RTN3调控巨噬细胞自噬对动脉粥样硬化影响的研究

Atherosclerosis (AS) is a cardiovascular disease with high morbidity and mortality. Later on, abnormality of autophagy of macrophage has been acknowledged as a significant event in AS development. However, the concrete molecular regulatory mechanism still remains elusive and needs further researches. In our previous studies, we found that: i) Reticulon3 (RTN3), a protein usually located in endoplasmic reticulum (ER), was also detected in autophagosomes and other intracellular vesicles in macrophages; ii) triggering autophagy in RTN3 over-expressed macrophages via treating the cells with oxLDL causes lower expression of autophagy-inducing protein Beclin1 and microtubule associated protein-1 light-chain 3(LC3-II), which are considered as autophagic markers, and more deposition of lipid droplets. iii) RTN3 can bind Bcl-2 or Beclin1, a key protein in autophagy regulation; and iv) RTN3 levels in AS patients' peripheral blood mononuclear cell are higher than that in normal controls, and it has a positive correlation with C response protein(CRP) and a negtive correlation with high-density lipoprotein(HDL). Based on these findings, we postulate that RTN3 plays an important role in macrophage attending AS development via interaction with Bcl-2 or Beclin1, regulating macrophage autophagy. We propose to clarify the molecular mechanism of autophagy in macrophages and its influence to AS through investigating the thogenesis from the aspects of cell, animal and human. Our study could potentially provide new thoughts into the prevention and treatment of AS by offering new molecular explanation to the autophagy of macrophage in AS.

动脉粥样硬化AS发病率和死亡率都很高。巨噬细胞自噬改变是AS进程中的重要事件，但具体分子调控机制亟待研究。申请者的前期研究发现：内质网膜蛋白RTN3，可定位于巨噬细胞自噬体等胞内小泡；RTN3高表达巨噬细胞中加入oxLDL，自噬蛋白Beclin1、LC3II表达明显减少，脂滴沉积较多；RTN3 knock down的巨噬细胞中，同浓度oxLDL刺激，LC3II表达升高，自噬体数目明显增加，脂滴沉积减少；RTN3可与自噬调节蛋白Beclin1、Bcl-2相互作用；AS病人外周血单核RTN3表达高于正常人，且与血清HDL呈负相关，与C反应蛋白呈正相关。因此推测：RTN3通过与Beclin1、Bcl2作用，调控巨噬细胞自噬影响AS进程。本研究拟从细胞、动物和人体水平，探讨RTN3调控巨噬自噬的分子机制及对AS的影响，从巨噬自噬层面对AS病理机制做进一步诠释，将为AS防治提供新的理论依据和手段。

动脉粥样硬化（AS）是冠心病、脑卒中等心脑血管疾病发病的共同基础。AS的发病机理非常复杂，其中巨噬细胞自噬改变及脂质代谢异常是AS发生过程中的重要事件。我们的研究证实：RTN3（reticulon3）作为一种内质网膜蛋白，可定位于巨噬细胞自噬体及脂滴等胞内小泡；通过构建RTN3转基因小鼠，我们发现RTN3过表达可以导致肥胖及甘油三酯的积累；在肥胖及高甘油三酯致AS患者中，也发现RTN3含量显著增加；通过构建线虫模型，我们发现RTN3在调节甘油三酯的生物合成、储存及控制脂滴扩张等方面起到关键作用；通过细胞实验，我们证实敲低RTN3会导致巨噬细胞自噬蛋白Beclin1、LC3II的表达水平升高，自噬体数目明显增加；在分子实验中，我们明确RTN3通过与HSPA5的相互作用，促进SREBP1-c及AMPK的激活，从而加速甘油三酯的合成；而且，我们构建了AS相关血脂异常的遗传资源库，通过对数百名AS相关血脂异常患者进行基因测序分析，我们新发现了数十个已知致病基因新的突变位点；与此同时，通过对我们构建的RTN3敲除小鼠模型的研究，我们发现RTN3敲除小鼠的BACE1蛋白水平显著上升；同时在阿尔兹海默症小鼠模型中敲除RTN3，可以促进淀粉样蛋白的沉积；而在高脂饮食诱导的肥胖小鼠模型中，我们发现了内质网应激的增强，且在脂肪组织中表现出慢性炎症反应，我们发现化学分子伴侣可以调节脂质代谢异常并降低高脂喂养肥胖小鼠的炎症因子水平及游离胆固醇水平。综上所述，我们的研究为RTN3调节自噬、导致AS、肥胖及甘油三酯的积累提供了证据；建立了AS相关遗传资源库，扩展了中国人群中AS相关致病突变的突变谱，也将有助于动脉粥样硬化相关的遗传诊断与咨询；揭示了RTN3与BACE1在促进阿尔兹海默症发病过程中的重要作用；明确了调控内质网应激水平的分子可能成为肥胖相关的代谢综合征的新药物靶点。