中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (29): 4620-4627.doi: 10.12307/2024.584

• 水凝胶材料Hydrogel materials • 上一篇    下一篇

氧化苦参碱水凝胶通过激活角化细胞Nrf2/HO-1通路促进创面愈合

刘  璐1，刘淑丹1，刘晓丹1，杨  丽2，凌  晨1，海小明1，马会明3，陈冬梅1   

  1. 1宁夏医科大学总医院，宁夏回族自治区干细胞与再生医学重点实验室，宁夏回族自治区银川市  750004；宁夏医科大学，2基础医学院，3生育力保持教育部重点实验室，宁夏回族自治区银川市  750004
  • 收稿日期:2023-11-01 接受日期:2023-12-14 出版日期:2024-10-18 发布日期:2024-03-22
  • 通讯作者: 陈冬梅，博士，副研究员，宁夏医科大学总医院，宁夏回族自治区干细胞与再生医学重点实验室，宁夏回族自治区银川市 750004
  • 作者简介:刘璐，男，1990年生，陕西省榆林市人，汉族，在读硕士，主治医师，主要从事创面愈合与瘢痕研究。
  • 基金资助:
    国家自然科学基金地区项目(81960355)，项目负责人：陈冬梅；宁夏自治区自然科学基金项目(2022AAC03500)，项目负责人：陈冬梅；宁夏回族自治区重点研发计划项目(2022BEG03147)，项目负责人：刘淑丹

Oxymatrine hydrogel promotes wound healing by activating Nrf2/HO-1 pathway in keratinocytes

Liu Lu1, Liu Shudan1, Liu Xiaodan1, Yang Li2, Ling Chen1, Hai Xiaoming1, Ma Huiming3, Chen Dongmei1   

  1. 1General Hospital of Ningxia Medical University, Key Laboratory of Stem Cell and Regenerative Medicine of Ningxia Hui Autonomous Region, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 2School of Basic Medicine, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 3Key Laboratory for Maintaining the Educational Ability of Medical College Students in Ningxia, Ministry of Education, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Received:2023-11-01 Accepted:2023-12-14 Online:2024-10-18 Published:2024-03-22
  • Contact: Chen Dongmei, PhD, Associate researcher, General Hospital of Ningxia Medical University, Key Laboratory of Stem Cell and Regenerative Medicine of Ningxia Hui Autonomous Region, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • About author:Liu Lu, Master candidate, Attending physician, General Hospital of Ningxia Medical University, Key Laboratory of Stem Cell and Regenerative Medicine of Ningxia Hui Autonomous Region, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Supported by:
    National Natural Science Foundation of China, No. 81960355 (to CDM); Natural Science Foundation of Ningxia Hui Autonomous Region, No. 2022AAC03500 (to CDM); Key Research & Development Plan Project of Ningxia Hui Autonomous Region, No. 2022BEG03147 (to LSD)

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摘要：


文题释义：

氧化苦参碱：是含有四环喹嗪啶类结构的典型生物碱之一(分子式：C15H24N2O2；分子质量：264.360 g/mol)，在自然界主要存在于豆科槐属Sophor L.植物苦参S.flavescens Ait.、越南槐S.tonkinensis Gagnep、苦豆子S.alopecuroides L.和白刺花S.viciifolia Hance中。氧化苦参碱具有广泛的药理作用和生物活性，如抗菌、抗病毒、抗氧化、抗炎、免疫调节、抗肿瘤等，具有保护心、肝、肺、肾、脑、血管等作用。
Nrf2：是一种亮氨酸拉链转录激活因子，可调节多种抗氧化途径。在生理条件下，Nrf2锚定在细胞质中，在氧化应激条件下，Nrf2从细胞质转移到细胞核，进而激活其下游多种分子并驱动多种功能，包括抗氧化应激、抗凋亡和抗炎作用。


背景：慢性创面炎症和氧化应激阻碍了角化细胞的再生，氧化苦参碱具有抗氧化、抗炎等多种生物活性，可能具有促进创面愈合的潜在效应。

目的：探讨氧化苦参碱对创面愈合的作用，以及对H2O2诱导人角质形成细胞系HaCaT细胞氧化应激损伤的保护作用。
方法：①体内实验：分别制备含0，0.05，0.1，0.2 g/L氧化苦参碱的甲基丙烯酰化透明质酸(HAMA)水凝胶。在75只糖尿病小鼠背中部制作直径12 mm的全层皮肤缺损模型，随机分5组干预，每组15只：模型组创面包扎固定，单纯水凝胶组以HAMA水凝胶覆盖创面，低、中、高剂量氧化苦参碱组分别以含0.05，0.1，0.2 g/L氧化苦参碱的HAMA水凝胶覆盖创面，光固化后包扎固定，14 d内进行相关指标的检测。②体外实验：将人角质形成细胞系HaCaT分5组培养，正常组常规培养，H2O2组及低、中、高浓度氧化苦参碱组均加入H2O2干预4 h，然后分别更换为含0，0.05，0.1，0.2 g/L氧化苦参碱的培养基，培养24 h后进行相关指标的检测。

