【摘要】：以脂多糖類(lèi)似物(KLA)誘導(dǎo)的RAW264.7細(xì)胞為研究對(duì)象,采用代謝組學(xué)研究手段,研究水飛薊素對(duì)脂多糖誘導(dǎo)炎癥模型中花生四烯酸代謝通路的影響。以超高效液相色譜-三重四極桿質(zhì)譜聯(lián)用為平臺(tái),對(duì)不同濃度水飛薊素作用下KLA誘導(dǎo)RAW264.7炎癥細(xì)胞分泌的類(lèi)二十烷酸代謝物進(jìn)行定量分析,通過(guò)考察主成分分析(PCA)、正交偏最小二乘法判別分析(OPLS-DA)的VIP值和Kruskal-Wallis秩和檢驗(yàn)結(jié)果顯著性差異(P)值篩選代謝標(biāo)記物。建立了59種類(lèi)二十烷酸(含15種同位素內(nèi)標(biāo))在5 min內(nèi)實(shí)現(xiàn)快速分離的液相色譜-質(zhì)譜聯(lián)用方法;確定了細(xì)胞存活率在58%~80%的水飛薊素濃度為50~150μmol/L;篩選出數(shù)據(jù)處理結(jié)果同時(shí)滿足變異權(quán)重參數(shù)(VIP)值1且結(jié)果P值0.05的類(lèi)二十烷酸代謝標(biāo)記物12-OxoLeukotriene B4(12-OxoLTB4);通過(guò)分析柱狀圖和炎癥信號(hào)通路,確定水飛薊素借助其抗氧自由基特性發(fā)揮抗炎作用,通過(guò)抑制脂氧合酶-5(5-LOX)的活性及阻斷5-LOX代謝通路中產(chǎn)生氧自由基的脂質(zhì)過(guò)氧化反應(yīng)來(lái)減少氧自由基及過(guò)氧化物的形成。綜上所述,所建立的方法能快速準(zhǔn)確地定量分析多種類(lèi)二十烷酸,并從代謝組學(xué)角度解釋了水飛薊素的抗炎機(jī)制。
[Abstract]:The effects of silymarin on arachidonic acid metabolic pathway in lipopolysaccharide induced inflammatory model were studied in RAW264.7 cells induced by lipopolysaccharide analogues (KLA). Using ultra-high performance liquid chromatography-quadrupole mass spectrometry as a platform, the eicosanoic acid metabolites secreted by RAW264.7 inflammatory cells induced by KLA with different concentrations of silymarin were quantitatively analyzed. The principal component analysis of (PCA), was carried out. The VIP value of orthogonal partial least squares discriminant analysis (OPLS-DA) was significantly different from that of Kruskal-Wallis rank sum test. (P) value was used to screen metabolic markers. A rapid separation method of 59 eicosanoic acids (including 15 isotopic internal standards) within 5 min was established by liquid chromatography-mass spectrometry, and the concentration of silymarin with cell survival rate of 58 ~ 80% was determined to be 50 ~ 150 渭 mol/L;. The eicosanoic acid metabolic marker 12-OxoLeukotriene B4 (12-OxoLTB4), which satisfied the variation weight parameter (VIP) value 1 and the result P value 0. 05, was screened out. By analyzing the histogram and the inflammatory signaling pathway, silymarin was determined to play an anti-inflammatory effect with the help of its anti-oxygen free radical properties. The formation of oxygen free radicals and peroxides was reduced by inhibiting the activity of lipoxygenase-5 (5-LOX) and blocking the lipid peroxidation of oxygen free radicals in the metabolic pathway of 5-LOX. In conclusion, the established method can be used for rapid and accurate quantitative analysis of many kinds of eicosanoic acids, and explains the anti-inflammatory mechanism of silymarin from the perspective of metabolomics.
【作者單位】： 上海交通大學(xué)藥學(xué)院;
【基金】：上海市科委科研計(jì)劃項(xiàng)目(15142200200,16142200300) 中國(guó)博士后科學(xué)基金(2015M581628) 上海市引進(jìn)技術(shù)的吸收與創(chuàng)新專(zhuān)項(xiàng)(XC-ZXSJ-02-2016-11)~~
【分類(lèi)號(hào)】：O657.63;R284

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