高脂血症脂联素活性受抑的分子成因及病理意义

Adiponectin (APN) is a novel adipocyte-derive molecule with strong cardiovascular protective actions. We have recently demonstrated that administration of APN protects vascular endothelial function in hyperlipidemic animals by reducing oxidative/nitrative stress; Additional study further demonstrated that APN might be modified/inhibited by unknown molecules present in hyperlipidemic plasma, suggesting that post-translational modification and functional inhibition of APN contribute to vascular endothelial dysfunction in hyperlipidemia. However, the identity of and mechanisms by which the hyperlipidemic plasma may bind and modify APN function remains completely unknown. Our most recent preliminary experiments suggest that multiple cytokines, particularly TNFα, can bind with APN. The specific aims of the current grant application are 1) defining the identity of those molecules that bind with APN by co-immunoprecipitation, mass spectrum analysis, and 2D gel electrophoresis; 2) determining the effect of this APN-binding/modification on APN biological function in cultured endothelial cells; and 3) identifying the molecular interventions that may block APN modification by hyperlipidemic plasma, and verifying their vascular protective effect in hyperlipidemic animals. Successful completion of these experiments will not only advance our understanding of molecular mechanisms of endothelial dysfunction in dyslipidemia, but may also provide experimental evidence supporting enhancement of APN function as a novel therapeutic strategy in hyperlipidemic patients.

脂联素（APN）是新近发现的脂肪细胞因子，具有明确的心血管保护作用。申请者前期系列研究已证实：APN可降低血管内皮氧化/硝化应激，改善高脂血症内皮功能；而高脂血浆可抑制APN活性，提示APN活性降低致其内皮保护作用丧失，可能参与高脂血症早期内皮功能障碍的形成。但高脂血浆抑制APN活性的分子机制目前尚一无所知。申请者最新预实验结果提示，高脂血浆中多种细胞因子特别是TNF-α与APN偶联并相互作用可能是抑制其活性的重要因素。本研究拟采用免疫共沉淀、质谱分析及双向凝胶电泳等技术，首先明确高脂血浆中与APN偶联的物质成分及其生化特性，进一步在细胞和整体水平验证其对APN活性的影响，最终确定可阻断高脂血浆对APN偶联抑制的分子靶点，并验证这些干预措施的内皮保护效应。本项目的完成将不仅为高脂血症血管内皮功能障碍提供全新的分子机制，且可望为以提高APN生物活性而保护血管的这一全新策略提供有力的实验证据。

高脂血症是代谢综合征的一个重要临床表现。由高脂血症导致的内皮功能失调是高脂血症引起各种心脑血管并发症的重要病理生理基础。探讨高脂血症性心血管疾病的发病机制，可为有效地防治心血管事件提供重要的理论和实验依据。APN是一种由脂肪细胞分泌的具有多种作用的蛋白质。研究表明APN具有促进胰岛素敏感性、保护心肌、改善内皮功能、抗炎等诸多重要的生理功能。我们前期的研究表明，补充外源性脂联素可改善高脂血症大鼠的血管内皮功能失调，进一步发现，高脂血浆可抑制APN活性并导致APN抵抗，且APN抵抗发生在受体前及受体水平，提示高脂血症早期APN抵抗可能是其导致内皮功能失调的重要因素之一。因此，本课题旨在明确"高脂血浆中是否存在抑制APN活性的血管活性物质，即探讨产生APN抵抗的受体前机制"这一目前尚未报道的科学问题。通过建立高脂血症动物模型，进行免疫沉淀、质谱分析等一系列分子生物学试验，发现高脂血症大鼠血浆甘油三酯、总胆固醇、低密度脂蛋白、高密度脂蛋白及APN的含量显著升高；载脂蛋白A1（APOA1）、载脂蛋白C1（APOC1）、PON1等十八种血管活性物质与高脂血浆中APN结合显著增多。其中APOA1在血浆中含量显著升高（P<0.05）,此外，APOA1及APOC1可显著抑制APN对AMPK的磷酸化水平。以上研究结果表明，高脂血症早期大鼠存在APN抵抗，与高脂血浆中载脂蛋白水平升高、结合并抑制APN活性有关。