Abstract
目的
探讨虾青素对高糖处理的人皮肤成纤维细胞（Fb）衰老的作用及其机制。
方法
该研究为实验研究。取人皮肤Fb，分为常规培养的对照组、用终物质的量浓度30 mmol/L的葡萄糖处理的高糖组及分别用终物质的量浓度25、50 μmol/L的虾青素预处理后再同高糖组处理的低虾青素组、高虾青素组。培养48 h后，采用细胞计数试剂盒-8检测细胞存活率，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丙二醛、谷胱甘肽、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）水平，采用蛋白质印迹法检测细胞中p53、磷酸化p53、p21、p16、Rb、磷酸化Rb、基质金属蛋白酶1（MMP1）、MMP3、MMP13蛋白表达水平及细胞质和细胞核中核转录因子红系2相关因子2（Nrf2）蛋白表达水平。另取人皮肤Fb，分为同前处理的对照组、高糖组、高虾青素组及用终物质的量浓度为50 μmol/L的虾青素和10 μmol/L的ML385预处理后再同高糖组细胞处理的高虾青素+ML385组。培养48 h后，同前检测细胞存活率，细胞中p53、磷酸化p53、p21、p16、Rb、磷酸化Rb、MMP1、MMP3、MMP13蛋白表达水平及细胞质和细胞核中Nrf2蛋白表达水平。以上实验中样本数均为3。
结果
培养48 h后，与对照组比较，高糖组细胞存活率明显降低（P < 0.05）；与高糖组比较，低虾青素组和高虾青素组细胞存活率均明显升高（P < 0.05）；与低虾青素组比较，高虾青素组细胞存活率明显升高（P < 0.05）。与对照组比较，高糖组细胞中活性氧、丙二醛水平均明显升高（P < 0.05），谷胱甘肽、SOD、CAT、GSH-Px水平均明显降低（P < 0.05）。与高糖组比较，低虾青素组细胞中活性氧、丙二醛水平均明显降低（P < 0.05），CAT、GSH-Px水平均明显升高（P < 0.05）；高虾青素组细胞中活性氧、丙二醛水平均明显降低（P < 0.05），谷胱甘肽、SOD、CAT、GSH-Px水平均明显升高（P < 0.05）。与低虾青素组比较，高虾青素组细胞中活性氧、丙二醛水平均明显降低（P < 0.05），谷胱甘肽、CAT、GSH-Px水平均明显升高（P < 0.05）。与对照组比较，高糖组细胞中p53、p21、磷酸化p53、p16、Rb、MMP1、MMP3、MMP13蛋白表达水平均明显升高（P < 0.05），磷酸化Rb蛋白表达水平明显降低（P < 0.05）。与高糖组比较，低虾青素组细胞中p53、p21、磷酸化p53、MMP13蛋白表达水平均明显降低（P < 0.05），磷酸化Rb蛋白表达水平明显升高（P < 0.05）；高虾青素组细胞中p53、p21、磷酸化p53、p16、Rb、MMP1、MMP3、MMP13蛋白表达水平均明显降低（P < 0.05），磷酸化Rb蛋白表达水平明显升高（P < 0.05）。与低虾青素组比较，高虾青素组细胞中p21、磷酸化p53、p16、Rb、MMP1蛋白表达水平均明显降低（P < 0.05）。与对照组、高虾青素组比较，高糖组细胞质中Nrf2蛋白表达水平明显升高（P值均 < 0.05），细胞核中Nrf2蛋白表达水平明显降低（P值均 < 0.05）。与低虾青素组比较，高糖组细胞质中Nrf2蛋白表达水平明显升高（P < 0.05）；高虾青素组细胞质中Nrf2蛋白表达水平明显降低（P < 0.05），细胞核中Nrf2蛋白表达水平明显升高（P < 0.05）。培养48 h后，与对照组[（100.0±6.0）%]比较，高糖组细胞存活率[（73.9±2.2）%]明显降低（P < 0.05）；与高糖组比较，高虾青素组细胞存活率[（93.8±1.5）%]明显升高（P < 0.05）；与高虾青素组比较，高虾青素+ML385组细胞存活率[（71.7±2.7）%]明显降低（P值均 < 0.05）。与对照组、高虾青素组比较，高糖组细胞中p53、磷酸化p53、p21、p16、Rb、MMP1、MMP3、MMP13蛋白表达水平均明显升高（P < 0.05），磷酸化Rb蛋白表达水平明显降低（P值均 < 0.05）；与高虾青素组比较，高虾青素+ML385组细胞中p53、磷酸化p53、p21、p16、Rb、MMP1、MMP13蛋白表达水平均明显升高（P < 0.05），磷酸化Rb蛋白表达水平明显降低（P < 0.05）。与对照组比较，高糖组细胞质中Nrf2蛋白表达水平明显升高（P < 0.05）；与高糖组比较，高虾青素组细胞质中Nrf2蛋白表达水平明显降低（P < 0.05），细胞核中Nrf2蛋白表达水平明显升高（P < 0.05）；与高虾青素组比较，高虾青素+ML385组细胞核中Nrf2蛋白表达水平明显降低（P < 0.05）。
结论
虾青素通过调节Nrf2核位移抑制高糖处理的人皮肤Fb氧化应激反应，下调细胞衰老相关蛋白的表达，从而缓解细胞衰老。
Keywords: 细胞衰老, 氧化性应激, 基因，肿瘤抑制, 基质金属蛋白酶类, 细胞周期, 成纤维细胞, 虾青素, 核转录因子红系2相关因子2
Abstract
Objective
To investigate the effect and mechanism of astaxanthin on the aging of high glucose-treated human skin fibroblasts (Fbs).
Methods
The study was an experimental study. Human skin Fbs were collected and divided into control group with conventional culture, high glucose group treated with glucose at a final molarity of 30 mmol/L, and low astaxanthin group and high astaxanthin group pretreated with astaxanthin at final molarities of 25 and 50 μmol/L respectively and then treated as that in high glucose group. After 48 h of culture, the cell survival rate was detected by the cell counting kit-8, the reactive oxygen species level in cells was detected by the fluorescent probe method, the levels of malondialdehyde, glutathione, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in cells were detected by colorimetry, and the protein expression levels of p53, phosphorylated p53, p21, p16, Rb, phosphorylated Rb, matrix metalloproteinase 1 (MMP1), MMP3, and MMP13 in cells and nuclear factor-erythroid 2-related factor 2 (Nrf2) in cytoplasm and nucleus were detected by Western blotting. Another batch of human skin Fbs were collected and divided into control group, high glucose group, and high astaxanthin group which were treated as before, and high astaxanthin+ML385 group, which were pretreated with astaxanthin at a final molarity of 50 μmol/L and ML385 at a final molarity of 10 μmol/L respectively and then treated as that in high glucose group. After 48 h of culture, the cell survival rate, the protein expression levels of p53, phosphorylated p53, p21, p16, Rb, phosphorylated Rb, MMP1, MMP3, and MMP13 in cells and Nrf2 in cytoplasm and nucleus were detected as before. The sample number in all experiments above was 3.
