Xu S Y, Gong Z, Han Y J, Wang J X, Yu L, Xu G
Department of Plastic and Burn Surgery, Northern Jiangsu People's Hospital Affiliated to Yangzhou University/Northern Jiangsu People's Hospital, Yangzhou 225009, China.
Zhonghua Shao Shang Yu Chuang Mian Xiu Fu Za Zhi. 2025 May 20;41(5):481-490. doi: 10.3760/cma.j.cn501225-20240525-00197.
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. 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. 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 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 (<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 (<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 (<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 (<0.05), while the protein expression levels of Bax, Cyt-C, cleaved caspase-9, and cleaved caspase-3 were significantly decreased (<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 (<0.05), while the protein expression levels of Bax, Cyt-C, cleaved caspase-9, and cleaved caspase-3 were significantly increased (<0.05). 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.
探讨普鲁士蓝纳米颗粒（PBNP）在过氧化氢处理的小鼠脂肪来源干细胞（ADSCs）凋亡中的作用及机制，为慢性伤口治疗提供参考。本研究为实验性研究。采用水热法合成了具有立方微形态的PBNP。采用酶消化法从6只6 - 8周龄雄性癌症研究所小鼠中分离ADSCs。将ADSCs分为正常培养的对照组、终浓度为200 μmol/L过氧化氢处理的过氧化氢组、终质量浓度分别为10和20 μg/mL PBNP预处理后再按过氧化氢组处理的低PBNP组和高PBNP组。培养24 h后，采用荧光探针法检测活性氧（ROS）水平，采用比色法测定超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、过氧化氢酶（CAT）、丙二醛（MDA）水平及乳酸脱氢酶（LDH）释放率，采用细胞计数试剂盒-8评估细胞存活率，采用蛋白质印迹法检测B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、细胞色素C（Cyt-C）、裂解的半胱氨酸天冬氨酸特异性蛋白酶-3（caspase-3）、裂解的caspase-9、磷脂酰肌醇3-激酶（PI3K）、磷酸化PI3K（p-PI3K）、蛋白激酶B（Akt）和磷酸化Akt（p-Akt）的蛋白表达水平，并计算p-PI3K/PI3K和p-Akt/Akt的比值。另一批ADSCs分为对照组、过氧化氢组、高PBNP组（处理同前）、N-乙酰-L-半胱氨酸（NAC）组、高PBNP+LY294002组和高PBNP+MK-2206组，分别用终浓度为5 mmol/L的NAC、终质量浓度为20 μg/mL的PBNP和终浓度为10 μmol/L的LY294002，以及终质量浓度为20 μg/mL的PBNP和终浓度为100 μmol/L的MK-2206预处理，然后按过氧化氢组处理。培养24 h后，检测并计算p-PI3K/PI3K和p-Akt/Akt比值，同前测定Bcl-2、Bax、Cyt-C、裂解的caspase-3和裂解的caspase-9的蛋白表达水平。所有实验均设3个样本。培养24 h后，过氧化氢组细胞内ROS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和高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组和高PBNP组细胞内SOD、CAT和GSH-Px水平、细胞存活率、Bcl-2蛋白表达水平以及p-PI3K/PI3K和p-Akt/Akt比值均显著升高（P<0.05），对照组和高PBNP组细胞内MDA水平和LDH释放率以及低PBNP组细胞内LDH释放率均显著降低（P<0.05），对照组、低PBNP组和高PBNP组细胞内Bax、Cyt-C、裂解的caspase-9和裂解的caspase-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组和高PBNP组细胞内p-PI3K/PI3K和p-Akt/Akt比值以及Bcl-2蛋白表达水平均显著升高（P<0.05），而Bax、Cyt-C、裂解的caspase-9和裂解的caspase-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组和高PBNP+MK-2206组细胞内p-PI3K/PI3K和p-Akt/Akt比值以及Bcl-2蛋白表达水平均显著降低（P<0.05），而Bax、Cyt-C、裂解的caspase-9和裂解的caspase-3蛋白表达水平均显著升高（P<0.05）。PBNP可通过激活PI3K/Akt信号通路并降低细胞内凋亡相关蛋白的表达水平，抑制氧化应激诱导的小鼠ADSCs凋亡。
