基于花生四烯酸代谢通路研究水飞蓟素对脂多糖诱导炎症模型影响

以脂多糖类似物(KLA)诱导的RAW264.7细胞为研究对象,采用代谢组学研究手段,研究水飞蓟素对脂多糖诱导炎症模型中花生四烯酸代谢通路的影响。以超高效液相色谱-三重四极杆质谱联用为平台,对不同浓度水飞蓟素作用下KLA诱导RAW264.7炎症细胞分泌的类二十烷酸代谢物进行定量分析,通过考察主成分分析(PCA)、正交偏最小二乘法判别分析(OPLS-DA)的VIP值和Kruskal-Wallis秩和检验结果显著性差异(P)值筛选代谢标记物。建立了59种类二十烷酸(含15种同位素内标)在5 min内实现快速分离的液相色谱-质谱联用方法;确定了细胞存活率在58%~80%的水飞蓟素浓度为50~150μmol/L;筛选出数据处理结果同时满足变异权重参数(VIP)值1且结果P值0.05的类二十烷酸代谢标记物12-OxoLeukotriene B4(12-OxoLTB4);通过分析柱状图和炎症信号通路,确定水飞蓟素借助其抗氧自由基特性发挥抗炎作用,通过抑制脂氧合酶-5(5-LOX)的活性及阻断5-LOX代谢通路中产生氧自由基的脂质过氧化反应来减少氧自由基及过氧化物的形成。综上所述,所建立的方法能快速准确地定量分析多种类二十烷酸,并从代谢组学角度解释了水飞蓟素的抗炎机制。

Investigation on silymarin impact on lipopolysaccharide induced inflammation model based on arachidonic acid metabolism pathway

The objective of this research is to investigate the suppressive effect of silymarin on vitro cell culture model of inflammatory macrophage RAW264.7 induced by Kdo2-Lipid A, and explore its mechanism based on cell metabonomics. Ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method was used in the cell metabonomic assay to quantitative analysis of metabolites related to eicosanoids pathway. Then chemometric approaches such as principal component analysis were used to process the metabolic data. Within the established method, a total of 59 eicosanoids standards (containing 15 deuterated internal standards) were simultaneously separated in a single 5 min run, and the analytical method is proved to be rapid, sensitive and accurate. Whereafter, the metabolites with VIP> 1 and P value< 0.05 were considered as biomarkers. 12-OxoLeukotriene B₄ (12-OxoLTB₄) was eventually identified as metabolic biomarkers of silymarin treatment group in this research, and according to the related inflammatory pathways, we speculated silymarin has anti-inflammatory activities by inhibiting the 5-lipoxygenase (5-LOX) activity and blocking lipid peroxidation in 5-LOX metabolic pathways to reduce the formation of peroxides and oxygen free radicals. This study provide a novel approach to the mechanism research on the silymarin treatment on RAW264.7 cells based on cell metabonomics.