芒果苷抑制脂多糖诱导小鼠肺泡巨噬细胞炎症反应的机制研究

Machenism of mangiferin suppressing the inflammatory response induced by lipopolysaccharide in mouse alveolar macrophage

【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100μg/mL,以正常培养的MH-S细胞为对照,使用10μg/mL LPS处理24 h构建体外炎症模型(LPS组),再以100μg/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Western blotting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287.60μg/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287.60μg/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。

【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 100μg/mL.With normal cultured MH-S as the control,10μg/mL LPS was used for 24 h to construct an in vitro inflammatory model(LPS group),and then 100μg/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 287.60μg/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 287.60μg/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.