Results
After 48 h of culture, compared with that in control group, the cell survival rate in high glucose group was significantly decreased (P < 0.05); compared with that in high glucose group, the cell survival rates in low astaxanthin group and high astaxanthin group were significantly increased (P < 0.05); compared with that in low astaxanthin group, the cell survival rate in high astaxanthin group was significantly increased (P < 0.05). Compared with those in control group, the levels of reactive oxygen species and malondialdehyde in cells were significantly increased (P < 0.05), while the levels of glutathione, SOD, CAT, and GSH-Px in cells were significantly decreased in high glucose group (P < 0.05). Compared with those in high glucose group, the levels of reactive oxygen species and malondialdehyde in cells were significantly decreased (P < 0.05), while the levels of CAT and GSH-Px in cells were significantly increased in low astaxanthin group (P < 0.05); the levels of reactive oxygen species and malondialdehyde in cells were significantly decreased (P < 0.05), while the levels of glutathione, SOD, CAT, and GSH-Px in cells were significantly increased in high astaxanthin group (P < 0.05). Compared with those in low astaxanthin group, the levels of reactive oxygen species and malondialdehyde in cells were significantly decreased (P < 0.05), while the levels of glutathione, CAT, and GSH-Px in cells were significantly increased in high astaxanthin group (P < 0.05). Compared with those in control group, the protein expression levels of p53, p21, phosphorylated p53, p16, Rb, MMP1, MMP3, and MMP13 in cells were significantly increased (P < 0.05), while the protein expression level of phosphorylated Rb in cells was significantly decreased in high glucose group (P < 0.05). Compared with those in high glucose group, the protein expression levels of p53, p21, phosphorylated p53, and MMP13 in cells were significantly decreased (P < 0.05), while the protein expression level of phosphorylated Rb in cells was significantly increased in low astaxanthin group (P < 0.05); the protein expression levels of p53, p21, phosphorylated p53, p16, Rb, MMP1, MMP3, and MMP13 in cells were significantly decreased (P < 0.05), while the protein expression level of phosphorylated Rb in cells was significantly increased in high astaxanthin group (P < 0.05). Compared with those in low astaxanthin group, the protein expression levels of p21, phosphorylated p53, p16, Rb, and MMP1 in cells in high astaxanthin group were significantly decreased (P < 0.05). Compared with those in control group and high astaxanthin group, the protein expression level of cytoplasmic Nrf2 was significantly increased (with P values both < 0.05), while the protein expression level of nuclear Nrf2 was significantly decreased in high glucose group (with P values both < 0.05). Compared with those in low astaxanthin group, the protein expression level of cytoplasmic Nrf2 was significantly increased in high glucose group (P < 0.05); the protein expression level of cytoplasmic Nrf2 was significantly decreased (P < 0.05), while the protein expression level of nuclear Nrf2 was significantly increased in high astaxanthin group (P < 0.05). After 48 h of culture, compared with (100.0±6.0)% in control group, the cell survival rate was significantly decreased in high glucose group ((73.9±2.2)%, P < 0.05); compared with that in high glucose group, the cell survival rate was significantly increased in high astaxanthin group ((93.8±1.5)%, P < 0.05); compared with that in high astaxanthin group, the cell survival rate was significantly decreased in high astaxanthin+ML385 group ((71.7±2.7)%, P < 0.05). Compared with those in control group and high astaxanthin group, the protein expression levels of p53, phosphorylated p53, p21, p16, Rb, MMP1, MMP3, and MMP13 in cells were significantly increased (P < 0.05), while the protein expression level of phosphorylated Rb in cells was significantly decreased in high glucose group (with P values both < 0.05); compared with those in high astaxanthin group, the protein expression levels of p53, phosphorylated p53, p21, p16, Rb, MMP1, and MMP13 in cells were significantly increased (P < 0.05), while the protein expression level of phosphorylated Rb in cells was significantly decreased in high astaxanthin+ML385 group (P < 0.05). Compared with that in control group, the protein expression level of cytoplasmic Nrf2 was significantly increased in high glucose group (P < 0.05); compared with those in high glucose group, the protein expression level of cytoplasmic Nrf2 was significantly decreased (P < 0.05), while the protein expression level of nuclear Nrf2 was significantly increased in high astaxanthin group (P < 0.05); compared with that in high astaxanthin group, the protein expression level of nuclear Nrf2 was significantly decreased in high astaxanthin+ML385 group (P < 0.05).
Conclusions
Astaxanthin can alleviate aging of high glucose-treated human skin Fbs by regulating Nrf2 nuclear translocation to inhibit oxidative stress and downregulate the expression of aging-related proteins.
