整合代谢组学与肠道菌群分析酸枣仁汤改善失眠大鼠的作用机制

Mechanism of Suanzaoren Decoction in improving insomnia rats by integrating metabolomics and intestinal flora analysis

为探讨酸枣仁汤(SZRD)改善对氯苯丙氨酸(PCPA)致失眠大鼠模型的作用机制,通过腹腔注射PCPA溶液(400 mg·kg^(-1))制备失眠大鼠模型。利用UPLC-Q-TOF-MS/MS技术对大鼠海马样本进行代谢产物轮廓分析,并结合多元统计分析结合筛选差异代谢物,借助MetaboAnalyst 5.0构建相关代谢通路。利用16S rRNA基因V3~V4区高通量测序,通过LEfSe、OPLS-DA、PICRUSt2预测功能分析肠道菌群的结构及相对丰度变化。在海马中共鉴定出22个差异代谢物,包括氨基酸类、脂肪酸类、核苷类、有机酸类、维生素类、其他类等;共涉及8条代谢通路,主要为丙氨酸、天冬氨酸和谷氨酸代谢,D-谷氨酰胺和D-谷氨酸代谢,苯丙氨酸、酪氨酸和色氨酸生物合成,精氨酸生物合成等。16S rRNA基因测序显示,瘤胃球菌属Ruminococcus、真杆菌属Eubacterium是SZRD组与模型组的差异菌群;PICRUSt2与LEfSe整合分析表明瘤胃球菌属Ruminococcus可能是SZRD改善PCPA失眠大鼠的标志性菌属。进一步采用Spearman相关性分析,结果显示SZRD干预的海马差异代谢物2-氧基-4-甲基硫代丁酸和棕榈烯酸与差异菌呈显著正相关。综上,SZRD通过影响氨基酸等代谢通路,调控菌群结构,从而间接发挥改善失眠作用。研究结果为从肠道菌群角度阐释经典名方酸枣仁汤改善失眠作用机制提供了新机制、新思路。

To explore the mechanism of Suanzaoren Decoction(SZRD) in improving the insomnia rat model induced by DL-4-chlorophenylalanine(PCPA). The insomnia model was established by single intraperitoneal injection with PCPA(400 mg·kg^(-1)), UPLC-Q-TOF-MS/MS was used to analyze the profile of metabolites in rat hippocampus samples, combined with multivariate statistical analysis and screening of differential metabolites, and related metabolic pathways were constructed with MetaboAnalyst 5.0. The high-throughput sequencing of V3-V4 regions of 16 S rRNA gene was used to predict the structure and relative abundance of intestinal flora by LEfSe, OPLS-DA and PICRUSt2. A total of 22 differential hippocampus metabolites were identified by metabolomics analysis, including amino acids, fatty acids, nucleosides, organic acids, vitamins, and others. Pathway analysis showed that alanine, aspartate and glutamic metabolism, D-glutamine and D-glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, arginine biosynthesis were the main pathways. 16 S rRNA gene sequencing showed that Ruminococcus and Eubacterium were the differences between SZRD group and model group. Ruminococcus might be the sign of SZRD improving PCPA insomnia on analysis of PICRUSt2 and LEfSe. Furthermore Spearman correlation analysis showed that the differential metabolites 2-oxo-4-methylthiobutyric acid and palmitic acid intervened by SZRD were significantly positively correlated with the differential flora. In conclusion, SZRD indirectly improves insomnia by affecting metabolic pathways such as amino acids metabolic pathways and regulating the structure of flora. The results of this study provide a new mechanism and new idea for elucidating the mechanism of classic famous prescription SZRD in improving insomnia from the perspective of intestinal flora.