巨噬细胞脂噬溶酶体通路的功能节点对动脉粥样硬化的调控

Autophagy, as a lysosomal degradative pathway critical for the removal and breakdown of cellular organelles and proteins, involves in the metabolism of lipid droplets (LD), described as lipophagy. Lipopphagy which considered to be a cellular protective mechanism by the regulation of lipid homeostasis, mediates the delivery of cytoplasmic LD to lysosomes and then lysosomal acid lipase (LAL) hydrolyzes LD-associated cholesteryl ester to generate free cholesterol for efflux. Aberrant lipopphagy may be involved in the foam cell formation and pathogenesis of atherosclerosis. Our previous work showed that in macrophages incubated with oxidative LDL or apolipoprotein E-deficient lipoprotein remnants or chloroquine, the expression of autophagic markers PI3K class III and LC3II/I ratio were increased or decreased in different time, while the levels of LAL in lysosomes were significantly reduced, thus the amount of LD was increased in macrophages. Autophagy and LAL expression level was reduced, while LD accumulation in macrophages from apolipoprotein E deficient (apoE-/-) mice and scavenger receptor BI deficient (SRBI-/-) mice, compared with wild type mice. However, the effects on foam cell formation was not equal when LAL upregulated. These intriguing findings suggest whether coordinate regulation of lipophagy lysosomal pathway is the key of foam cells formation. In this current proposal, We will investigate the relationship between the change of targeting molecules in lipophagy lysosomal pathway and LD accumulation in macrophages treated with various conditions (loading with oxidiative LDL or lipoprotein remnants or Free cholesterol, cells with siRNA or overexpression of LAL) and explore the key targeting protein of lipopphagy pathway. We will also perform in vivo studies using macrophage overexpression of targeting LAL in apoE-/- or SRBI-/- mice to study the effects on foam cell formation and atherogenesis. The results of this study will provide a basis for developing new preventive or therapautic strategies and targeting markers for atherosclerosis by regulating LD metabolism.

自噬作为一种溶酶体依赖的物质降解途径参与脂滴(LD)代谢——近年命名为脂噬，是细胞脂代谢的重要调节机制，脂噬缺陷与动脉粥样硬化(As)进程密切联系。我们前期工作观察到：ox-LDL、脂蛋白残粒或氯喹处理小鼠巨噬细胞，自噬标志PI3K Ⅲ或LC3 II/I可在不同时间升高或降低，但溶酶体酸脂酶(LAL)减少，结果胞内LD蓄积明显；而在不同脂血症的As模型：apoE-/-小鼠和SRBI-/-小鼠中，巨噬细胞的自噬及LAL表达水平均较野生型的低，细胞脂质积聚，这是否说明脂噬溶酶体通路的协调性可能是细胞泡沫化的关键？故本课题采用不同自噬诱导条件（ox-LDL、脂蛋白残粒或异常HDL等）或转基因手段处理小鼠巨噬细胞，从自噬潮的不同阶段及溶酶体功能探讨调控LD代谢的功能节点，并利用apoE-/-小鼠和SRBI-/-小鼠探讨靶向巨噬细胞LAL表达对As进程的干预，为As提供防治的新思路及药物靶标。

自噬作为一种溶酶体依赖的物质降解途径参与脂滴(LD)代谢——近年命名为脂噬，是细胞脂代谢的重要调节机制，脂噬缺陷与动脉粥样硬化(AS)进程密切联系。已知LAL是溶酶体中降解胆固醇酯的关键酶，我们前期发现apoE-/-小鼠脂蛋白残粒（LR）诱发自噬而抑制溶酶体LAL，故提出：协调调控脂噬溶酶体通路的关键节点对巨噬细胞泡沫化及AS进程的干预有重要意义。本研究通过选择不同脂噬诱导条件(ox-LDL、LR等）作用于小鼠腹腔巨噬细胞，发现脂质明显聚集在溶酶体，自噬标志蛋白(PI3K III、LC3II/I)增高，而溶酶体LAL的表达水平降低。因此构建Lv-LAL-GFP重组慢病毒载体和LAL shRNA慢病毒载体，转染Raw264.7巨噬细胞或HepG2肝细胞，通过LAL高表达或沉默，发现LAL与细胞脂质沉积有密切关系，可改变炎症相关基因（TNFα、IL-6）、溶酶体LAMP1及自噬途径标志分子(LC3II/I、Beclin1、ATG5、P62)的表达水平，以及细胞胆固醇代谢及其调节相关信号分子(NPC1、PPARγ、CD36、ABCA1、ABCG8等）的水平。进一步建立LAL高表达的apoE-/-或SR-BI-/-小鼠模型，研究结果表明，与Lv-GFP 组小鼠相比，小鼠LAL肝脏高表达使血浆apoAI、抗氧化蛋白 PON1 活性显著上升，并上调肝脏脂代谢相关基因(LXRa、ABCA1、ABCG1、ABCG8、PON1、apoAI）及自噬相关基因（LC3、Beclin1、Atg5/7）的表达水平。Lv-LAL组小鼠肝脏脂质沉积减少，胆汁和粪便中TC和FC含量增多，主动脉油红O染色显示血管壁AS斑块也显著减少。以上结果表明肝细胞LAL水平对细胞脂质代谢、转运和沉积有重要的调节意义，其机制可能与自噬通路的顺应性相关。此外，我们正在利用apoE-/-、SR-BI-/-小鼠为受体鼠进行骨髓移植实验，探讨靶向巨噬细胞LAL高表达对高脂血症及异常HDL代谢模型鼠AS发展进程的影响及机制。本研究重点阐明了溶酶体中LAL在细胞脂噬通路中的调控地位及其对细胞氧还状态和炎症信号的影响，从调节LD代谢的角度为开发新的动脉粥样硬化预防或治疗策略提供基础。

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