Keywords: Cellular senescence; Oxidative stress; Genes, tumor suppressor; Matrix metalloproteinases; Cell cycle; Fibroblasts; Astaxanthin; Nuclear factor-erythroid 2-related factor 2
糖尿病是一种常见代谢紊乱性疾病[1-2]。持续性高血糖常导致创面愈合障碍，大约15%的糖尿病患者会发展为下肢慢性溃疡[3-4]，其中14%~24%的患者最终需要截肢[5]，对患者身心及社会经济造成沉重负担。
创面愈合是一个多阶段重叠的复杂过程，受氧化应激、炎症反应、胶原合成减少等多因素影响[6]。高糖环境引发的氧化应激被认为是驱动糖尿病创面难愈的核心环节，其通过产生大量活性氧破坏细胞功能、加剧细胞凋亡，并阻碍创面修复进程[7-8]。核转录因子红系2相关因子2（nuclear factor-erythroid 2-related factor 2，Nrf2）作为调控氧化还原平衡的关键转录因子，在拮抗氧化损伤中发挥核心作用，其活化可有效促进创面愈合[9-11]。虾青素是一种来源于藻类、细菌和真菌的类胡萝卜素[12-13]，具有极强的抗氧化活性，且能通过调节Nrf2信号通路增强细胞防御能力[14-15]。本实验研究通过采用高糖处理人皮肤Fb模拟糖尿病创面微环境，探讨虾青素是否通过调控Nrf2信号通路影响细胞衰老进程，为其在糖尿病创面治疗中的应用提供理论依据。
1. 材料与方法
1.1. 细胞及主要试剂与仪器来源
人皮肤Fb购自中国医学科学院基础医学研究所细胞资源中心。胎牛血清、DMEM培养基、二氧化碳培养箱、Multiskan FC型酶标仪购自美国赛默飞公司，细胞计数试剂盒-8、2'，7'-二氯二氢荧光素二乙酸酯荧光探针、丙二醛检测试剂盒、谷胱甘肽检测试剂盒、SOD检测试剂盒、过氧化氢酶（catalase，CAT）检测试剂盒、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）检测试剂盒购自北京索莱宝科技有限公司，小鼠源性p53、磷酸化p53、p21、p16、Rb、磷酸化Rb、基质金属蛋白酶1（matrix metalloproteinase 1，MMP1）、MMP3、MMP13、Nrf2、GAPDH、β肌动蛋白、组蛋白H3单克隆抗体及辣根过氧化物酶标记的山羊抗小鼠IgG单克隆抗体购自艾博抗（上海）贸易有限公司，Nrf2抑制剂ML385购自上海阿拉丁生化科技股份有限公司。LSM 880NLO型双光子激光共聚焦荧光显微镜购自德国卡尔蔡司公司，Tanon 5200系列全自动化学发光仪购自上海天能生命科学有限公司。
1.2. 虾青素对高糖处理的细胞的影响
1.2.1. 细胞分组与处理
将人Fb以每孔1×105个接种于6孔板中，每孔加入2 mL DMEM培养基，待细胞生长达50%融合时，将细胞分成对照组、高糖组、低虾青素组、高虾青素组。对照组细胞用DMEM培养基常规培养，高糖组细胞用含终物质的量浓度30 mmol/L葡萄糖的DMEM培养基培养[16]，低虾青素组、高虾青素组细胞分别用含终物质的量浓度25、50 μmol/L虾青素的DMEM培养基预处理12 h后再同高糖组细胞处理[17]。将各组细胞置于37 ℃、含体积分数5%二氧化碳的培养箱中培养48 h后进行后续实验。所有实验样本数均为3。
1.2.2. 细胞存活率检测
取4组细胞，以每孔1×104个接种于96孔板中，每孔加入10 μL细胞计数试剂盒-8溶液，37 ℃下避光孵育2 h后，使用酶标仪测定波长450 nm处的吸光度值，以此反映细胞存活情况。以对照组其中1个样本中细胞存活率为100%，计算其他3组细胞存活率。
1.2.3. 细胞氧化损伤相关指标水平检测
取4组细胞，以每孔1×104个接种于96孔板中，加入2'，7'-二氯二氢荧光素二乙酸酯荧光探针，用DMEM培养基洗涤细胞3次去除游离探针及水解产物，于激光共聚焦荧光显微镜100倍放大倍数下观察并拍照，用ImageJ图像分析软件（美国国立卫生研究院）分析荧光强度，反映活性氧水平（绿色荧光）。按照相应试剂盒说明书，采用比色法于酶标仪中检测细胞中丙二醛、谷胱甘肽、SOD、CAT、GSH-Px水平。
1.2.4. 细胞衰老相关蛋白表达水平检测
提取4组细胞的总蛋白，采用蛋白质印迹法检测细胞中p53、磷酸化p53、p21、p16、Rb、磷酸化Rb蛋白表达水平，一抗为小鼠源性p53、磷酸化p53、p21、p16、Rb、磷酸化Rb、β肌动蛋白单克隆抗体（稀释比均为1∶1 000），二抗为辣根过氧化物酶标记的山羊抗小鼠IgG单克隆抗体（稀释比为1∶10 000）。另提取4组细胞的总蛋白，同前检测细胞中MMP1、MMP3、MMP13蛋白表达水平，一抗为小鼠源性MMP1、MMP3、MMP13、GAPDH单克隆抗体（稀释比均为1∶1 000），二抗为辣根过氧化物酶标记的山羊抗小鼠IgG单克隆抗体（稀释比为1∶10 000）。另提取4组细胞的细胞质和细胞核蛋白，同前检测细胞质和细胞核中Nrf2蛋白表达水平，一抗为小鼠源性Nrf2、β肌动蛋白、组蛋白H3单克隆抗体（稀释比均为1∶1 000），二抗为辣根过氧化物酶标记的山羊抗小鼠IgG单克隆抗体（稀释比为1∶10 000）。采用全自动化学发光仪对目的蛋白显影，用ImageJ图像分析软件分析蛋白条带灰度值。以β肌动蛋白为内参照，检测细胞中p53、磷酸化p53、p21、p16、Rb、磷酸化Rb和细胞质中Nrf2蛋白表达水平；以GAPDH为内参照，检测细胞中MMP1、MMP3、MMP13蛋白表达水平；以组蛋白H3为内参照，检测细胞核中Nrf2蛋白表达水平。计算目的蛋白的相对表达水平。
1.3. 抑制Nrf2核位移后虾青素对高糖处理的细胞的影响
另取人Fb，分成对照组、高糖组、高虾青素组、高虾青素+ML385组，前3组细胞同1.2.1中相同组别进行处理。高虾青素+ML385组细胞用含终物质的量浓度为50 μmol/L的虾青素和10 μmol/L的ML385的DMEM培养基预处理12 h后再同高糖组细胞处理[18]。同1.2.2检测4组细胞存活率，同1.2.4检测4组细胞中p53、磷酸化p53、p21、p16、Rb、磷酸化Rb、MMP1、MMP3、MMP13蛋白表达水平及细胞质和细胞核中Nrf2蛋白表达水平。
1.4. 统计学处理
采用SPSS 26.0统计软件进行数据分析。计量资料数据均符合正态分布，以 x±s表示。多组间总体比较行单因素方差分析，组间多重比较行Bonferroni校正。P < 0.05为差异有统计学意义。
2. 结果
2.1. 虾青素对高糖处理的细胞的影响
2.1.1. 细胞存活率
培养48 h后，对照组、高糖组、低虾青素组、高虾青素组细胞存活率分别为（100.0±5.7）%、（75.3±1.7）%、（83.8±2.8）%、（94.8±2.5）%，组间总体比较，差异有统计学意义（F=30.13，P < 0.001）。与对照组比较，高糖组细胞存活率明显降低（P < 0.001）；与高糖组比较，低虾青素组和高虾青素组细胞存活率均明显升高（P < 0.001）；与低虾青素组比较，高虾青素组细胞存活率明显升高（P=0.031）。
2.1.2. 细胞氧化损伤相关指标水平
培养48 h后，与对照组比较，高糖组细胞中活性氧、丙二醛水平均明显升高（P < 0.05），谷胱甘肽、SOD、CAT、GSH-Px水平均明显降低（P < 0.05）。与高糖组比较，低虾青素组细胞中活性氧、丙二醛水平均明显降低（P < 0.05），CAT、GSH-Px水平均明显升高（P < 0.05）；高虾青素组细胞中活性氧、丙二醛水平均明显降低（P < 0.05），谷胱甘肽、SOD、CAT、GSH-Px水平均明显升高（P < 0.05）。与低虾青素组比较，高虾青素组细胞中活性氧、丙二醛水平均明显降低（P < 0.05），谷胱甘肽、CAT、GSH-Px水平均明显升高（P < 0.05）。见图 1、表 1。
图 1.