结果与结论：①体内实验：与模型组比较，单纯水凝胶组小鼠创面愈合率无明显变化，低、中、高剂量氧化苦参碱组治疗后7，14 d的创面愈合率升高(P < 0.05)。治疗后14 d的创面样本切片病理观察显示，与模型组相比，中、高剂量氧化苦参碱组再生表皮层厚度、显微血管数量与胶原沉积均增加(P < 0.05)。术后7 d的创面样本Western blotting检测显示，与模型组相比，中、高剂量氧化苦参碱组肿瘤坏死因子α与白细胞介素6的蛋白表达降低(P < 0.05)。②体外实验：CCK-8检测、EdU及Ki67染色显示，与H2O2组相比，中、高浓度氧化苦参碱组细胞增殖能力明显升高(P < 0.05)。与H2O2组相比，中、高浓度氧化苦参碱组线粒体膜电位升高(P < 0.05)、活性氧含量降低(P < 0.05)。Western blotting检测显示，与H2O2组相比，高浓度氧化苦参碱组Nrf2核蛋白、Nrf2总蛋白、HO-1蛋白及超氧化物歧化酶1蛋白的表达增加(P < 0.05)。③结果表明：氧化苦参碱能够通过上调Nrf2、HO-1蛋白减轻HaCat细胞的氧化应激损伤，加速创面愈合。

https://orcid.org/0000-0002-8285-2674(陈冬梅)

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料；口腔生物材料；纳米材料；缓释材料；材料相容性；组织工程

关键词: 氧化苦参碱, 水凝胶, 抗氧化, 创面愈合, Nrf2/HO-1信号通路, HaCat细胞

Abstract: BACKGROUND: Inflammation and oxidative stress contribute to the barriers of regeneration in chronic wound. Oxymatrine has various biological activities, such as anti-oxidation, anti-inflammation and so on, which may have the potential effect of promoting wound healing.
OBJECTIVE: To investigate the effect of oxymatrine on wound healing and the protective effect on H2O2-induced oxidative stress injury in human keratinoid cell line HaCaT cells.
METHODS: (1) In vivo experiment: Hyaluronic acid methacryloyl hydrogels containing 0, 0.05, 0.1, 0.2 g/L oxymatrine were prepared. A full-layer skin defect model with a diameter of 12 mm was made in the back of 75 diabetic mice and randomly divided into five groups for intervention, with 15 mice in each group. The wounds of the model group were bandaged and fixed. The wounds of the hydrogel group were covered with hyaluronic acid methacryloyl hydrogel. The wounds of the low-dose, moderate-dose and high-dose oxymatrine groups were covered with hyaluronic acid methacryloyl hydrogel containing 0.05, 0.1, and 
0.2 g/L oxymatrine, respectively, and then bandaged and fixed after light curing. Relevant indicators were detected within 14 days. (2) In vitro experiment: Human keratinocyte line HaCaT was divided into five groups. The normal group was cultured conventionally. H2O2 group and low-, moderate- and high-concentration oxymatrine groups were treated with H2O2 for 4 hours, and then the medium was replaced with medium containing 0, 0.05, 0.1, and 0.2 g/L oxymatrine, respectively, and the relevant indexes were detected after 24 hours of culture.
RESULTS AND CONCLUSION: (1) In vivo experiment: Compared with the model group, the wound healing rate of mice in the hydrogel group had no significant change. The wound healing rate of mice in the low-, moderate- and high-dose oxymatrine group was increased at 7 and 14 days after treatment (P < 0.05). Pathological observation of wound section 14 days after treatment showed that compared with the model group, the thickness of regenerated epidermal layer, the number of microvessels, and collagen deposition in the moderate- and high-dose oxymatrine groups were increased (P < 0.05). Western blot assay analysis of wound samples 7 days after surgery showed that compared with the model group, the protein expressions of tumor necrosis factor α and interleukin 6 in the moderate- and high-dose oxymatrine groups were decreased (P < 0.05). (2) In vitro experiment: CCK8 assay, EdU and Ki67 staining showed that compared with the H2O2 group, the cell proliferation ability of the moderate- and high-concentration oxymatrine groups was significantly increased (P < 0.05). Compared with the H2O2 group, mitochondrial membrane potential was increased (P < 0.05) and reactive oxygen species content was decreased (P < 0.05) in the moderate- and high-concentration oxymatrine groups. Western blot assay results showed that compared with the H2O2 group, the expression levels of Nrf2 nuclear protein, Nrf2 total protein, HO-1 protein, and superoxide dismutase 1 protein were increased in the high-concentration oxymatrine group (P < 0.05). (3) These findings confirm that oxymatrine can alleviate oxidative stress damage in HaCat cells and accelerate wound healing by upregulating the levels of Nrf2 and HO-1 protein. 

Key words: oxymatrine, hydrogel, antioxidant, wound healing, Nrf2/HO-1 signaling pathway, HaCat cell

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