褪黑素调控Nrf2/ARE信号通路延缓髓核间充质干细胞退变的实验研究

李才; 许盼盼; 胡捷; 朱坤; 叶雨辰; 张亚伟; 张长春

doi:10.16766/j.cnki.issn.1674-4152.002713

褪黑素调控Nrf2/ARE信号通路延缓髓核间充质干细胞退变的实验研究

doi: 10.16766/j.cnki.issn.1674-4152.002713

李才^{1, 2},
许盼盼^{1, 2},
胡捷^{1, 2},
朱坤^{1, 2},
叶雨辰^{1, 2},
张亚伟^{1, 2},
张长春^{1, 2, ,}

1.
蚌埠医学院第一附属医院骨科，安徽蚌埠 233004
2.
蚌埠医学院安徽省组织移植重点实验室，安徽蚌埠 233030

基金项目:

安徽省自然科学重点项目 1908085MC90

安徽省教育厅自然科学重点项目 KJ2021A0743

蚌埠医学院研究生科研创新计划 Byycx20029

蚌埠医学院大学生创新创业训练计划 bydc2021017

详细信息

通讯作者:
张长春，E-mail：zccanhui@sina.com

中图分类号: R681.53 R-332
计量
- 文章访问数: 275
- HTML全文浏览量: 68
- PDF下载量: 5
- 被引次数: 0
出版历程
- 收稿日期: 2022-02-06
- 网络出版日期: 2022-12-30

Melatonin regulates Nrf2/ARE signal pathway to delay the degeneration of nucleus pulposus mesenchymal stem cells

LI Cai^{1, 2},
XU Pan-pan^{1, 2},
HU Jie^{1, 2},
ZHU Kun^{1, 2},
YE Yu-chen^{1, 2},
ZHANG Ya-wei^{1, 2},
ZHANG Chang-chun^{1, 2
, ,}

1.
Department of Orthopaedics, the First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, China

摘要

摘要: 目的探讨褪黑素对氧化应激条件下的大鼠髓核间充质干细胞(NPMSCs)的影响及调节机制。方法使用差速贴壁法获取大鼠NPMSCs。显微镜下观察细胞状态，鉴定第三代细胞三系分化潜能和细胞表面免疫标志物；采用CCK-8法检测不同浓度过氧化氢和褪黑素诱导对大鼠NPMSCs活力的影响确定最佳浓度和实验分组；免疫荧光和流式细胞学技术检测NPMSCs活性氧水平；免疫印迹分析外基质标志蛋白(Ⅱ型胶原、糖胺聚糖)和通路核心蛋白(Nrf2、HO-1)表达情况。结果褪黑素在氧化应激下保护了NPMSCs的活性，且1 μmol/L浓度保护效应最为明显(0.717±0.018，t=7.102, P＜0.01)。褪黑素可抑制NPMSCs在H₂O₂诱导后的活性氧水平(阳性率70.0% vs. 32.7%)。此外，褪黑素还能显著提高Nrf2/ARE信号通路中关键蛋白核因子E2相关因子(1.925±0.024，t=13.150，P＜0.01)和血红素氧合酶-1(1.605±0.019，t=12.940, P＜0.01)的活性，改善NPMSCs的功能。褪黑素治疗后，细胞基质蛋白Ⅱ型胶原(0.850±0.010，t=25.200，P＜0.01)和糖胺聚糖(0.335±0.013，t=10.640，P＜0.01)表达增加。结论褪黑素通过调控Nrf2/ARE信号通路可以影响NPMSCs退变的生物学特性。
- 椎间盘退变 /
- 褪黑素 /
- 氧化应激 /
- 髓核间充质干细胞 /
- 活性氧
Abstract: Objective To explore the effect of melatonin on rat nucleus pulposus mesenchymal stem cells (NPMSCs) under oxidative stress and its regulatory mechanism. Methods Rat nucleus pulposus mesenchymal stem cells were isolated and purified by differential adhesion method. The state of cells was observed under the microscope to identify the differentiation potential of the third generation cells and cell surface immune markers. CCK-8 method was used to detect the effect of different concentrations of hydrogen peroxide and melatonin on the activity of NPMSCs in rats to determine the optimal concentration and experimental grouping. The level of reactive oxygen species in NPMSCs were detected by immunofluorescence and flow cytometry. The expressions of extracellular matrix marker proteins (collagen Ⅱ and glycosaminoglycan) and key proteins (Nrf2, HO-1) in Nrf2/ARE signaling pathway were analyzed by Western blotting. Results Melatonin protected the activity of NPMSCs under oxidative stress, and the protective effect of 1 μmol/L concentration was the most obvious (0.717±0.018, t=7.102, P < 0.01). Melatonin could inhibit the level of reactive oxygen species induced by H₂O₂ (positive rate: 70.0% vs. 32.7%). In addition, melatonin significantly increased the activities of Nrf2 (1.925±0.024, t=13.150, P < 0.01) and HO-1 (1.605±0.019, t=12.940, P < 0.01), and improved the function of NPMSCs. After treatment with melatonin, the expression of Collagen Ⅱ (0.850±0.010, t=25.200, P < 0.01) and glycosaminoglycan (0.335±0.013, t=10.640, P < 0.01) increased. Conclusion Melatonin affects the biological characteristics of NPMSCs degeneration by regulating Nrf2/ARE signal pathway.
- Intervertebral disc degeneration /
- Melatonin /
- Oxidative stress /
- Nucleus pulposus mesenchymal stem cells /
- Reactive oxygen species

HTML全文

图 1 NPMSCs表型鉴定

注：A为第3代NPMSCs细胞形态观察(×100)；B~D为28 d诱导后茜素红染色、油红O染色和阿利辛蓝染色(×100)；E为流式检测显示细胞高表达CD73、CD90，低表达CD34、CD45。

Figure 1. Phenotypic identification of NPMSCs

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图 2 褪黑素和H₂O₂对NPMSCs增殖的影响

注: A为不同浓度褪黑素对NPMSCs的细胞毒性检测；B为不同浓度H₂O₂和褪黑素预处理后诱导下细胞活力检测。与对照组比较，^aP < 0.01；与H₂O₂组比较，^bP < 0.01。

Figure 2. Effects of melatonin and H2O2 on proliferation of NPMSCs

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图 3 褪黑素改善NPMSCs氧化应激下的ROS水平

注：A为不同处理组明场形态学观察(×100)；B为不同处理组ROS在细胞内荧光分布(×100)；C为各处理组ROS水平流式细胞学检测。

Figure 3. Melatonin improves Ros levels of NPMSCs under oxidative stress

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图 4 褪黑素激活Nrf2/ARE通路改善NPMSCs退变

注：Western blotting检测各处理组外基质成分蛋白Ⅱ型胶原、糖胺聚糖及核蛋白Nrf2和下游靶蛋白HO-1相对表达水平；与对照组比较，^aP < 0.01；与H₂O₂组比较，^bP < 0.01。

Figure 4. Melatonin activates Nrf2/ARE pathway to ameliorate NPMSCs degeneration

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