4组人成纤维细胞培养48 h后的活性氧水平2'，7'-二氯二氢荧光素二乙酸酯×100。1A、1B、1C、1D.分别为对照组、高糖组、低虾青素组、高虾青素组，图 1B活性氧水平明显高于图 1A、1C、1D，图 1C活性氧水平明显高于图 1D
The reactive oxygen species level of human fibroblasts in 4 groups after 48 h of culture
注：对照组细胞行常规培养，高糖组细胞用终物质的量浓度30 mmol/L葡萄糖处理，低虾青素组、高虾青素组细胞分别用终物质的量浓度25、50 μmol/L虾青素预处理后再同高糖组处理；绿色荧光表示活性氧
表 1.
4组人成纤维细胞培养48 h后氧化损伤相关指标水平比较（x±s）
Comparison of the levels of indicators related to oxidative damage of human fibroblasts in 4 groups after 48 h of culture
| 组别 | 样本数 | 活性氧 | 丙二醛 | 谷胱甘肽 | SOD | CAT | GSH-Px |
| 注：对照组细胞行常规培养，高糖组细胞用终物质的量浓度30 mmol/L葡萄糖处理，低虾青素组、高虾青素组细胞分别用终物质的量浓度25、50 μmol/L虾青素预处理后再同高糖组处理；SOD为超氧化物歧化酶，CAT为过氧化氢酶，GSH-Px为谷胱甘肽过氧化物酶；F值、P值为组间各指标总体比较所得；P1值、P2值、P3值、P4值分别为对照组与高糖组、高糖组与低虾青素组、高糖组与高虾青素组、低虾青素组与高虾青素组各指标比较所得 | |||||||
| 对照组 | 3 | 3.3±0.6 | 4.0±0.6 | 22.0±3.1 | 30.3±4.6 | 37.0±1.3 | 181±12 |
| 高糖组 | 3 | 29.6±1.0 | 11.4±1.1 | 7.8±2.3 | 11.3±1.0 | 15.4±3.3 | 74±4 |
| 低虾青素组 | 3 | 20.8±1.4 | 7.7±0.6 | 14.4±0.6 | 17.5±1.1 | 23.4±2.4 | 108±9 |
| 高虾青素组 | 3 | 7.6±0.7 | 4.7±0.4 | 21.7±3.0 | 24.4±2.7 | 36.4±0.8 | 173±8 |
| F值 | 473.30 | 62.48 | 22.74 | 27.39 | 69.06 | 105.40 | |
| P值 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | |
| P1值 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | |
| P2值 | < 0.001 | 0.002 | 0.066 | 0.144 | 0.013 | 0.008 | |
| P3值 | < 0.001 | < 0.001 | < 0.001 | 0.002 | < 0.001 | < 0.001 | |
| P4值 | < 0.001 | 0.007 | 0.040 | 0.091 | < 0.001 | < 0.001 | |
2.1.3. 细胞衰老相关蛋白表达水平
培养48 h后，与对照组比较，高糖组细胞中p53、p21、磷酸化p53、p16、Rb、MMP1、MMP3、MMP13蛋白表达水平均明显升高（P < 0.05），磷酸化Rb蛋白表达水平明显降低（P < 0.05）。与高糖组比较，低虾青素组细胞中p53、p21、磷酸化p53、MMP13蛋白表达水平均明显降低（P < 0.05），磷酸化Rb蛋白表达水平明显升高（P < 0.05）；高虾青素组细胞中p53、p21、磷酸化p53、p16、Rb、MMP1、MMP3、MMP13蛋白表达水平均明显降低（P < 0.05），磷酸化Rb蛋白表达水平明显升高（P < 0.05）。与低虾青素组比较，高虾青素组细胞中p21、磷酸化p53、p16、Rb、MMP1蛋白表达水平均明显降低（P < 0.05）。见图 2、表 2。
图 2.
蛋白质印迹法检测的4组人成纤维细胞培养48 h后部分衰老相关蛋白表达水平。2A.细胞中p53、磷酸化p53、p21、p16、Rb、磷酸化Rb；2B.细胞中MMP1、MMP3、MMP13
The protein expression levels of certain aging-related proteins of human fibroblasts in 4 groups after 48 h of culture detected by Western blotting
注：条带上方1~3、4~6、7~9、10~12分别指示对照组、高糖组、低虾青素组、高虾青素组；对照组细胞行常规培养，高糖组细胞用终物质的量浓度30 mmol/L葡萄糖处理，低虾青素组、高虾青素组细胞分别用终物质的量浓度25、50 μmol/L虾青素预处理后再同高糖组处理；MMP为基质金属蛋白酶，GAPDH为3-磷酸甘油醛脱氢酶
表 2.
4组人成纤维细胞培养48 h后部分衰老相关蛋白表达水平比较（x±s）
Comparison of the protein expression levels of certain aging-related proteins of human fibroblasts in 4 groups after 48 h of culture
| 组别 | 样本数 | p53 | p21 | 磷酸化p53 | p16 | Rb | 磷酸化Rb | MMP1 | MMP3 | MMP13 |
| 注：对照组细胞行常规培养，高糖组细胞用终物质的量浓度30 mmol/L葡萄糖处理，低虾青素组、高虾青素组细胞分别用终物质的量浓度25、50 μmol/L的虾青素预处理后再同高糖组处理；F值、P值为组间各指标总体比较所得；P1值、P2值、P3值、P4值分别为对照组与高糖组、高糖组与低虾青素组、高糖组与高虾青素组、低虾青素组与高虾青素组各指标比较所得；MMP为基质金属蛋白酶 | ||||||||||
| 对照组 | 3 | 1.00±0.05 | 1.00±0.08 | 1.00±0.26 | 1.00±0.12 | 1.000±0.127 | 1.00±0.12 | 1.00±0.19 | 1.00±0.29 | 1.00±0.13 |
| 高糖组 | 3 | 4.39±0.17 | 5.30±0.15 | 6.95±0.11 | 3.71±0.36 | 1.915±0.186 | 0.36±0.05 | 2.71±0.45 | 2.37±0.13 | 2.20±0.23 |
| 低虾青素组 | 3 | 3.29±0.24 | 3.53±0.10 | 3.69±0.23 | 3.61±0.30 | 1.838±0.154 | 0.96±0.04 | 2.00±0.35 | 1.69±0.37 | 1.38±0.27 |
| 高虾青素组 | 3 | 3.35±0.57 | 1.01±0.45 | 0.94±0.14 | 1.05±0.17 | 0.868±0.190 | 0.87±0.05 | 0.88±0.38 | 1.13±0.33 | 1.08±0.11 |
| F值 | 59.29 | 221.48 | 630.87 | 106.64 | 32.50 | 51.87 | 17.66 | 13.49 | 23.52 | |
| P值 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | 0.002 | < 0.001 | |
| P1值 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | 0.002 | 0.003 | < 0.001 | |
| P2值 | 0.018 | < 0.001 | < 0.001 | > 0.999 | > 0.999 | < 0.001 | 0.246 | 0.127 | 0.005 | |
| P3值 | 0.025 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | 0.001 | 0.005 | < 0.001 | |
| P4值 | > 0.999 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | 0.870 | 0.030 | 0.305 | 0.601 | |
培养48 h后，与对照组、高虾青素组比较，高糖组细胞质中Nrf2蛋白表达水平明显升高（P值均 < 0.05），细胞核中Nrf2蛋白表达水平明显降低（P值均 < 0.05）。与低虾青素组比较，高糖组细胞质中Nrf2蛋白表达水平明显升高（P < 0.05）；高虾青素组细胞质中Nrf2蛋白表达水平明显降低（P < 0.05），细胞核中Nrf2蛋白表达水平明显升高（P < 0.05）。见图 3、表 3。
图 3.
