Nogo-B抑制Ox-LDL诱导的巨噬细胞泡沫化

Nogo-B在血管损伤、组织修复和炎症反应中发挥重要作用。然而，Nogo-B在动脉粥样硬化中的作用仍不明确。本研究拟在巨噬细胞中探讨Nogo-B对巨噬细胞泡沫化的影响。在RAW264.7细胞中沉默Nogo-B后，采用氧化低密度脂蛋白(Ox-LDL)或DiI修饰的Ox-LDL诱导巨噬细胞泡沫化；通过激光共聚焦显微镜观察巨噬细胞中荧光脂质，并在透射电镜下观察各组细胞中自噬泡；采用Western 印迹分析Plin2、p62和LC3-II的蛋白质水平；采用实时荧光定量PCR检测p62 mRNA水平；采用氯喹处理以及mRFP-GFP-LC3双荧光体系分析自噬流功能；进一步过表达Nogo-B后，比较巨噬细胞中脂质负荷程度以及Plin2、p62和LC3-II的蛋白质水平。结果显示，DiI-Ox-LDL处理后，Nogo-B沉默组细胞中脂质负荷程度高于对照组（2.34±0.67 vs. 0.69±0.14，P＜0.05）；Ox-LDL处理后，Nogo-B沉默组细胞中自噬泡数量（8.67±0.58 vs. 4.33±0.58，P＜0.01）、Plin2（4.65±0.50 vs. 3.24±0.71，P＜0.05）、p62（10.13±1.79 vs. 5.76±1.84，P＜0.05）和LC3-II（4.38±0.20 vs. 2-33±1.56，P＜0.01）的蛋白质水平均显著高于对照组，而p62 mRNA水平无差异（P＞0.05）；进一步研究发现，Nogo-B沉默组的自噬流被抑制了；过表达Nogo-B后，虽然p62蛋白质水平无明显变化，但是细胞中脂质负荷程度显著低于对照组（1.68±1.06 vs. 4.94±0.70，P＜0.05），Plin2和LC3-II的蛋白质水平也明显降低。上述结果表明，Nogo-B通过促进自噬流抑制了Ox-LDL诱导的巨噬细胞泡沫化，Nogo-B可能具有抗动脉粥样硬化的作用。

Nogo-B Inhibits Ox-LDL Induced Macrophage Foam Cell Formation

Nogo-B plays a key role in the vascular injury, tissue repair and inflammation process. However, its role in atherosclerosis is unclear. The aim of this study was to investigate the role of Nogo-B in macrophage foam cell formation. RAW264.7 cells were stimulated by oxidized low density lipoprotein (Ox-LDL) or DiI-Ox-LDL to establish the formation of macrophage foam cells. The lipid droplets in cells were examined by confocal microscopy, and autophagic vesicles were observed by transmission electron microscopy. The protein expressions of Plin2, p62 and LC3-II were detected by Western blotting, and the p62 mRNA levels were detected by real-time PCR. Autophagy flux was measured by Western blotting and the mRFP.GFP.LC3 double fluorescence system. The results showed that the levels of lipid loading were increased in the Nogo-B knockdown group (2.34±0.67 vs. 0.69±0.14，P＜0.05). The numbers of autophagic vesicles (8.67±0.58 vs. 4.33±0.58，P＜0.01)，the protein expressions of Plin2 (4.65±0.50 vs. 3.24±0.71，P＜0.05), p62 (10.13±1.79 vs. 5.76±1.84，P＜0.05) and LC3-II (4.38±0.20 vs. 2.33±1.56，P＜0.01) were significantly increased. In addition, no differences were detected in mRNA expression levels of p62 (P＞0.05). Further studies showed that autophagy flux was inhibited in the Nogo-B knockdown group. After treated with Ox-LDL, the levels of lipid loading (1.68±1.06 vs. 4.94±0.70，P＜0.05), the protein expressions of Plin2 and LC3-II were decreased in the Nogo-B overexpression group, while p62 protein levels were not changed. In conclusion, Nogo-B inhibited Ox-LDL-induced macrophage foam cell formation by promoting autophagy flux and may play a protective role in atherosclerosis.