中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (26): 4115-4120.doi: 10.3969/j.issn.2095-4344.1398

• 组织工程骨及软骨材料 tissue-engineered bone and cartilage materials • 上一篇    下一篇

白藜芦醇调节氧化应激减少磨损颗粒诱导巨噬细胞肿瘤坏死因子α的表达

刘文华，梁锦峰，邓少杰
  

  1. 深圳市宝安区人民医院骨关节科，广东省深圳市  518101
  • 收稿日期:2019-05-04
  • 通讯作者: 梁锦峰，硕士，副主任医师，深圳市宝安区人民医院骨关节科，广东省深圳市 518101
  • 作者简介:刘文华，男，1980年生，广东省梅州市人，汉族，2013年南方医科大学毕业，硕士，副主任医师，主要从事关节骨科、创伤骨科研究。

Resveratrol reduces tumor nuclear factor-α expression in wear-particle-treated macrophages by regulating the level of intracellular oxidative stress

Liu Wenhua, Liang Jinfeng, Deng Shaojie
  

  1. Department of Bone and Joint Diseases, Shenzhen Baoan District People's Hospital, Shenzhen 518101, Guangdong Province, China
  • Received:2019-05-04
  • Contact: Liang Jinfeng, Master, Associate chief physician, Department of Bone and Joint Diseases, Shenzhen Baoan District People's Hospital, Shenzhen 518101, Guangdong Province, China
  • About author:Liu Wenhua, Master, Associate chief physician, Department of Bone and Joint Diseases, Shenzhen Baoan District People's Hospital, Shenzhen 518101, Guangdong Province, China

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摘要：

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文题释义：
磨损颗粒：人工关节各个部件之间反复的摩擦可产生磨损颗粒，这些磨损颗粒引起的慢性炎症是导致假体松动的关键原因。磨损颗粒诱导的假体周围炎症性骨溶解是一个复杂的过程；磨损颗粒可被假体周围组织的巨噬细胞识别并吞噬，引起ERK、NF-κB等信号蛋白和转录因子的活化，进而诱导肿瘤坏死因子α、白细胞介素1β、白细胞介素6等促炎因子的产生和释放，这一系列的病理生理过程可导致假体周围组织发生慢性炎症反应，诱发假体周围骨溶解。
氧化应激：是由于氧化物的形成和降解平衡紊乱引起，由氧化应激产生的活性氧自由基和活性氮自由基与炎症发生发展过程密切相关。活性氧自由基和活性氮自由基的产生主要由氧化应激相关酶和抗氧化应激相关酶调控，前者包括烟酰胺腺嘌呤二核苷酸磷酸氧化酶和一氧化氮合成酶，后者主要包括过氧化氢酶、超氧化物歧化酶、谷胱甘肽过氧化物酶和谷胱甘肽还原酶。
 
 
背景：大量体外细胞实验和动物实验表明，白藜芦醇不仅具有抗氧化应激、抗炎、抗病毒、抗肿瘤和延缓衰老等生理活性，还具有骨保护作用。
目的：观察白藜芦醇对钛磨损颗粒诱导小鼠RAW264.7巨噬细胞氧化应激水平与炎症反应的调节作用。
方法：以不同浓度(0，10，20，40，80，160 μmol/L)的白藜芦醇溶液干预小鼠RAW264.7巨噬细胞，培养24 h后，WST-1法检测细胞存活率，选择对细胞活性影响较小的浓度进行后续实验。将小鼠RAW264.7巨噬细胞分5组干预：对照组常规培养；钛颗粒组加入钛颗粒刺激细胞12 h；低、中、高浓度白藜芦醇组分别加入10，20，40 μmol/L的白藜芦醇溶液预刺激细胞4 h，随后各组加入钛颗粒刺激细胞12 h。钛颗粒刺激12 h后，收集细胞与细胞培养上清，利用流式细胞仪检测细胞内活性氧水平，荧光定量PCR检测抗氧化应激相关酶(铜/锌超氧化物歧化酶、锰超氧化物歧化酶、谷胱甘肽过氧化物酶、谷胱甘肽还原酶、过氧化氢酶)和肿瘤坏死因子αmRNA水平，利用ELISA检测肿瘤坏死因子α质量浓度。
结果与结论：①10，20，40 μmol/L白藜芦醇对小鼠RAW264.7巨噬细胞活性无明显抑制作用，故选择此3组浓度进行后续实验；②与对照组比较，钛颗粒组细胞内活性氧水平升高(P < 0.05)；与钛颗粒组比较，白藜芦醇呈浓度依赖性降低细胞内活性氧水平(P < 0.05)；③与对照组比较，钛颗粒组锰超氧化物歧化酶、谷胱甘肽过氧化物酶mRNA表达下降(P < 0.05)，谷胱甘肽还原酶、过氧化氢酶mRNA表达升高(P < 0.05)；与钛颗粒组比较，中、高浓度白藜芦醇组锰超氧化物歧化酶、铜/锌超氧化物歧化酶mRNA表达升高(P < 0.05)，低、中、高浓度白藜芦醇组过氧化氢酶mRNA表达升高(P < 0.05)；④与对照组比较，钛颗粒组肿瘤坏死因子α mRNA水平与肿瘤坏死因子α的分泌均升高(P < 0.05)；与钛颗粒组比较，低、中、高浓度白藜芦醇呈浓度依赖性降低肿瘤坏死因子α mRNA水平与肿瘤坏死因子α的分泌(P < 0.05)；⑤结果表明，白藜芦醇能够有效提高金属钛颗粒刺激后巨噬细胞内抗氧化应激相关酶的转录，降低细胞内氧化应激水平，减少炎症因子肿瘤坏死因子α的释放。