蛋白质印迹法检测的4组人成纤维细胞培养48 h后细胞质和细胞核中Nrf2蛋白表达水平。3A.细胞质；3B.细胞核
The protein expression levels of Nrf2 in cytoplasm and nucleus of human fibroblasts in 4 groups after 48 h of culture detected by Western blotting
注：Nrf2为核转录因子红系2相关因子2；条带上方1~3、4~6、7~9、10~12分别指示对照组、高糖组、低虾青素组、高虾青素组；对照组细胞行常规培养，高糖组细胞用终物质的量浓度30 mmol/L葡萄糖处理，低虾青素组、高虾青素组细胞分别用终物质的量浓度25、50 μmol/L虾青素预处理后再同高糖组处理
表 3.
4组人成纤维细胞培养48 h后细胞质和细胞核中Nrf2蛋白表达水平比较（x±s）
Comparison of the protein expression levels of Nrf2 in cytoplasm and nucleus of human fibroblasts in 4 groups after 48 h of culture
| 组别 | 样本数 | 细胞质中Nrf2 | 细胞核中Nrf2 |
| 注：对照组细胞行常规培养，高糖组细胞用终物质的量浓度30 mmol/L葡萄糖处理，低虾青素组、高虾青素组细胞分别用终物质的量浓度25、50 μmol/L虾青素预处理后再同高糖组处理；Nrf2为核转录因子红系2相关因子2；F值、P值为组间各指标总体比较所得；P1值、P2值、P3值、P4值分别为对照组与高糖组、高糖组与低虾青素组、高糖组与高虾青素组、低虾青素组与高虾青素组各指标比较所得 | |||
| 对照组 | 3 | 1.000±0.147 | 1.000±0.018 |
| 高糖组 | 3 | 1.717±0.144 | 0.585±0.097 |
| 低虾青素组 | 3 | 0.906±0.206 | 0.795±0.056 |
| 高虾青素组 | 3 | 0.397±0.077 | 1.139±0.141 |
| F值 | 39.04 | 21.59 | |
| P值 | < 0.001 | < 0.001 | |
| P1值 | 0.002 | 0.003 | |
| P2值 | 0.001 | 0.130 | |
| P3值 | < 0.001 | < 0.001 | |
| P4值 | 0.020 | 0.010 | |
2.2. 抑制Nrf2核位移后虾青素对高糖处理的细胞的影响
2.2.1. 细胞存活率
培养48 h后，对照组、高糖组、高虾青素组、高虾青素+ML385组细胞存活率分别为（100.0±6.0）%、（73.9±2.2）%、（93.8±1.5）%、（71.7±2.7）%（F=48.22，P < 0.001）。与对照组比较，高糖组细胞存活率明显降低（P < 0.001）；与高糖组比较，高虾青素组细胞存活率明显升高（P < 0.001）；与高虾青素组比较，高虾青素+ML385组细胞存活率明显降低（P < 0.001）。
2.2.2. 细胞衰老相关蛋白表达水平
培养48 h后，与对照组、高虾青素组比较，高糖组细胞中p53、磷酸化p53、p21、p16、Rb、MMP1、MMP3、MMP13蛋白表达水平均明显升高（P < 0.05），磷酸化Rb蛋白表达水平明显降低（P值均 < 0.05）；与高虾青素组比较，高虾青素+ML385组细胞中p53、磷酸化p53、p21、p16、Rb、MMP1、MMP13蛋白表达水平均明显升高（P < 0.05），磷酸化Rb蛋白表达水平明显降低（P < 0.05）。见图 4、表 4。
图 4.
蛋白质印迹法检测的4组人成纤维细胞培养48 h后部分衰老相关蛋白表达水平。4A.细胞中p53、磷酸化p53、p21、p16、Rb、磷酸化Rb；4B.细胞中MMP1、MMP3、MMP13
The protein expression levels of certain aging-related proteins of human fibroblasts in 4 groups after 48 h of culture detected by Western blotting
注：条带上方1~3、4~6、7~9、10~12分别指示对照组、高糖组、高虾青素组、高虾青素+ML385组；对照组细胞行常规培养，高糖组细胞用终物质的量浓度30 mmol/L葡萄糖处理，高虾青素组和高虾青素+ML385组细胞分别用终物质的量浓度50 μmol/L虾青素、终物质的量浓度50 μmol/L虾青素+10 μmol/L ML385预处理后再同高糖组处理；MMP为基质金属蛋白酶，GAPDH为3-磷酸甘油醛脱氢酶
表 4.
