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

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

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(0,10,20,40,80,160μmol/L)的白藜芦醇溶液干预小鼠RAW264.7巨噬细胞,培养24h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10,20,40μmol/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12h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αm RNA水平,利用ELISA检测肿瘤坏死因子α质量浓度。结果与结论:(1)10,20,40μmol/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m RNA表达下降(P <0.05),谷胱甘肽还原酶、过氧化氢酶m RNA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m RNA表达升高(P <0.05),低、中、高浓度白藜芦醇组过氧化氢酶m RNA表达升高(P<0.05);(4)与对照组比较,钛颗粒组肿瘤坏死因子αm RNA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αm RNA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。

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.