海带多糖干预动脉粥样硬化发生的结构基础与作用机理

Atherosclerosis （AS） is the primary cause of cardiovascular and cerebrovascular diseases, both of which are the two causes of death around the world and thus seriously affect the human health and life quality. However, there is no effective solution to treat this disease until today. Recently, there is considerable interest in identifying preventive factors that may delay the onset or progression of atherosclerosis, which will have great significance for the prevention of cardiovascular and cerebrovascular diseases. Our previous experimental findings revealed that polysaccharides are the bioactive compounds in Laminaria japonica for preventing onset and progression of atherosclerosis, and the differences were obviously observed in pharmacological activities among the polysaccharide fractions with different molecular weight. Accordingly, this project aims to study the structural basis and mechanism of anti-atherosclerosis of polysaccharides from Laminaria japonica (LP). Firstly, the crude polysaccharides will be extracted from the raw materials of Laminaria japonica, and the active and homogenous polysaccharide fractions will be obtained from the crude LP after separation and purification. At the same time, the atherosclerosis model of LDL receptor-dificient (LDLr-/-) mice induced by high fat diet will be applied to assessment of anti-atherosclerosis of polysaccharides during these processes. Secondly, the structures of active and homogenous polysaccharides will be characterized. Based on these results, the structure-function relationships of LP will be studied by molecular modifications (e.g. desulfation modification, sulfation modification and enzymatic degradation),and so the structure basis of LP for anti-atherosclerosis will be further determined. At last, the mechanisms of anti-atherosclerosis of LP will be investigated through testing the phosphorylation and dephosphorylation of some key proteins in MAPK, JAK-STAT and NF-κB signal transduction pathways. Moreover, the expression of some related proinflammatory cytokines, anti-inflammatory cytokines and adhesion molecules will be analyzed by western blotting, RT-PCR and ELISA methods. The accomplishment of this project will supply the original information for the deep research of studying anti-atherosclerosis mechanisms of polysaccharides and oriented screening and designning the new functional factors of polysaccharide analogues with high activity of anti-atherosclerosis.

动脉粥样硬化(AS)是心脑血管疾病发生的主要病理基础，严重威胁着人类健康和生活质量，但至今无有效的治疗措施，寻找有效的预防AS发生发展的功能因子，对解决心脑血管疾病的防治问题具有十分重要的意义。我们前期的研究发现，多糖是海带发挥抗AS功能的活性化学成分，且不同分子量多糖组分的活性差异较大。本项目将以此为基础，以高脂饲料诱导LDLr-/-小鼠动脉粥样硬化为活性评价模型，从海带中提取分离纯化活性多糖单一组分，解析其化学结构；在此基础上，借助多糖分子修饰技术，通过研究抗AS活性多糖的构效关系，阐明多糖抗AS功能所需的结构基础；以MAPK、JAK-STAT和NF-κB信号转导通路为切入点，通过研究多糖对信号通路关键蛋白的调控作用，明确多糖干预AS病变发生的信号转导机制，探讨多糖干预AS发生的作用机理。研究成果将为深入研究多糖抗AS的作用机理和目的性筛选与设计高抗AS活性多糖类功能因子奠定理论基础。

本项目在分离纯化活性海带多糖的基础上，重点对抗动脉粥样硬化活性多糖、免疫调节活性多糖的化学结构、作用机理及构效关系展开了研究，主要获得如下研究结果：（1）利用离子交换色谱、分子排阻色谱等分离纯化技术，从海带水溶性总多糖中纯化获得4种多糖均一组分LJP-11、LJP-12、LJP-31和LJP61A，并分析鉴定了它们的理化性质和化学结构。（2）活性筛选研究发现，LJP-11和LJP-31具有较强的免疫调节活性，LJP-12具有较强的抗动脉粥样硬化活性，LJP61A不仅抗动脉粥样硬化活性，而且对慢性肾功能衰竭及并其发性血管钙化具有较强的干预作用。（3）采用oxLDL诱导泡沫化细胞模型，发现LJP61A不仅显著降低PPARγ、CD36、ACAT1的mRNA表达，而且能显著增强ABCA1、SR-B1的mRNA与蛋白水平。同时，LJP61A通过mTOR显著抑制了ox-LDL对TLR2/4介导的MAPK和NFκB信号通路的激活作用，进而抑制了其下游效应分子TNF-α和IL-1β的mRNA的表达和分泌。（4）采用高脂饮食（HFD）诱导LDLr-/-小鼠动脉粥样硬化模型，发现LJP61A能显著改善HFD诱导的胰岛素抵抗、肝性脂肪变性和VLDL的过量分泌，且调控了IRS1和IRS2介导的PI3K-AKT-mTORC1与PI3K-AKT-Foxo1信号通路及其下游效应分子的表达。在此基础上，利用高糖、高胰岛素诱导HepG2细胞产生胰岛素抵抗的细胞模型，应用mTORC1与Foxo1抑制剂，进一步证实LJP61A通过调控PI3K-AKT-mTORC1与PI3K-AKT-Foxo1信号通路，影响TG合成、apoB降解和apoB 脂化，进而抑制VLDL的过度分泌。（5）LJP12能显著抑制高脂饮食诱导的主动脉TNF-α、IL-1β、ICAM-1和MCP-1的mRNA表达及NF-κB p65、JNK、ERK1/2和p38等蛋白的磷酸化。这些结果提示，海带多糖主要是从调控炎性和血脂代谢两方面抑制高脂饮食诱导的动脉粥样硬化发生。（6）构效关系研究表明，多糖主链中含有→6)-β-D-Galp-(1→，且其C-4位被硫酸化，或多糖存在α-D-Glcp-(1→末端基可能是多糖抗动脉粥样硬化活性所需要基本化学结构。研究还发现，硫酸基团含量是决定抗动脉粥样硬化活性高低的关键因素，取代度越大，其活性越强。