4组人成纤维细胞培养48 h后部分衰老相关蛋白表达水平比较（x±s）
Comparison of the protein expression levels of certain aging-related proteins of human fibroblasts in 4 groups after 48 h of culture
| 组别 | 样本数 | p53 | p21 | 磷酸化p53 | p16 | Rb | 磷酸化Rb | MMP1 | MMP3 | MMP13 |
| 注：对照组细胞行常规培养，高糖组细胞用终物质的量浓度30 mmol/L葡萄糖处理，高虾青素组和高虾青素+ML385组细胞分别用终物质的量浓度50 μmol/L虾青素、终物质的量浓度50 μmol/L虾青素+10 μmol/L ML385预处理后再同高糖组处理；F值、P值为组间各指标总体比较所得；P1值、P2值、P3值分别为对照组与高糖组、高糖组与高虾青素组、高虾青素组与高虾青素+ML385组各指标比较所得；MMP为基质金属蛋白酶 | ||||||||||
| 对照组 | 3 | 1.00±0.07 | 1.00±0.21 | 1.00±0.03 | 1.00±0.26 | 1.000±0.061 | 1.00±0.10 | 1.00±0.27 | 1.00±0.12 | 1.00±0.12 |
| 高糖组 | 3 | 3.31±0.36 | 7.30±1.04 | 2.46±0.12 | 2.97±0.13 | 4.175±0.070 | 0.38±0.04 | 5.24±0.30 | 5.14±0.12 | 4.53±0.29 |
| 高虾青素组 | 3 | 2.16±0.56 | 2.60±0.10 | 0.90±0.09 | 1.29±0.27 | 1.036±0.008 | 0.91±0.09 | 1.54±0.22 | 1.55±0.37 | 1.24±0.34 |
| 高虾青素+ML385组 | 3 | 4.97±0.29 | 6.54±0.77 | 2.15±0.40 | 2.27±0.22 | 3.371±0.489 | 0.40±0.10 | 4.10±0.43 | 3.82±1.56 | 2.75±0.81 |
| F值 | 63.55 | 64.54 | 41.77 | 48.19 | 128.00 | 42.20 | 122.50 | 17.26 | 36.44 | |
| P值 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | |
| P1值 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | 0.001 | < 0.001 | |
| P2值 | 0.031 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | 0.004 | < 0.001 | |
| P3值 | < 0.001 | < 0.001 | < 0.001 | 0.004 | < 0.001 | < 0.001 | < 0.001 | 0.053 | 0.025 | |
培养48 h后，与对照组比较，高糖组细胞质中Nrf2蛋白表达水平明显升高（P < 0.05）；与高糖组比较，高虾青素组细胞质中Nrf2蛋白表达水平明显降低（P < 0.05），细胞核中Nrf2蛋白表达水平明显升高（P < 0.05）；与高虾青素组比较，高虾青素+ML385组细胞核中Nrf2蛋白表达水平明显降低（P < 0.05）。见图 5、表 5。
图 5.
蛋白质印迹法检测的4组人成纤维细胞培养48 h后细胞质和细胞核中Nrf2蛋白表达水平。5A.细胞质；5B.细胞核
The protein expression levels of Nrf2 in cytoplasm and nucleus of human fibroblasts in 4 groups after 48 h of culture detected by Western blotting
注：Nrf2为核转录因子红系2相关因子2；条带上方1~3、4~6、7~9、10~12分别指示对照组、高糖组、高虾青素组、高虾青素+ML385组；对照组细胞行常规培养，高糖组细胞用终物质的量浓度30 mmol/L葡萄糖处理，高虾青素组和高虾青素+ML385组细胞分别用终物质的量浓度50 μmol/L虾青素、终物质的量浓度50 μmol/L虾青素+10 μmol/L ML385预处理后再同高糖组处理
表 5.
4组人成纤维细胞培养48 h后细胞质和细胞核中Nrf2蛋白表达水平比较（x±s）
Comparison of the protein expression levels of Nrf2 in cytoplasm and nucleus of human fibroblasts in 4 groups after 48 h of culture
| 组别 | 样本数 | 细胞质中Nrf2 | 细胞核中Nrf2 |
| 注：对照组细胞行常规培养，高糖组细胞用终物质的量浓度30 mmol/L葡萄糖处理，高虾青素组和高虾青素+ML385组细胞分别用终物质的量浓度50 μmol/L虾青素、终物质的量浓度50 μmol/L虾青素+10 μmol/L ML385预处理后再同高糖组处理；Nrf2为核转录因子红系2相关因子2；F值、P值为组间各指标总体比较所得；P1值、P2值、P3值分别为对照组与高糖组、高糖组与高虾青素组、高虾青素组与高虾青素+ML385组各指标比较所得 | |||
| 对照组 | 3 | 1.000±0.172 | 1.000±0.030 |
| 高糖组 | 3 | 1.983±0.311 | 0.931±0.187 |
| 高虾青素组 | 3 | 0.917±0.275 | 1.494±0.129 |
| 高虾青素+ML385组 | 3 | 1.513±0.077 | 0.969±0.103 |
| F值 | 14.19 | 13.34 | |
| P值 | 0.001 | 0.002 | |
| P1值 | 0.004 | 0.999 | |
| P2值 | 0.003 | 0.004 | |
| P3值 | 0.075 | 0.005 | |
3. 讨论
糖尿病创面愈合延迟与高血糖引发的氧化应激密切相关。高糖环境通过多种途径促进活性氧大量生成，引起脂质过氧化，同时消耗SOD等抗氧化物质，破坏氧化-抗氧化平衡[19-22]，进而直接损伤创面修复细胞，抑制其增殖、迁移并促进凋亡[23-24]。
在糖尿病创面微环境中，氧化应激诱发DNA损伤可激活p53信号，上调p21表达。p21通过抑制周期蛋白依赖性激酶4和周期蛋白依赖性激酶6与细胞周期蛋白D形成的复合物，以及周期蛋白依赖性激酶2与细胞周期蛋白E形成的复合物的活性，从而阻滞细胞周期[25-31]。同时，p16协同增强该效应，维持Rb蛋白低磷酸化状态，阻碍细胞G1/S期转换[32-34]。周期停滞细胞转化为衰老细胞，分泌MMP1、MMP3等蛋白酶，破坏ECM，最终阻碍肉芽组织形成与再上皮化，导致创面迁延不愈[35-36]。
本研究表明糖尿病创面的高糖环境使细胞存活率降低，细胞中活性氧、丙二醛水平及p53、磷酸化p53、p21、p16、Rb、MMP1、MMP3、MMP13蛋白表达水平均明显升高，而谷胱甘肽、SOD、CAT、GSH-Px水平均明显降低，表明高糖环境产生的氧化应激反应会破坏细胞周期，阻碍细胞完成自我修复，进而降低细胞抗氧化能力，诱导细胞衰老，加快细胞凋亡进程。
虾青素是一种强效酮式类胡萝卜素，其独特的共轭双键结构具备卓越的氧化还原能力，可保护线粒体并提高能量生产效率[37]。在分子机制上，虾青素通过修饰Kelch样ECH相关蛋白1的半胱氨酸残基，激活Nrf2和抗氧化反应元件信号通路，促进Nrf2入核，进而上调多种抗氧化酶表达，增强细胞整体抗氧化能力，维持细胞稳态与功能[38-40]。上述机制表明虾青素对糖尿病创面具有潜在治疗价值，但其保护创面Fb抵抗氧化损伤的具体机制仍有待进一步明确。
本研究首先对Fb进行高糖处理，再以不同浓度虾青素进行干预，检测细胞存活率、氧化损伤指标及衰老相关蛋白表达水平。结果显示，虾青素能明显提高高糖条件下细胞的存活率，上调衰老相关蛋白表达水平，逆转细胞周期停滞；同时降低细胞中活性氧和丙二醛水平，并提升谷胱甘肽、CAT和GSH-Px等抗氧化因子的表达。进一步研究显示，虾青素可激活Nrf2信号通路，表现为随着其浓度增加，Nrf2从Kelch样ECH相关蛋白1-Nrf2复合物中解离并转移至细胞核，提示虾青素促进了Nrf2核移位，可能与其缓解氧化应激有关。为验证Nrf2信号通路是否介导虾青素的保护作用，本研究联合使用Nrf2抑制剂ML385与高浓度虾青素预处理细胞。结果显示，ML385明显抑制了Nrf2核转移，并伴随衰老相关蛋白表达上升，说明ML385通过阻断Nrf2信号通路削弱了虾青素的抗凋亡与抗衰老作用。
本研究结果表明，糖尿病创面愈合延迟与高糖诱导的氧化应激密切相关，其通过激活p53、p21和p16信号通路促使细胞周期阻滞与衰老，从而导致组织修复受阻。虾青素作为一种天然抗氧化剂，通过促进Nrf2核转位激活Nrf2-抗氧化反应元件信号通路，增强细胞内抗氧化防御能力，并逆转高糖所致的周期停滞与细胞衰老。本研究从细胞周期调控与抗氧化协同角度，揭示了虾青素在糖尿病创面治疗中的潜在价值，为后续开发以Nrf2为靶点的治疗策略提供了实验依据。
Funding Statement
国家临床重点专科建设项目（Z155080000004）；上海市康复医学重中之重研究中心（2023ZZ02027）
National Key Clinical Specialty Discipline Construction Project of China (Z155080000004); Shanghai Research Center of Rehabilitation Medicine (Top Priority Research Center of Shanghai) (2023ZZ02027)
本文亮点
(1) 证明虾青素作为一种强效天然抗氧化剂，可有效减轻高糖引起的人皮肤成纤维细胞衰老。
(2) 揭示了虾青素通过激活核转录因子红系2相关因子2抗氧化信号通路进而影响衰老相关蛋白的表达，为针对糖化衰老的干预策略提供了新思路。
Highlights
(1) It was confirmed that astaxanthin, as a potent natural antioxidant, could effectively alleviate high glucose-induced aging of human skin fibroblasts.
