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

Abstract

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.