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.
目的： 探讨普鲁士蓝纳米粒（PBNP）对经过氧化氢处理的小鼠脂肪间充质干细胞（ADSC）凋亡的作用及其机制，以为慢性创面的治疗提供参考依据。 方法： 该研究为实验研究。采用水热合成法制备微观形貌为正方体的PBNP。取6只6~8周龄雄性美国癌症研究所小鼠，采用酶消化法提取ADSC。取ADSC，分为常规培养的对照组、用终物质的量浓度200 μmol/L过氧化氢处理的过氧化氢组及分别用终质量浓度10、20 μg/mL PBNP预处理后同过氧化氢组处理的低PBNP组、高PBNP组。培养24 h后，采用荧光探针法检测细胞中活性氧水平，采用比色法检测细胞中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、过氧化氢酶（CAT）、丙二醛水平及乳酸脱氢酶（LDH）释放率，采用细胞计数试剂盒-8检测细胞存活率，采用蛋白质印迹法检测细胞中B细胞淋巴瘤2（Bcl-2）、Bcl-2相关X蛋白（Bax）、细胞色素C（Cyt-C）、剪切型胱天蛋白酶3、剪切型胱天蛋白酶9、磷脂酰肌醇3-激酶（PI3K）、磷酸化PI3K（p-PI3K）、蛋白激酶B（Akt）、磷酸化Akt（p-Akt）的蛋白表达水平，并计算p-PI3K与PI3K比值、p-Akt与Akt比值。另取ADSC，分为同前处理的对照组、过氧化氢组、高PBNP组，以及分别用终物质的量浓度5 mmol/L的N-乙酰半胱氨酸（NAC）、终质量浓度20 μg/mL PBNP联合终物质的量浓度10 μmol/L LY294002、终质量浓度20 μg/mL PBNP联合终物质的量浓度100 nmol/L MK-2206预处理后同过氧化氢组处理的NAC组、高PBNP+LY294002组、高PBNP+MK-2206组。培养24 h后，同前检测并计算细胞中p-PI3K与PI3K比值、p-Akt与Akt比值及检测Bcl-2、Bax、Cyt-C、剪切型胱天蛋白酶3、剪切型胱天蛋白酶9的蛋白表达水平。所有实验样本数均为3。 结果： 培养24 h后，过氧化氢组细胞中活性氧水平为29.0±1.1，显著高于对照组的2.6±1.1、低PBNP 组的16.5±0.9、高PBNP组的5.3±0.9（P值均<0.05）。与过氧化氢组相比，对照组、低PBNP组、高PBNP组细胞中SOD、CAT、GSH-Px水平及细胞存活率、Bcl-2蛋白表达水平、p-PI3K与PI3K比值、p-Akt与Akt比值均显著升高（P<0.05），对照组、高PBNP组细胞中丙二醛水平、LDH释放率及低PBNP组细胞中LDH释放率均显著降低（P<0.05），对照组、低PBNP组、高PBNP组细胞中Bax、Cyt-C、剪切型胱天蛋白酶9、剪切型胱天蛋白酶3蛋白表达水平均显著降低（P<0.05）。培养24 h后，与过氧化氢组相比，对照组、NAC组、高PBNP组细胞中p-PI3K与PI3K比值、p-Akt与Akt比值、Bcl-2蛋白表达水平均显著升高（P<0.05），Bax、Cyt-C、剪切型胱天蛋白酶9、剪切型胱天蛋白酶3蛋白表达水平均显著降低（P<0.05）；与高PBNP组相比，高PBNP+LY-294002组及高PBNP+MK-2206组细胞中p-PI3K与PI3K比值、p-Akt与Akt比值、Bcl-2蛋白表达水平均显著降低（P<0.05），Bax、Cyt-C、剪切型胱天蛋白酶9、剪切型胱天蛋白酶3蛋白表达水平均显著升高（P<0.05）。 结论： PBNP能激活PI3K/Akt信号通路，降低细胞中凋亡相关蛋白表达水平，抑制氧化应激导致的小鼠ADSC凋亡。.