目的 利用尿液代谢组学结合网络药理学研究山茱萸多糖改善2型糖尿病合并非酒精性脂肪性肝病（type 2 diabetes mellitus-non-alcoholic fatty liver disease，T2DM-NAFLD）的作用机制。方法 通过气相色谱法测定山茱萸多糖的单糖组成。采用高糖高脂饲料喂养联合ip链脲佐菌素的方法构建T2DM-NAFLD大鼠，给予山茱萸多糖或二甲双胍干预28 d后，考察山茱萸多糖对T2DM-NAFLD模型大鼠空腹胰岛素、糖化血清蛋白、胰岛素抵抗指数以及胰腺组织病理变化的影响。采用超高效液相色谱-线性离子阱串联静电场轨道高分辨质谱技术检测大鼠尿液代谢物水平，利用主成分分析、偏最小二乘-判别分析和正交偏最小二乘法-判别分析等多元统计方法，结合MetaboAnalyst数据库寻找各组差异代谢物与相关代谢通路。采用网络药理学方法探究山茱萸多糖的靶点及相关疾病靶点，构建交集靶点的蛋白-蛋白相互作用网络，筛选的靶点网络结合代谢组学筛选的交集靶点联合分析相同代谢通路，并采用Western blotting检测核心靶点的蛋白表达水平。结果 与模型组比较，山茱萸多糖显著降低大鼠血清中空腹胰岛素、糖化血清蛋白水平和胰岛素抵抗指数（P＜0.05、0.01），改善胰腺组织病理变化。山茱萸多糖可有效调节T2DM-NAFLD大鼠尿液代谢轮廓。结合多元统计分析和在线数据库，共筛选出62个模型组和对照组组间显著变化的代谢标志物；山茱萸多糖可显著回调其中的14个代谢标志物，主要富集到乙醛酸和二羧酸代谢、甘氨酸、丝氨酸和苏氨酸代谢等21条代谢通路。代谢组学与网络药理学联合分析显示，二者均涉及甘氨酸、丝氨酸和苏氨酸代谢、嘌呤代谢2条代谢通路。Western blotting结果显示，山茱萸多糖干预后，大鼠肝脏中丝氨酸羟甲基转移酶2和脱氧胞苷激酶蛋白表达水平显著降低（P＜0.01），丙氨酸乙醛酸转氨酶、甘氨酸-N-甲基转移酶和含漆酶结构域 1蛋白表达水平显著升高（P＜0.01）。结论 山茱萸多糖能通过氨基酸代谢和嘌呤代谢等糖脂代谢通路改善T2DM-NAFLD，为山茱萸多糖干预T2DM-NAFLD的临床应用提供理论依据。

Objective To investigate the mechanism of Shanzhuyu (Corni Fructus) polysaccharides in improving type 2 diabetes mellitus combined with non-alcoholic fatty liver disease (T2DM-NAFLD) using urinary metabolomics combined with network pharmacology. Methods Gas chromatography was adopted to determine the monosaccharide composition of Corni Fructus polysaccharides. T2DM-NAFLD rats were constructed by feeding high sugar and high-fat diet combined with ip streptozotocin. After intervention with Corni Fructus polysaccharides or metformin for 28 d, the effects of Corni Fructus polysaccharides on fasting insulin, glycosylated serum protein, insulin resistance index and pancreatic tissue pathological changes in T2DM-NAFLD model rats were investigated. Ultra-high performance liquid chromatography-linear ion trap combined with electrostatic field orbit high-resolution mass spectrometry technology was applied to detect the levels of urinary metabolites in rats. Multivariate statistical methods such as principal component analysis, partial least squares-discriminant analysis and orthogonal partial least squares-discriminant analysis were used, and combined with MetaboAnalyst database to find the differential metabolites and related metabolic pathways among groups. Network pharmacology method was used to explore the component targets of Corni Fructus polysaccharides and the targets of related diseases, and to construct the protein-protein interaction network of intersection targets. The target network screened was combined with the intersection targets screened by metabolomics for joint analysis of the same metabolic pathways, and Western blotting was used to detect the protein expression levels of core targets. Results Compared with model group, Corni Fructus polysaccharides significantly reduced fasting insulin, glycosylated serum protein levels and insulin resistance index in serum of rats (P < 0.05, 0.01), and improved pancreatic tissue pathological changes. Corni Fructus polysaccharides could effectively regulate the urinary metabolic profile of T2DM-NAFLD rats. Combined with multivariate statistical analysis and online database, a total of 62 metabolic markers with significant changes between model group and control group were screened out. Corni Fructus polysaccharides could significantly adjust 14 metabolic markers of them, which were mainly enriched in 21 metabolic pathways such as glyoxylate and dicarboxylic acid metabolism, glycine, serine and threonine metabolism. The combined analysis of metabolomics and network pharmacology showed that both involved two metabolic pathways such as glycine, serine and threonine metabolism and purine metabolism. Western blotting results showed that after the intervention of Corni Fructus polysaccharides, the protein expressions of serine hydroxymethyltransferase 2 and deoxycytidine kinase in liver of rats were significantly decreased (P < 0.01), and the protein expressions of alanine glyoxylate transaminase, glycine-N-methyltransferase and laccase domain-containing protein 1 were significantly increased (P < 0.01). Conclusion Corni Fructus polysaccharides could improve T2DM-NAFLD through glycolipid metabolic pathways such as amino acid metabolism and purine metabolism, providing a theoretical basis for the clinical application of Corni Fructus polysaccharides in intervention of T2DM-NAFLD.

R285.5

国家重点研发计划（2023YFC3504000）；河南省中药材产业技术体系项目（豫农科教[2018]14号）；河南省重大科技专项（221100310400）；河南省2024年科技发展计划（242102310524）；郑州市科技协同创新项目（2023XTCX053）；河南中医药大学博士科研基金（BSJJ2022-04）