Fig. 2.
CeONP60/40 and CeONP20/80 reduce IR-induced intracellular ROS generation, and increase mitochondrial O2•- scavenging in primary hBMSCs 24h post-irradiation. The CeONP60/40 nanozyme selectively and significantly upregulates SOD1 and SOD2 gene expression. [A] Representative confocal microscope images of intracellular ROS and mitochondrial counter-staining in living hBMSCs. After IR-exposure at a dose of 7 Gy, a 160 kV tube voltage, 4 mA tube current, at a distance of 30 cm between the source and the surface (SC 500 smart controller, KIMTRON, USA), cells were stained with ROS/DCFDA (green), and MitoSpy™ Red CMXRos (red). An IR-induced increase in ROS is observed in the untreated control cell group with less expression identified in both groups following 24h pre-treatment with 10 μg/mL of CeONPs and followed by exposure to 7 Gy of irradiation. [B] Flow cytometry results showing a significant decrease in the fold-change of O2•- levels after 10 μg/mL of CeONP treatment at 24 h. Similar amounts of mitochondrial O2•- scavenging were observed following 10 μg/mL of nanozyme treatment. [C] Expression of antioxidation-related Catalase, GPX, SOD1 and SOD2 genes after 10 μg/mL CeONP treatment and 7 Gy radiation at 24 h and quantified using qRT-PCR. CeONP60/40 selectively and significantly upregulated SOD1 and SOD2 expression when compared with CeONP20/80 and the control, untreated cells. No other significant differences in gene expression were found. Experiments were carried out in triplicate. All values are given as the mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001. [D] Western blot analysis of SOD1. Endogenous α-Tubulin expression was shown as control. **p < 0.01. [E] Catalase activity was measured using an Amplite® Fluorimetric Catalase Assay Kit.