基于内源性代谢产物解析桂枝汤解热作用的分子机制

Analysis of molecular mechanism of Guizhi Decoction based on endogenous metabolites

目的:在对桂枝汤解热作用评价的基础上,利用GC-MS代谢组学技术探索其解热作用的分子机制。方法:SD大鼠随机分为桂枝汤组、模型组、空白组,注射10%酵母悬液诱导发热,桂枝汤组分2次给药,空白组给予等量蒸馏水,分别于造模4、5、6、7、8h后测定体温并记录;同时采集大鼠血液,经处理后获得血清,进行GC-MS代谢组学分析,PCA、PLS-DA等分析方法进行数据模式识别,并依据VIP值>1和手动积分计算筛选出差异代谢物并进行通路分析;运用Cytoscape中的MetScape插件对差异代谢物进行网络模块化分析;筛选出节点度不小于平均度值的相关靶点。结果:桂枝汤具有一定的解热效果,与模型组比较,桂枝汤组大鼠体温下降,在造模5h后的各时间点均低于模型组大鼠体温,在造模6h与7h检测时间点出现显著性差异(P<0.05)。通过代谢组学分析技术找出与桂枝汤解热相关的9个差异代谢物,分别为Stearic acid、L-Lactic acid、Glycerol、Palmitic acid、Linoleic acid、D-Glucose、Urea、Arachidonic acid、L-Threonine,以差异代谢物进行通路分析发现桂枝汤的解热作用可能与亚油酸代谢通路、花生四烯酸代谢通路、淀粉和蔗糖的代谢通路以及甘油酯代谢通路有关;模块化分析获得其中最大的2个模块是花生四烯酸代谢通路和亚油酸代谢通路,根据度值得到潜在靶点为CYP450与PFKFB1、PFKFB2。结论:桂枝汤具有较好的解热作用,可能是从氨基酸、糖类代谢、脂肪类代谢等多层面协同发挥作用,具有多靶点性。

Objective: Based on the evaluation of the antipyretic effect of Guizhi Decoction, the molecular mechanism of its antipyretic effect was explored using GC-MS metabolomics technology. Methods: SD rats were randomly divided into Guizhi Decoction group, model group and blank group, and 10% yeast suspension was injected to induce fever. Guizhi Decoction group was administered twice, and the blank group was given the same amount of distilled water, the body temperature was measured and recorded 4, 5, 6, 7, 8 h after modeling;at the same time, blood samples of rats in each group were collected respectively, then serum samples were obtained after treatment and used to GC-MS metabolomics analysis. Data pattern recognition was performed using PCA, PLS-DA and other analytical methods. Differential metabolites were screened based on VIP>1 and manual integration calculation and pathway analysis. Network modular analysis of differential metabolites was performed using MetScape plugin in Cytoscape. Relevant targets whose node degrees were greater than the average degree value were selected. Results: Guizhi Decoction had a certain antipyretic effect. Compared with the model group, the Guizhi Decoction group’s body temperature decreased. At 5 hours after modeling, the temperature of the rats in the Guizhi Decoction group was lower than that of the model group. There was a significant difference at the 6 h and 7 h detection time point(P<0.05). Nine differential metabolites related to the antipyretic of Guizhi Decoction were found by metabolomics analysis technology: Stearic acid, L-Lactic acid, Glycerol, Palmitic acid, Linoleic acid, D-Glucose, Urea, Arachidonic acid and L-Threonine. The pathway analysis using differential metabolites was found that the antipyretic effect of Guizhi Decoction may be related to Linoleic acid metabolism pathway, Arachidonic acid metabolism pathway, Starch and sucrose metabolism pathway, and Glyceride metabolism pathway. The two largest modules were obtained, wh ich were Arachidonic acid metabolism pathway and Linoleic acid metabolism pathway by modular analysis.According to the degree value, the screened potential targets were CYP450 and PFKFB1, PFKFB2. Conclusion: Guizhi Decoction has a good antipyretic effect, which may be synergistic from multiple aspects such as amino acid, sugar metabolism, and fat metabolism, with multiple targets.