vivo models, revealing the relationship between the ALI protection capability and P-selectin antagonism; then discuss the mechanism of molecular recognition between P-selectin and carbohydrate ligands during P-selectin mediated the interaction and the progress of ALI with the oligosaccharide fragment from BCPS-2. The present project will provide the theoretical basis for the screening and development of new efficient anti-inflammatory and ALI protective natural carbohydrate lead compounds, and also provide a scientific basis for rational development and utilization of B. chinense TCM resources.

柴胡是我国传统中药材，但对柴胡多糖的研究相对薄弱。作为中药重要活性物质，从多糖组分中挖掘新型先导化合物，对于中药资源的合理开发与利用具有现实意义。急性肺损伤（ALI）是危急重症的研究热点和难点，中性粒细胞（PMN）向肺组织过度浸润引发失控的炎症是ALI的主要成因，黏附分子P-selectin被证实是治疗ALI的潜在靶点。课题组前期研究发现柴胡多糖BCPS-2可拮抗P-selectin与生理配体的结合，阻断PMN组织浸润，抑制炎症发展，提示我们BCPS-2中存在特定糖苷键型的片段能够拮抗P-selectin生理功能。本项目将对BCPS-2进行可控性降解，纯化与P-selectin具有高亲和力的功能性寡糖片段，系统评价其干预ALI的作用效果，揭示糖链配基与ALI潜在靶标间的分子识别模式，为筛选具有防治ALI的天然糖类先导化合物以及新药研发奠定理论基础，为中药柴胡资源的高值化利用提供科学依据。

柴胡是我国传统中药材，但其多糖药理研究相对薄弱。作为中药重要活性物质，从多糖组分中挖掘新型先导化合物，对于中药资源的合理开发与利用具有现实意义。急性肺损伤（ALI）是危急重症的研究热点和难点，中性粒细胞（PMN）向肺组织过度浸润引发失控的炎症是ALI的主要成因，黏附分子P-selectin被证实是治疗ALI的潜在靶点。课题组研究发现柴胡多糖BCPS-2可拮抗P-selectin与生理配体的结合，阻断PMN组织浸润，抑制炎症发展，其中存在特定糖苷键型的片段能够拮抗P-selectin生理功能。本项目通过对柴胡多糖BCPS-2进行可控性降解，并对降解工艺进行优化；制备硫酸化修饰样品BCPS-2S；制备P-selectin高亲和力层析介质，并纯化制备与P-selectin具有高亲和力的核心糖链基序BCPS-cm；在整体水上评价BCPS-cm和BCPS-2S阻断中性粒细胞浸润、抑制炎症信号放大以及对急性肺损伤的保护作用；从分子细胞水平上评价BCPS-cm和BCPS-2拮抗P-selectin介导的中性粒细胞黏附作用的能力，深入挖掘BCPS-cm和BCPS-2S与P-selectin互作的关键结构域信息。通过上述研究系统评价核心糖链基序BCPS-cm干预ALI的效果，揭示天然糖链配基与潜在ALI靶标间的分子识别模式，为筛选具有防治ALI的天然糖类先导化合物以及新药研发奠定理论基础，为中药柴胡资源的高值化利用提供科学依据。

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