巨噬源性泡沫细胞中p62蛋白上调作用和机制的研究

A study on the role and mechanism of upregulated p62 protein in macrophage-derived foam cells

目的探讨巨噬源性泡沫细胞中p62蛋白对脂质代谢相关的自噬以及炎症因子表达的影响,为p62在抗动脉粥样硬化中的应用提供参考。方法利用Ox-LDL刺激RAW264.7细胞的方法模拟巨噬源性泡沫细胞的形成,通过Western blot及实时荧光定量PCR检测巨噬源性泡沫细胞中p62蛋白和mRNA水平。通过Western blot比较p62 siRNA组和对照组中Ox-LDL诱导的LC3剪切、脂滴相关蛋白Plin2和过氧化物酶体相关蛋白PEX2的蛋白水平,通过实时荧光定量PCR比较TNFα和IL-6 mRNA表达水平。结果 Ox-LDL对RAW264.7细胞中p62蛋白以及mRNA水平均有上调作用。干扰p62的表达之后,LC3-Ⅱ蛋白水平降低,Plin2的蛋白水平并无明显变化,PEX2的蛋白水平升高,TNFα和IL-6 mRNA表达升高。进一步研究发现,干扰Nrf2后能明显抑制Ox-LDL对p62的上调作用。结论巨噬源性泡沫细胞中Ox-LDL通过Nrf2介导p62蛋白的上调,p62可能具有抗动脉粥样硬化的作用。

Objective To investigate the effect of p62 protein on the lipid autophagy and the expression of inflammatory cytokines in macrophage-derived foam cells. Methods RAW264.7 cells were stimulated by oxidized low density lipoprotein to mimic the formation of macrophage-derived foam cells.The levels of p62 protein and mRNA in macrophage-derived foam cells were detected by Western blot and real-time qPCR.The protein levels of LC3,Plin2 and PEX2 were measured by Western blot in Ox-LDL treated Raw264.7 cells to determine the effects of p62 on autophagy in macrophage-derived foam cells.The TNFα and IL-6 mRNA levels were detected by real-time PCR. Results Ox-LDL up-regulated both autophagy levels and p62 protein levels in RAW264.7 cells.After interfering the expression of p62,the level of LC3-II protein decreased,but the protein levels of lipid droplet-related protein Plin2 did not change significantly.The peroxisome-related protein PEX2 level increased.The expression of TNFα and IL-6 also increased.Further studies showed that Nrf2 interference significantly inhibited the up-regulation of p62 by Ox-LDL. Conclusion Ox-LDL mediates the upregulation of p62 through Nrf2 in macrophage-derived foam cells,and p62 may play a protective role in atherosclerosis.