Abstract
Objective: To explore the role and mechanism of Prussian blue nanoparticle (PBNP) in the apoptosis of mouse adipose-derived stem cells (ADSCs) treated with hydrogen peroxide, providing a reference for chronic wound treatment. Methods: This research was an experimental research. PBNP with a cubic micromorphology was synthesized via the hydrothermal method. ADSCs were isolated from 6 male 6-8 weeks old Institute of Cancer Research mice using enzymatic digestion. ADSCs were divided into control group with normal culture, hydrogen peroxide group treated with hydrogen peroxide at final molarity of 200 μmol/L, and low PBNP group and high PBNP group pretreated with PBNP at final mass concentration of 10 and 20 μg/mL respectively and then treated as that in hydrogen peroxide group. After 24 h of culture, the reactive oxygen species (ROS) level was detected by fluorescence probe method, the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), malondialdehyde (MDA) levels, and lactate dehydrogenase (LDH) release rate were measured by colorimetric method, the cell survival rate was assessed by cell counting kit-8, and the protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), cytochrome C (Cyt-C), cleaved cysteinyl aspartate specific protease-3 (caspase-3), cleaved caspase-9, phosphatidylinositide 3-kinase (PI3K), phospho-PI3K (p-PI3K), protein kinase B (Akt), and phospho-Akt (p-Akt) were detected by Western blotting, with ratios of p-PI3K/PI3K and p-Akt/Akt being calculated. Another batch of ADSCs were divided into control group, hydrogen peroxide group, high PBNP group, which were treated as before, and N-acetyl-L-cysteine (NAC) group, high PBNP+LY294002 group, and high PBNP+MK-2206 group pretreated with NAC at final molarity of 5 mmol/L, PBNP at final mass concentration of 20 μg/mL and LY294002 at final molarity of 10 μmol/L, and PBNP at final mass concentration of 20 μg/mL and MK-2206 at final molarity of 100 μmol/L, respectively, and then treated as that in hydrogen peroxide group. After 24 h of culture, the p-PI3K/PI3K and p-Akt/Akt ratios were detected and calculated, and protein expression levels of Bcl-2, Bax, Cyt-C, cleaved caspase-3, and cleaved caspase-9 were measured as before. There were 3 samples in all experiments. Results: After 24 h of culture, the ROS level in cells in hydrogen peroxide group was 29.0±1.1, which was significantly higher than 2.6±1.1 in control group, 16.5±0.9 in low PBNP group, and 5.3±0.9 in high PBNP group (with P values all <0.05). Compared with those in hydrogen peroxide group, the levels of SOD, CAT, and GSH-Px, the cell survival rate, the Bcl-2 protein expression level, and the ratios of p-PI3K/PI3K and p-Akt/Akt were markedly increased in cells in control group, low PBNP group, and high PBNP group (P<0.05), the MDA level and LDH release rate in cells in control group and high PBNP group and the LDH release rate in cells in low PBNP group were significantly decreased (P<0.05), and the protein expression levels of Bax, Cyt-C, cleaved caspase-9, and cleaved caspase-3 in cells in control group, low PBNP group, and high PBNP group were significantly decreased (P<0.05). After 24 h of culture, compared with those in hydrogen peroxide group, the ratios of p-PI3K/PI3K and p-Akt/Akt, as well as Bcl-2 protein expression level were significantly increased in cells in control group, NAC group, and high PBNP group (P<0.05), while the protein expression levels of Bax, Cyt-C, cleaved caspase-9, and cleaved caspase-3 were significantly decreased (P<0.05). Compared with those in high PBNP group, the ratios of p-PI3K/PI3K and p-Akt/Akt, as well as Bcl-2 protein expression level were significantly decreased in cells in high PBNP+LY-294002 group and high PBNP+MK-2206 group (P<0.05), while the protein expression levels of Bax, Cyt-C, cleaved caspase-9, and cleaved caspase-3 were significantly increased (P<0.05). Conclusions: PBNP can inhibit apoptosis of mouse ADSCs caused by oxidative stress through activating the PI3K/Akt signaling pathway and reducing the expression level of apoptosis-related proteins in cells.