羟氯喹通过抑制MEK/ERK/ROS通路减少中性粒细胞胞外诱捕网形成改善DSS诱导小鼠结肠炎
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1.南京医科大学第一附属医院消化科;2.南京医科大学第一附属医院消化科 江苏省南京市 210029

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国家自然科学基金(82070568)


Hydroxychloroquine alleviates intestinal inflammation in DSS induced colitis mice by inhibiting MEK/ERK pathway and reducing the formation of ROS-dependent neutrophil extracellular trapsJiang Jingyue1,Zhang Hongjie1*
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Department of Gastroenterology,the First Affiliated Hospital of Nanjing Medical University

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    摘要:

    目的:探讨羟氯喹(Hydroxychloroquine, HCQ)对葡聚糖硫酸钠(Dextran Sulfate Sodium Salt,DSS)诱导的结肠炎模型鼠肠道炎症的影响及其影响中性粒细胞胞外诱捕网(neutrophil extracellular traps, NETs)形成的机制。方法:雄性C57BL/6鼠随机分为正常组、DSS模型组(自由饮用3%DSS溶液+200μL纯水灌胃/每天)、HCQ+DSS组(自由饮用3%DSS溶液+ 200μL HCQ(60mg/kg)灌胃/每天)。评估小鼠疾病活动指数(Disease Activity Index,DAI),造模7天后处死小鼠,分析小鼠结肠长度,肠组织HE 染色评估肠道炎症水平。荧光显微镜及荧光酶标仪检测各组小鼠结肠组织NETs水平。收集志愿者外周血提取中性粒细胞进行细胞实验。荧光显微镜及荧光酶标仪检测NETs水平。DCFH-DA荧光探针孵育后荧光酶标仪检测活性氧(reactive oxygen species, ROS)水平。蛋白质印迹法检测p-MEK、MEK、p-ERK、ERK蛋白水平。结果:HCQ减轻DSS诱导的小鼠结肠炎并可抑制结肠炎模型鼠肠组织中NETs形成。在细胞实验,HCQ可抑制体外培养中性粒细胞活性氧释放和NETs形成,同时抑制MEK/ERK信号通路的激活。MEK激动剂C16-PAF可逆转HCQ对MEK/ERK信号通路的抑制作用,增强活性氧的释放,并增加NETs的形成。结论:HCQ可减轻DSS小鼠肠道炎症,其机制可能是通过抑制中性粒细胞MEK/ERK信号通路,减少活性氧释放,从而减轻NETs导致的肠道损伤。

    Abstract:

    Objective: To explore the effect and mechanism of hydroxychloroquine (HCQ) on lipopolysaccharide (LPS) induced neutrophil extracelluar traps (NETs) Methods: Mice were randomly divided into control group, DSS group (3% DSS drinking water+200 μ L pure water by gavage), HCQ+DSS group(3% DSS drinking water+200 μ L HCQ(60mg/kg)by gavage). The disease activity index (DAI) score were evaluated every day. The mice were killed after 7 days, colon length of mice in each group were counted. The pathological changes of colon tissue of mice in each group were observed by HE staining;NETs level was detected by fluorescence microscope and fluorescence microplate reader; Collect the peripheral blood of volunteers to extract neutrophils for experiments in vitro, and detect the level of NETs with fluorescence microscope and fluorescence microplate analyzer;The level of reactive oxygen species (ROS) was detected by fluorescence microplate analyzer after incubation with DCFH-DA fluorescence probe;The protein expression levels of p-MEK, MEK, p-ERK and ERK were detected by Western blot.Results: HCQ alleviates intestinal inflammation in DSS model mice by reduceing the formation of NETs in vivo and in vitro.In vitro, HCQ decreased the release of reactive oxygen species, and inhibited the activation of MEK/ERK signaling pathway. The MEK agonist C16-PAF reversed the inhibition of HCQ on MEK/ERK signaling pathway, enhanced the release of reactive oxygen species, and then increased the formation of NETs.Conclusion: HCQ can reduce DSS induced colitis in mice, and its mechanism may be that it can reduce the release of reactive oxygen species by inhibiting the MEK/ERK signal pathway, thereby reducing the intestinal damage caused by NETs.

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  • 收稿日期:2022-11-30
  • 最后修改日期:2023-02-01
  • 录用日期:2023-05-23
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