关键词: 白藜芦醇, 钛颗粒, 巨噬细胞, 氧化应激, 假体松动, 骨溶解, 肿瘤坏死因子α, 活性氧

Abstract:

BACKGROUND: Numerous in vitro cell and animal experiments have shown that resveratrol exhibits anti-oxidative stress, anti-inflammatory, anti-viral, anti-tumor and anti-aging activities and it also has bone-protecting effect.
OBJECTIVE: To investigate the regulatory effect of resveratrol on oxidative stress and inflammatory response of RAW264.7 macrophages induced by titanium wear-particles.
METHODS: The RAW264.7 macrophages were treated with different doses of resveratrol (0, 10, 20, 40, 80, 160 μmol/L) for 24 hours. Cell viability was detected using WST-1 method. Resveratrol doses that did not produce significant change in cell viability were used in the subsequent experiments. RAW264.7 cells were divided into five groups. In the control group, cells were routinely cultured. In the titanium group, RAW264.7 macrophages were treated with titanium particles for 12 hours. In the low-, medium-, and high-dose resveratrol groups, RAW264.7 macrophages were pre-treated with 10, 20, 40 μmol/L resveratrol for 4 hours, and then they were treated with titanium particles for 12 hours. Thereafter, cells and cell supernatant were collected for measurement of intracellular reactive oxygen species generation using flow cytometry. Total RNA was extracted from cells for measuring mRNA level of anti-oxidative enzymes (Cu/Zn superoxide dismutase, Mn superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase) and tumor necrosis factor-α. Cell supernatant was used for measuring the level of TNF-α using ELISA.
RESULTS AND CONCLUSIONS: Treatment with resveratrol (10, 20, and 40 μmol/L) did not affect the viability of RAW264.7 macrophages. Therefore, these doses (10, 20, and 40 μmol/L) were used in the following experiments. Intracellular reactive oxygen species generation in the titanium group was significantly increased compared with the control group (P < 0.05). Resveratrol decreased reactive oxygen species generation in a dose-dependent manner compared with the titanium group (P < 0.05). Compared with the control group, Mn superoxide dismutase and glutathione peroxidase mRNA levels significantly decreased (P < 0.05), glutathione reductase and catalase mRNA levels significantly increased (P < 0.05) in the titanium group. Compared with the titanium group, Mn superoxide dismutase and Cu/Zn superoxide dismutase mRNA levels significantly increased in the medium- and high-dose resveratrol groups (P < 0.05), and catalase mRNA level significantly increased in the low-, medium-, and high-dose resveratrol groups (P < 0.05). The mRNA level and secretion of tumor necrosis factor-α in the titanium group were significantly increased than in the control group (P < 0.05). Compared with the titanium group, low-, medium-, and high-dose resveratrol decreased the mRNA level and secretion of tumor necrosis factor-α (P < 0.05). These results indicate that resveratrol can effectively enhance the transcription of antioxidant stress-related enzymes in macrophages stimulated by titanium particles, reduce the level of intracellular oxidative stress, and reduce the release of inflammatory factor tumor necrosis factor-α.

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