代谢组学结合网络药理学技术探究“瓜蒌-薤白”药对抗动脉粥样硬化的配伍机制

Compatibility mechanism of Trichosanthis Fructus-Allii Macrostemonis Bulbus combination against atherosclerosis: based on metabolomics and network pharmacology

从代谢组学的角度,研究“瓜蒌-薤白”药对治疗载脂蛋白E基因敲除(ApoE-/-)小鼠动脉粥样硬化(AS)的配伍机制。利用高脂饮食建立AS小鼠模型,苏木素-伊红(HE)染色与Masson染色评价小鼠主动脉病理学形态;采用超高效液相色谱-四极杆飞行时间质谱技术对小鼠血清样品进行代谢轮廓分析,结合偏最小二乘判别分析与正交偏最小二乘判别分析等多元数据统计方法,筛选小鼠体内潜在生物标志物;运用网络药理学技术进一步筛选AS相关的代谢物及其代谢通路的干预靶点,Gene Ontology(GO)生物功能富集分析得到与代谢通路干预靶点相关的生物学途径。结果显示,瓜蒌及“瓜蒌-薤白”可不同程度地减少AS小鼠主动脉窦斑块(P<0.05)及胶原面积(P<0.05);与瓜蒌、薤白相比,“瓜蒌-薤白”减少主动脉窦斑块(P<0.05)及胶原面积(P<0.05)更为显著;代谢组学检测出16种小鼠体内潜在生物标志物,瓜蒌可回调甘油磷脂代谢途径中4种代谢物,薤白可回调花生四烯酸代谢途径中2种代谢物,“瓜蒌-薤白”药对可回调甘油磷脂、亚油酸、花生四烯酸及嘧啶代谢途径中8种代谢物;网络药理学筛选与AS相关的代谢物,表明瓜蒌回调2种AS相关代谢物,与24个代谢靶点关联,涉及甘油磷脂代谢相关生物途径;薤白回调2种AS相关代谢物,与40个代谢靶点关联,涉及花生四烯酸代谢相关生物途径;“瓜蒌-薤白”药对回调6种AS相关代谢物,与57个代谢靶点关联,涉及亚油酸、甘油磷脂、花生四烯酸代谢相关的生物途径。表明“瓜蒌-薤白”配伍后影响AS小鼠体内更多的代谢途径,增强对亚油酸代谢、甘油磷脂及花生四烯酸代谢的影响,相互协同改善AS小鼠体内脂质紊乱及炎症反应,发挥抗AS的作用。

This study aims to investigate the compatibility mechanism of Trichosanthis Fructus-Allii Macrostemonis Bulbus combination against atherosclerosis(AS) in apolipoprotein E-deficient(ApoE-/-) mice. To be specific, high-fat diet was used to induce AS in mice. The pathological morphology of mice aorta was evaluated based on hematoxylin-eosin(HE) staining and Masson staining. The metabolic profiling of mouse serum samples was performed with ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Multiple statistical analysis methods including partial least squares-discriminant analysis and orthogonal partial least squares-discriminant analysis were employed to screen potential biomarkers in mice. With the techniques in network pharmacology, the metabolites related to AS and the targets in the metabolic pathways were screened out. The results showed that Trichosanthis Fructus alone and the pair all reduced the plaque area of aortic sinus(P<0.05) and collagen area(P<0.05). Compared with the Trichosanthis Fructus alone and Allii Macrostemonis Bulbus alone, the combination significantly decreased the plaque area of aortic sinus(P<0.05) and collagen area(P<0.05). Metabolomics revealed 16 biomarkers in mice. Trichosanthis Fructus re-gulated the abnormal levels of 4 metabolites in glycerophosphatide metabolic pathway. Allii Macrostemonis Bulbus modulated the abnormal levels of 2 metabolites in arachidonic acid metabolic pathway and the combination recovered the levels of 8 metabolites in glycerophosphatide, linoleic acid, arachidonic acid, and pyrimidine metabolic pathways. Network pharmacology suggested that Trichosanthis Fructus regulated 24 targets which related to 2 AS-associated metabolites and involved glycerophosphatide metabolic pathway. Allii Macroste-monis Bulbus modulated 40 targets which related to 2 AS-associated metabolites and involved the arachidonic acid metabolic pathway. The combination regulated 57 targets which related to 6 AS-metabolites and involved linoleic acid metabolic pathway, glycerophosphatide metabolic pathway, and arachidonic acid metabolic pathway. These results indicate that the Trichosanthis Fructus-Allii Macrostemonis Bulbus combination enhances the regulation of linoleic acid metabolism, glycerophosphatide metabolism, and arachido-nic acid metabolism, thereby synergistically alleviating lipid disorder and inflammatory response in AS mice.