Role and mechanism of Prussian blue nanoparticles in the apoptosis of mouse adipose-derived mesenchymal stem cells treated with hydrogen peroxide
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摘要:
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。 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. -
参考文献
(40) [1] SenooM.Epidermal stem cells in homeostasis and wound repair of the skin[J].Adv Wound Care (New Rochelle),2013,2(6):273-282.DOI: 10.1089/wound.2012.0372. [2] 王一希,陈俊杰,岑瑛,等.脂肪间充质干细胞外泌体促进糖尿病创面愈合的研究进展[J].中华烧伤与创面修复杂志,2022,38(5):491-495.DOI: 10.3760/cma.j.cn501120-20210218-00057. [3] BydonM,QuW,MoinuddinFM,et al.Intrathecal delivery of adipose-derived mesenchymal stem cells in traumatic spinal cord injury: phase I trial[J].Nat Commun,2024,15(1):2201.DOI: 10.1038/s41467-024-46259-y. [4] HoseinzadehA,EsmaeiliSA,SahebiR,et al.Fate and long-lasting therapeutic effects of mesenchymal stromal/stem-like cells: mechanistic insights[J].Stem Cell Res Ther,2025,16(1):33.DOI: 10.1186/s13287-025-04158-z. [5] ZhangZ,ZhangM,ZhangZ,et al.ADSCs combined with melatonin promote peripheral nerve regeneration through autophagy[J].Int J Endocrinol,2022:5861553.DOI: 10.1155/2022/5861553. [6] KangY,XuL,DongJ,et al.Programmed microalgae-gel promotes chronic wound healing in diabetes[J].Nat Commun,2024,15(1):1042.DOI: 10.1038/s41467-024-45101-9. [7] BusquetsMA,EstelrichJ.Prussian blue nanoparticles: synthesis, surface modification, and biomedical applications[J].Drug Discov Today,2020,25(8):1431-1443.DOI: 10.1016/j.drudis.2020.05.014. [8] XuZ,LiuY,MaR,et al.Thermosensitive hydrogel incorporating prussian blue nanoparticles promotes diabetic wound healing via ROS scavenging and mitochondrial function restoration[J].ACS Appl Mater Interfaces,2022,14(12):14059-14071.DOI: 10.1021/acsami.1c24569. [9] GuoN,WangX,XuM,et al.PI3K/AKT signaling pathway: molecular mechanisms and therapeutic potential in depression[J].Pharmacol Res,2024,206:107300.DOI: 10.1016/j.phrs.2024.107300. [10] GlavianoA, FooASC, LamHY, et al. PI3K/AKT/mTOR signaling transduction pathway and targeted therapies in cancer[J]. Mol Cancer, 2023,22(1):138. DOI: 10.1186/s12943-023-01827-6. [11] 孙宝俊,赵风景,章文锋,等.普鲁士蓝纳米粒对过氧化氢诱导脂肪间充质干细胞凋亡的影响[J].中华实验外科杂志,2023,40(12):2548-2553.DOI: 10.3760/cma.j.cn421213-20230416-00217. [12] LinF,YangY,WeiS,et al.Hydrogen sulfide protects against high glucose-induced human umbilical vein endothelial cell injury through activating PI3K/Akt/eNOS pathway[J].Drug Des Devel Ther,2020,14:621-633.DOI: 10.2147/DDDT.S242521. [13] DuanMX,ZhouH,WuQQ,et al.Andrographolide protects against hg-induced inflammation, apoptosis, migration, and impairment of angiogenesis via PI3K/AKT-eNOS signalling in HUVECs[J].Mediators Inflamm,2019:6168340.DOI: 10.1155/2019/6168340. [14] ParkJS,PiaoJ,ParkG,et al.Substance-P restores cellular activity of ADSC impaired by oxidative stress[J].Antioxidants (Basel),2020,9(10):978.DOI: 10.3390/antiox9100978. [15] 唐茂涵,张艳杰,刘银,等. 三甲氧苄嗪对脂肪来源间充质干细胞氧化应激损伤的影响及机制研究[J]. 