(2) It was revealed that through activating the nuclear factor-erythroid 2-related factor 2 antioxidant signaling pathway, astaxanthin thereby influenced the expression of aging-related proteins, providing new insights for intervention strategies targeting glycation-induced aging.
利益冲突 所有作者声明不存在利益冲突
作者贡献声明 唐黎珺：实验操作、论文撰写；樊亮花、王静荣、罗蒙：统计学分析；杨小鑫、仲瑞青：指导论文撰写；高红艳：研究指导、论文修改、经费支持
References
- 1.Tomic D, Shaw JE, Magliano DJ. The burden and risks of emerging complications of diabetes mellitus. Nat Rev Endocrinol. 2022;18(9):525–539. doi: 10.1038/s41574-022-00690-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.李 威杰, 秦 晓光, 朱 甜, et al. 减重与代谢外科新进展. 中华消化外科杂志. 2023;22(8):958–964. doi: 10.3760/cma.j.cn115610-20230711-00399. [DOI] [Google Scholar]
- 3.Burgess JL, Wyant WA, Abdo Abujamra B, et al. Diabetic wound-healing science. Medicina (Kaunas) 2021;57(10):1072. doi: 10.3390/medicina57101072. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Wolf SJ, Melvin WJ, Gallagher K. Macrophage-mediated inflammation in diabetic wound repair. Semin Cell Dev Biol. 2021;119:111–118. doi: 10.1016/j.semcdb.2021.06.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Stone A, Donohue CM. Diabetic foot ulcers in geriatric patients. Clin Geriatr Med. 2024;40(3):437–447. doi: 10.1016/j.cger.2024.03.002. [DOI] [PubMed] [Google Scholar]
- 6.Wilkinson HN, Hardman MJ. Wound healing: cellular mechanisms and pathological outcomes. Open Biol. 2020;10(9):200223. doi: 10.1098/rsob.200223. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Deng L, Du C, Song P, et al. The role of oxidative stress and antioxidants in diabetic wound healing. Oxid Med Cell Longev. 2021;2021:8852759. doi: 10.1155/2021/8852759. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Mu X, Wu X, He W, et al. Pyroptosis and inflammasomes in diabetic wound healing. Front Endocrinol (Lausanne) 2022;13:950798. doi: 10.3389/fendo.2022.950798. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Neagu M, Constantin C, Surcel M, et al. Diabetic neuropathy: a NRF2 disease? J Diabetes. 2024;16(9):e13524. doi: 10.1111/1753-0407.13524. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Zhang DD. Thirty years of NRF2: advances and therapeutic challenges. Nat Rev Drug Discov. 2025;24(6):421–444. doi: 10.1038/s41573-025-01145-0. [DOI] [PubMed] [Google Scholar]
- 11.郭 佳, 张 军霞. 核转录因子红系2相关因子2在创面愈合中的作用研究进展. 中华烧伤与创面修复杂志. 2023;39(1):91–95. doi: 10.3760/cma.j.cn501225-20220531-00209. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Urakaze M, Kobashi C, Satou Y, et al. The beneficial effects of astaxanthin on glucose metabolism and modified low-density lipoprotein in healthy volunteers and subjects with prediabetes. Nutrients. 2021;13(12):4381. doi: 10.3390/nu13124381. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Nishida Y, Berg PC, Shakersain B, et al. Astaxanthin: past, present, and future. Mar Drugs. 2023;21(10):514. doi: 10.3390/md21100514. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Zhang Q, Luo C, Li Z, et al. Astaxanthin activates the Nrf2/Keap1/HO-1 pathway to inhibit oxidative stress and ferroptosis, reducing triphenyl phosphate (TPhP) -induced neurodevelopmental toxicity. Ecotoxicol Environ Saf. 2024;271:115960. doi: 10.1016/j.ecoenv.2024.115960. [DOI] [PubMed] [Google Scholar]
- 15.Hafez MH, El-Far AH, Elblehi SS. Astaxanthin alleviates fipronil-induced neuronal damages in male rats through modulating oxidative stress, apoptosis, and inflammatory markers. Sci Rep. 2025;15(1):14299. doi: 10.1038/s41598-025-95447-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Zhu Y, Ruan CX, Wang J, et al. High glucose inhibits the survival of HRMCs and its mechanism. Eur Rev Med Pharmacol Sci. 2022;26(16):5683–5688. doi: 10.26355/eurrev_202208_29502. [DOI] [PubMed] [Google Scholar]
- 17.Zhang Z, Qiu Y, Li W, et al. Astaxanthin alleviates foam cell formation and promotes cholesterol efflux in ox-LDL-induced RAW264.7 cells via circTPP2/miR-3073b-5p/ABCA1 pathway. Molecules. 2023;28(4):1701. doi: 10.3390/molecules28041701. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Yang CS, Guo XS, Yue YY, et al. Astaxanthin promotes the survival of adipose-derived stem cells by alleviating oxidative stress via activating the Nrf2 signaling pathway. Int J Mol Sci. 2023;24(4):3850. doi: 10.3390/ijms24043850. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Ju CC, Liu XX, Liu LH, et al. Epigenetic modification: a novel insight into diabetic wound healing. Heliyon. 2024;10(6):e28086. doi: 10.1016/j.heliyon.2024.e28086. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Wang G, Yang F, Zhou W, et al. The initiation of oxidative stress and therapeutic strategies in wound healing. Biomed Pharmacother. 2023;157:114004. doi: 10.1016/j.biopha.2022.114004. [DOI] [PubMed] [Google Scholar]
