氧化铜纳米颗粒经p38和ERK信号通路诱导巨噬细胞自噬性损伤

Copper oxide nanoparticles induce autophagic injury in macrophages via the p38 and ERK signaling pathways

摘要:

目的：分析氧化铜纳米颗粒（copper oxide nanoparticles，CuONPs）对Raw264.7巨噬细胞自噬的影响，并探讨调节自噬的相关信号通路。方法：用透射电子显微镜（transmission electron microscopy，TEM）观察CuONPs的形态特征；利用Raw264.7巨噬细胞进行体外实验；不同浓度梯度的CuONPs处理巨噬细胞24 h，用MTS法检测细胞活力；TEM观察自噬小体的形成情况；自噬小体报告质粒GFP-LC3瞬时转染细胞，激光共聚焦显微镜观察CuONPs处理前后Raw264.7细胞胞质中GFP-LC3蛋白绿色点状聚集情况；利用Western blot检测细胞中自噬相关蛋白LC3-Ⅱ/Ⅰ的表达情况及相关的p38和ERK信号通路磷酸化水平。结果：CuONPs可显著抑制Raw264.7细胞活力，并呈一定的剂量依赖关系（F=35.987，P=0.000）；TEM结果显示，CuONPs颗粒沉积于自噬体；激光共聚焦结果表明GFP-LC3转染细胞后，CuONPs处理组GFP-LC3点状聚集明显多于对照组，此外Western blot结果显示LC3-Ⅱ/Ⅰ表达水平明显呈剂量依赖性上升（F=156.585，P=0.000），上述结果说明CuONPs能促进巨噬细胞中自噬小体的生成；而且Western blot结果表明10 μg/mL的CuONPs处理细胞，能明显上调p-p38、p-ERK蛋白水平，并呈现时间依赖性升高（p-p38：F=72.899，P=0.000；p-ERK：F=123.300，P=0.000）。结论：CuONPs能抑制细胞活力，并诱导巨噬细胞自噬活化，并且p38和ERK通路可能参与了自噬的分子调控。

Abstract:

Objective：To investigate the effect of copper oxide nanoparticles（CuONPs） on autophagy of Raw264.7 macrophages and related signaling pathways involved in the regulation of autophagy. Methods：Transmission electron microscopy（TEM） was used to observe the morphological characteristics of CuONPs. Raw264.7 macrophages were used for in vitro experiment. Raw264.7 macrophages were exposed to different concentrations of CuONPs for 24 hours，and then MTS assay was used to measure cell viability. TEM was used to observe the formation of autophagosome. After transient transfection of macrophages with green fluorescent protein-light chain 3（GFP-LC3） plasmid，a laser scanning confocal microscope was used to observe GFP-LC3 green fluorescent puncta in the cytoplasm of Raw264.7 macrophages before and after CuONPs treatment. Western blot was used to measure the expression of the autophagy-related protein microtubule-associated protein I light chain 3（LC3-Ⅱ/Ⅰ） and the level of phosphorylation of the p38 and ERK signaling pathways in macrophages. Results：CuONPs significantly inhibited the viability of Raw264.7 macrophages in a dose-dependent manner（F=35.987，P=0.000）. TEM images showed the deposition of CuONPs in autophagosome. The laser scanning confocal microscope showed that after the macrophages were transfected with GFP-LC3，the CuONPs treatment group had a significantly higher number of GFP-LC3 green fluorescence puncta than the control group. In addition，Western blot showed a significant dose-dependent increase in LC3-Ⅱ/Ⅰ after CuONPs treatment（F=156.585，P=0.000）. The above results indicated that CuONPs promoted the formation of autophagosome in macrophages. Western blot also showed that CuONPs at a concentration of 10 μg/mL significantly upregulated the protein levels of p-p38 and p-ERK in a time-dependent manner（p-p38：F=72.899，P=0.000；p-ERK：F=123.300，P=0.000）. Conclusion：CuONPs can reduce cell viability and induce the activation of autophagy in macrophages，and the p38 and ERK signaling pathways may be involved in the molecular regulation of autophagy