四川大学学报(医学版),2017,48(2):173-178. [16] YangCS,GuoXS,YueYY,et al.Astaxanthin promotes the survival of adipose-derived stem cells by alleviating oxidative stress via activating the Nrf2 signaling pathway[J].Int J Mol Sci,2023,24(4):3850.DOI: 10.3390/ijms24043850. [17] ZhangW,HuS,YinJJ,et al.Prussian blue nanoparticles as multienzyme mimetics and reactive oxygen species scavengers[J].J Am Chem Soc,2016,138(18):5860-5865.DOI: 10.1021/jacs.5b12070. [18] XieX,GaoW,HaoJ,et al.Self-synergistic effect of Prussian blue nanoparticles for cancer therapy: driving photothermal therapy and reducing hyperthermia-induced side effects[J].J Nanobiotechnology,2021,19(1):126.DOI: 10.1186/s12951-021-00819-2. [19] ZhouT,YangX,ChenZ,et al.Prussian blue nanoparticles stabilize SOD1 from ubiquitination-proteasome degradation to rescue intervertebral disc degeneration[J].Adv Sci (Weinh),2022,9(10):e2105466.DOI: 10.1002/advs.202105466. [20] SahooBM,BanikBK,BorahP,et al.Reactive oxygen species (ROS): key components in cancer therapies[J].Anticancer Agents Med Chem,2022,22(2):215-222.DOI: 10.2174/1871520621666210608095512. [21] DarenskayaMA,KolesnikovaLI,KolesnikovSI.Oxidative stress: pathogenetic role in diabetes mellitus and its complications and therapeutic approaches to correction[J].Bull Exp Biol Med,2021,171(2):179-189.DOI: 10.1007/s10517-021-05191-7. [22] IrazabalMV,TorresVE.Reactive oxygen species and redox signaling in chronic kidney disease[J].Cells,2020,9(6):1342.DOI: 10.3390/cells9061342. [23] BhattS,PuliL,PatilCR.Role of reactive oxygen species in the progression of Alzheimer's disease[J].Drug Discov Today,2021,26(3):794-803.DOI: 10.1016/j.drudis.2020.12.004. [24] JiJY,RenDY,WengYZ.Efficiency of multifunctional antibacterial hydrogels for chronic wound healing in diabetes: a comprehensive review[J].Int J Nanomedicine,2022,17:3163-3176.DOI: 10.2147/IJN.S363827. [25] 魏科东,吴婉婉,任涵,等.基于PI3K/Akt信号通路探讨肉桂酸保护H2O2诱导H9c2细胞损伤的作用机制[J].海南医学院学报,2024,30(14):1048-1058.DOI: 10.13210/j.cnki.jhmu.20240408.001. [26] GahlRF,DwivediP,TjandraN.Bcl-2 proteins bid and bax form a network to permeabilize the mitochondria at the onset of apoptosis[J].Cell Death Dis,2016,7(10):e2424.DOI: 10.1038/cddis.2016.320. [27] SusnowN,ZengL,MargineantuD,et al.Bcl-2 family proteins as regulators of oxidative stress[J].Semin Cancer Biol,2009,19(1):42-49.DOI: 10.1016/j.semcancer.2008.12.002. [28] TiongYL,NgKY,KohRY,et al.Melatonin prevents oxidative stress-induced mitochondrial dysfunction and apoptosis in high glucose-treated Schwann cells via upregulation of Bcl2, NF-κB, mTOR, Wnt signalling pathways[J].Antioxidants (Basel),2019,8(7):198.DOI: 10.3390/antiox8070198. [29] ZhangM,ZhengJ,NussinovR,et al.Release of cytochrome C from Bax pores at the mitochondrial membrane[J].Sci Rep,2017,7(1):2635.DOI: 10.1038/s41598-017-02825-7. [30] JulienO,WellsJA.Caspases and their substrates[J].Cell Death Differ,2017,24(8):1380-1389.DOI: 10.1038/cdd.2017.44. [31] NoorZM,AhmadH, Ul AinQ, et al. Caspase 3 and its role in the pathogenesis of cancer[J]. Clin Oncol, 2022, 7. [32] NuutinenU,PostilaV,MättöM,et al.Inhibition of PI3-kinase-Akt pathway enhances dexamethasone-induced apoptosis in a human follicular lymphoma cell line[J].Exp Cell Res,2006,312(3):322-330.DOI: 10.1016/j.yexcr.2005.10.023. [33] RevathideviS,MunirajanAK.Akt in cancer: mediator and more[J].Semin Cancer Biol,2019,59:80-91.DOI: 10.1016/j.semcancer.2019.06.002. [34] KaleJ,KutukO,BritoGC,et al.Phosphorylation switches Bax from promoting to inhibiting apoptosis thereby