- 21.Liu Y, Liu Y, He W, et al. Fibroblasts: immunomodulatory factors in refractory diabetic wound healing. Front Immunol. 2022;13:918223. doi: 10.3389/fimmu.2022.918223. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Feng J, Wang J, Wang Y, et al. Oxidative stress and lipid peroxidation: prospective associations between ferroptosis and delayed wound healing in diabetic ulcers. Front Cell Dev Biol. 2022;10:898657. doi: 10.3389/fcell.2022.898657. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Huang K, Mi B, Xiong Y, et al. Angiogenesis during diabetic wound repair: from mechanism to therapy opportunity[J/OL]. Burns Trauma, 2025, 13: tkae052[2025-08-18]. https://pubmed.ncbi.nlm.nih.gov/39927093/. DOI: 10.1093/burnst/tkae052.
- 24.Citrin KM, Chaube B, Fernández-Hernando C, et al. Intracellular endothelial cell metabolism in vascular function and dysfunction. Trends Endocrinol Metab. 2025;36(8):744–755. doi: 10.1016/j.tem.2024.11.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Gasek NS, Yan P, Zhu J, et al. Clearance of p21 highly expressing senescent cells accelerates cutaneous wound healing. Nat Aging. 2025;5(1):21–27. doi: 10.1038/s43587-024-00755-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Zhang S, Meng N, Liu S, et al. Targeting senescent HDF with the USP7 inhibitor P5091 to enhance DFU wound healing through the p53 pathway. Biochem Biophys Res Commun. 2024;722:150149. doi: 10.1016/j.bbrc.2024.150149. [DOI] [PubMed] [Google Scholar]
- 27.Samarawickrama PN, Zhang G, Zhu E, et al. Clearance of senescent cells enhances skin wound healing in type 2 diabetic mice. Theranostics. 2024;14(14):5429–5442. doi: 10.7150/thno.100991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.McElhinney K, Irnaten M, O'Brien C. P53 and myofibroblast apoptosis in organ fibrosis. Int J Mol Sci. 2023;24(7):6737. doi: 10.3390/ijms24076737. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Ma Z, Ding Y, Ding X, et al. PDK4 rescues high-glucose-induced senescent fibroblasts and promotes diabetic wound healing through enhancing glycolysis and regulating YAP and JNK pathway. Cell Death Discov. 2023;9(1):424. doi: 10.1038/s41420-023-01725-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Qin Y, Wu K, Zhang Z, et al. NLRC3 deficiency promotes cutaneous wound healing due to the inhibition of p53 signaling. Biochim Biophys Acta Mol Basis Dis. 2022;1868(11):166518. doi: 10.1016/j.bbadis.2022.166518. [DOI] [PubMed] [Google Scholar]
- 31.Jiang G, Jiang T, Chen J, et al. Mitochondrial dysfunction and oxidative stress in diabetic wound. J Biochem Mol Toxicol. 2023;37(7):e23407. doi: 10.1002/jbt.23407. [DOI] [PubMed] [Google Scholar]
- 32.Qin Y, Liu H, Wu H. Cellular senescence in health, disease, and lens aging. Pharmaceuticals (Basel) 2025;18(2):244. doi: 10.3390/ph18020244. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Choi H, Kang C. Living beyond restriction: LBR promotes cellular immortalization by suppressing genomic instability and senescence. FEBS J. 2024;291(10):2091–2093. doi: 10.1111/febs.17141. [DOI] [PubMed] [Google Scholar]
- 34.Mahiny-Shahmohammady D, Hauck L, Billia F. Defining the molecular underpinnings controlling cardiomyocyte proliferation. Clin Sci (Lond) 2022;136(12):911–934. doi: 10.1042/CS20211180. [DOI] [PubMed] [Google Scholar]
- 35.O'Reilly S, Markiewicz E, Idowu OC. Aging, senescence, and cutaneous wound healing-a complex relationship. Front Immunol. 2024;15:1429716. doi: 10.3389/fimmu.2024.1429716. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Ågren MS, Litman T, Eriksen JO, et al. Gene expression linked to reepithelialization of human skin wounds. Int J Mol Sci. 2022;23(24):15746. doi: 10.3390/ijms232415746. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Jacquier EF, Kassis A, Marcu D, et al. Phytonutrients in the promotion of healthspan: a new perspective. Front Nutr. 2024;11:1409339. doi: 10.3389/fnut.2024.1409339. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Kanwugu ON, Glukhareva TV, Danilova IG, et al. Natural antioxidants in diabetes treatment and management: prospects of astaxanthin. Crit Rev Food Sci Nutr. 2022;62(18):5005–5028. doi: 10.1080/10408398.2021.1881434. [DOI] [PubMed] [Google Scholar]
- 39.商 冠华, 田 春梅. Nrf2信号通路作为虾青素治疗新靶点的研究进展. 国际医药卫生导报. 2024;30(1):20–24. doi: 10.3760/cma.j.issn.1007-1245.2024.01.004. [DOI] [Google Scholar]
- 40.Lv B, Xing S, Wang Z, et al. NRF2 inhibitors: recent progress, future design and therapeutic potential. Eur J Med Chem. 2024;279:116822. doi: 10.1016/j.ejmech.2024.116822. [DOI] [PubMed] [Google Scholar]