Machenism of mangiferin suppressing the inflammatory response induced by lipopolysaccharide in mouse alveolar macrophage
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摘要: 【目的】探究芒果苷抑制脂多糖（LPS）诱导的炎症反应作用机制，为筛选治疗脓毒症的候选药物提供科学依据。【方法】选取小鼠肺泡巨噬细胞（MH-S）为研究对象，通过CCK-8试剂检测芒果苷对MH-S细胞的毒性作用，根据细胞毒性试验确定芒果苷的作用浓度为100 μg/mL，以正常培养的MH-S细胞为对照，使用10 μg/mL LPS处理24 h构建体外炎症模型（LPS组），再以100 μg/mL芒果苷干预24 h（干预组）；通过ELISA检测MH-S细胞上清液中的炎症因子（IL-1β、TNF-α、IL-6和IL-18）含量，采用实时荧光定量PCR检测IL-1β、TNF-α、IL-6和IL-18基因表达情况，并以Westernblotting和免疫荧光法检测TLR4/NF-κB信号通路的变化。【结果】芒果苷浓度低于287.60 μg/mL时，对MH-S细胞无明显的毒性作用。与对照组相比，LPS组MH-S细胞中的IL-1β、TNF-α、IL-6和IL-18基因相对表达量极显著升高（P<0.01，下同），细胞上清液中的IL-1β、TNF-α、IL-6和IL-18含量显著（P<0.05，下同）或极显著升高；但以芒果苷干预后，IL-1β、TNF-α、IL-6和IL-18基因的相对表达量及其分泌水平均显著降低，表明芒果苷能有效抑制LPS诱导MH-S细胞的炎症反应。此外，LPS组MH-S细胞中的NF-κB基因相对表达量与蛋白表达水平极显著高于对照组，经芒果苷干预后NF-κB基因相对表达量与蛋白表达水平均显著下降，表明芒果苷能有效抑制LPS诱导MH-S细胞中核转录因子的表达；LPS组MH-S细胞表面的TLR4表达水平明显升高，但经芒果苷干预后MH-S细胞表面的TLR4表达水平明显降低，说明芒果苷能有效抑制LPS诱导MH-S细胞表面TLR4的表达。【结论】芒果苷对MH-S细胞的增殖无明显抑制作用，其半数增殖抑制率（IC50）为287.60 μg/mL；芒果苷对LPS诱导的MH-S细胞炎症反应具有良好的保护作用，其作用机制可能与抑制TLR4/NF-κB信号通路激活而减少炎症因子的分泌有关。
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关键词:
- 芒果苷 /
- 小鼠肺泡巨噬细胞（MH-S） /
- 脂多糖（LPS） /
- 炎症因子 /
- TLR4/NF-κB信号通路
Abstract: 【Objective】The objective of this study was to analyze the inhibition mechanisms of mangiferin on lipopolysaccharide（LPS）-induced inflammatory response，with the goal of identifying a potential therapeutic agent for sepsis. 【Method】In this research，mouse alveolar macrophage（MH-S）were chosen as the subject of investigation，and the cytotoxic impact of mangiferin on MH-S was assessed using the CCK-8 assay. According to the cytotoxicity test，the concentration of mangiferin was 100 μg/mL. With normal cultured MH-S as the control，10 μg/mL LPS was used for 24 h to construct an in vitro inflammatory model（LPS group），and then 100 μg/mL mangiferin was intervened for 24 h（intervention group）. The contents of inflammatory factors（IL-1β，TNF-α，IL-6 and IL-18）in MH-S supernatants were measured by ELISA，gene expression of IL-1β，TNF-α，IL-6 and IL-18 was assessed using real-time fluorescence quantitative PCR， and changes in TLR4/NF-κB signaling pathway were measured by Western blotting and immunofluorescence.【Result】 Mangiferin did not have obvious toxic effect on MH-S when its concentration was below 287.60 μg/mL. Compared with the control group，the relative expression of IL-1β，TNF-α，IL-6 and IL-18 was extremely significantly increased in the LPS group（P<0.01，the same below），and the contents of IL-1β，TNF-α，IL-6 and IL-18 in the cell supernatant was significantly（P<0.05，the same below）or extremely significantly increased. Following treatment with mangiferin intervention，there was significant decrease in both relative expression levels of IL-1β，TNF-α，IL-6 and IL-18 genes and their secretion levels，suggesting that mangiferin could alleviate inflammatory response in MH-S induced by LPS. In addition， relative expression of NF-κB gene and protein expression level in MH-S of LPS group were extremely significantly higher than that of the control group，and the relative expression of NF-κB gene and protein expression level decreased significantly after mangiferin intervention，indicating that mangiferin could effectively inhibit expression of nuclear transcription factors in MH-S induced by LPS；the TLR 4 expression level on the surface of MH-S of LPS group was greatly increased，but the TLR 4 expression level on MH-S surface decreased greatly after mangiferin intervention，indicating that mangiferin could effectively inhibit TLR 4 expression on the surface of MH-S induced by LPS.【Conclusion】Mangiferin has no obvious inhibitory effect on the proliferation of MH-S cells，and the half proliferation inhibition rate（IC50）of mangiferin is 287.60 μg/mL. Mangiferin has fine protective effect against the LPS-induced inflammatory response in MH-S， and the mechanism of action may be related to the reduction of inflammatory factor secretion by inhibiting the activation of TLR4/NF-κB signaling pathway. -
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