increasing drug resistance[J].EMBO Rep,2018,19(9):e45235.DOI: 10.15252/embr.201745235. [35] KangJ,WangY,GuoX,et al.N-acetylserotonin protects PC12 cells from hydrogen peroxide induced damage through ROS mediated PI3K/AKT pathway[J].Cell Cycle,2022,21(21):2268-2282.DOI: 10.1080/15384101.2022.2092817. [36] ZhangE,GaoB,YangL,et al.Notoginsenoside Ft1 promotes fibroblast proliferation via PI3K/Akt/mTOR signaling pathway and benefits wound healing in genetically diabetic mice[J].J Pharmacol Exp Ther,2016,356(2):324-332.DOI: 10.1124/jpet.115.229369. [37] LiJY,RenKK,ZhangWJ,et al.Human amniotic mesenchymal stem cells and their paracrine factors promote wound healing by inhibiting heat stress-induced skin cell apoptosis and enhancing their proliferation through activating PI3K/AKT signaling pathway[J].Stem Cell Res Ther,2019,10(1):247.DOI: 10.1186/s13287-019-1366-y. [38] LiD,LiuM,LiW,et al.Synthesis of Prussian blue nanoparticles and their antibacterial, antiinflammation and antitumor applications[J].Pharmaceuticals (Basel),2022,15(7):769.DOI: 10.3390/ph15070769. [39] 刘颖,程凤,王泽薇,等. 负载小鼠脂肪干细胞的甲壳素/透明质酸/胶原水凝胶的制备及其对大鼠全层皮肤缺损创面愈合的作用[J]. 中华烧伤与创面修复杂志,2024,40(1):50-56. DOI: 10.3760/cma.j.cn501225-20230928-00101. [40] 潘泽平,石云龙,袁志强,等.负载锌离子的复合水凝胶对糖尿病小鼠全层皮肤缺损感染创面的作用及机制[J].中华烧伤与创面修复杂志,2024,40(9):866-875.DOI: 10.3760/cma.j.cn501225-20231120-00200. -
图 2 4组小鼠脂肪间充质干细胞培养24 h后的活性氧水平 2',7'-二氯二氢荧光素二乙酸酯×100。2A、2B、2C、2D.分别为对照组、过氧化氢组、低PBNP组、高PBNP组,图2B中活性氧水平显著高于图 2A、2C、2D,图2C中活性氧水平显著高于图2D
注：对照组细胞常规培养,过氧化氢组细胞用终物质的量浓度200 μmol/L过氧化氢培养,低普鲁士蓝纳米粒（PBNP）组、高PBNP组细胞分别用终质量浓度10、20 μg/mL PBNP预处理后同过氧化氢组培养；绿色荧光表示活性氧
图 3 4组小鼠脂肪间充质干细胞培养24 h后氧化损伤相关指标水平及细胞存活率（样本数均为3,
注：横坐标中1、2、3、4分别表示行常规培养的对照组,用终物质的量浓度200 μmol/L过氧化氢处理的过氧化氢组,分别为用终质量浓度10、20 μg/mL普鲁士蓝纳米粒（PBNP）预处理后同过氧化氢组处理的低PBNP组、高PBNP组；4组细胞中超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）、丙二醛水平及乳酸脱氢酶（LDH）释放率、细胞存活率总体比较,差异均有统计学意义（F值分别为34.80、26.43、56.08、24.56、234.70、39.27,P<0.001）；与对照组比较,aP<0.05；与过氧化氢组比较,bP<0.05；与低PBNP组比较,cP<0.05
图 4 4组小鼠脂肪间充质干细胞培养24 h后凋亡相关蛋白的表达水平（样本数均为3）
注：图4A、4B条带上方1~3、4~6、7~9、10~12及图4C~4G横坐标中1、2、3、4分别表示行常规培养的对照组,用终物质的量浓度200 μmol/L过氧化氢处理的过氧化氢组,分别用终质量浓度10、20 μg/mL普鲁士蓝纳米粒（PBNP）预处理后同过氧化氢组处理的低PBNP组、高PBNP组；图4C~4G中数据以
图 5 4组小鼠脂肪间充质干细胞培养24 h后PI3K/Akt信号通路中相关蛋白的表达水平（样本数均为3）
注：图5A、5B条带上方1~3、4~6、7~9、10~12及图5B、5C横坐标中1、2、3、4分别表示行常规培养的对照组,用终物质的量浓度200 μmol/L过氧化氢处理的过氧化氢组,分别用终质量浓度10、20 μg/mL普鲁士蓝纳米粒（PBNP）预处理后同过氧化氢组处理的低PBNP组、高PBNP组；图5B和5C中数据以
图 6 6组小鼠脂肪间充质干细胞培养24 h后PI3K/Akt信号通路中相关蛋白的表达水平（样本数均为3）
注：图6A条带上方1~3、4~6、7~9、10~12、13~15、16~18及图6B、6C横坐标中1、2、3、4、5、6分别表示行常规培养的对照组,用终物质的量浓度200 μmol/L过氧化氢处理的过氧化氢组,用终质量浓度20 μg/mL普鲁士蓝纳米粒（PBNP）预处理后同过氧化氢组处理的高PBNP组,用终物质的量浓度5 mmol/L的N-乙酰半胱氨酸（NAC）预处理后同过氧化氢组处理的NAC组,用终质量浓度20 μg/mL PBNP联合终物质的量浓度10 μmol/L LY294002预处理后同过氧化氢组处理的高PBNP+LY294002组,用终质量浓度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高PBNP+MK-2206组；图6B、6C中数据以
图 7 6组小鼠脂肪间充质干细胞培养24 h后凋亡相关蛋白的表达水平（样本数均为3）
注：7A、7B条带上方1~3、4~6、7~9、10~12、13~15、16~18及图7C~7G横坐标中1、2、3、4、5、6分别表示行常规培养的对照组,用终物质的量浓度200 μmol/L过氧化氢处理的过氧化氢组,用终质量浓度20 μg/mL 普鲁士蓝纳米粒（PBNP）预处理后同过氧化氢组处理的高PBNP组,用终物质的量浓度5 mmol/L的N-乙酰半胱氨酸（NAC）预处理后同过氧化氢组处理的NAC组,用终质量浓度20 μg/mL PBNP联合终物质的量浓度10 μmol/L LY294002预处理后同过氧化氢组处理的高PBNP+LY294002组,用终质量浓度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高PBNP+MK-2206组；图7C~7G中数据以
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