蛇床子素对脂多糖诱导的巨噬细胞极化和炎症反应的影响

The effect of osthole on lipopolysaccharide-induced macrophages polarization and inflammatory reaction

目的 :探讨蛇床子素(osthole, OST)对脂多糖(lipopolysaccharide, LPS)诱导的巨噬细胞极化、炎症反应的影响。方法:以CCK-8法检测不同浓度OST对RAW264.7巨噬细胞增殖活性的影响;使用细胞内活性氧(reactive oxygen species, ROS)检测试剂盒(DCFH-DA)、免疫荧光染色、q-PCR、流式细胞术明确不同浓度(6.25、12.5、25μmol/L)OST对巨噬细胞极化和炎症反应的影响。采用Graphpad prism 8.0软件包对数据进行统计学分析。结果:CCK-8结果显示,在25μmol/L以下时,OST对RAW264.7无明显细胞毒性。免疫荧光染色及q-PCR结果显示,6.25、12.5、25μmol/L的OST均能抑制M1型巨噬细胞炎症因子的表达,iNOS、TNF-α、CCR7呈浓度依赖性降低,并且有效上调M2抑炎因子IL-10、Arg-1及CD206的表达。流式细胞仪分析表明,OST可有效抑制LPS诱导的巨噬细胞M1型标志物CD86的表达。结论:OST调节LPS诱导的M1型巨噬细胞极化,减轻炎症反应。

PURPOSE: To investigate the effect of Osthole(OST) on lipopolysaccharide(LPS)-induced macrophage polarization and inflammatory reaction. METHODS: The effect of different concentrations of OST on proliferative activity of RAW264.7 macrophages was examined by CCK-8 method;the effect of OST at different concentrations(6.25, 12.5 and25 μmol/L) on macrophage polarization and inflammation was investigated by using intracellular reactive oxygen species(ROS) detection kit(DCFH-DA), immunofluorescence staining, q-PCR and flow cytometry. The effects of OST on macrophage polarization and inflammatory responses were investigated by immunoblotting of proteins. Graphpad prism 8.0 software package was used for statistical analysis of the data. RESULTS: CCK-8 results showed that OST was not significantly cytotoxic to RAW264.7 at less than 25 μmol/L, immunofluorescence and q-PCR results showed that OST at 6.25,12.5 and 25 μmol/L inhibited the expression of inflammatory factors in M1 macrophages, and iNOS, TNF-α, CCR7 were reduced in a concentration-dependent manner, and effectively upregulated the expression of M2 inflammatory factors IL-10,Arg-1 and CD206. Flow cytometry showed that OST effectively inhibited the expression of LPS-induced M1 marker CD86 in macrophages. CONCLUSIONS: OST can regulate lipopolysaccharide-induced M1 macrophages polarization and reduce inflammatory reaction.