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

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