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This study aimed to explore the effects of Ziziphi Spinosae Seme (ZSS) and Fried Ziziphi Spinosae Semen (FZSS) on metabolites and intestinal flora structure in insomnia mice by combining metabolomics analysis and 16S rRNA gene sequencing. Animal experimentation was approved according to the Committee on the Ethics of Animal Experiments of Shanxi University of Chinese Medicine (approval number: 2021DW172). The prediction targets were validated in para-chlorophenylalanine (PCPA) induced insomnia mice with administration of ZSS and FZSS for 5 days, respectively. Then pentobarbital sodium induced sleeping test were performed to evaluate the synergistic sleep-aiding effect of ZSS and FZSS. The metabolic profile of serum from insomnia mice was analyzed by UPLC-Q-Orbitrap MS. Different metabolites were screened combined with multivariate statistical analysis. The relevant metabolic pathways and networks were constructed by MetaboAnalyst 5.0. Intestinal flora changes were detected by 16S rRNA sequencing technology. Animal study indicated that, compared to PCPA-induced insomnia model, ZSS and FZSS shortened the sleeping latency and increased the sleeping duration. The serum metabolomics results showed that, there are 36 potential biomarkers associated with insomnia were identified. Compared with the model group, 25 and 27 differential metabolites were identified in the ZSS and FZSS groups, respectively. Both ZSS and FZSS groups could significantly adjust to the common 20 differential metabolites. A total of 10 pathways are closely related to insomnia, which including amino acid metabolism, energy metabolism and lipid metabolism. 5 metabolic pathways are shared in ZSS and FZSS, including phenylalanine, tyrosine and tryptophan biosynthesis, cysteine and methionine metabolism and so on. Arachidonic acid metabolism is the unique metabolic pathway for ZSS to improve sleep; 3 metabolic pathways including glutamate and glutamine are the unique regulatory pathway for FZSS to improve sleep. PCoA analysis showed that the structure of ZSS and FZSS recovered to blank group, ZSS and FZSS can decrease the abundance of Pvotella and increase the abundance of Lactobacillus. LEfSe and correlation analysis between metabolites and differential intestinal flora showed that kynurenic acid, L-glutamic acid, D-proline are significantly positively associated with Gammaproteobacteria in ZSS and 5-HT, acetylcholine, L-methionine are significantly positively associated with Lactobacillus in FZSS. In conclusion, both ZSS and FZSS can influence metabolic pathways such as amino acids, energy and lipids and regulate the structure of intestinal flora, and then play a part in treating insomnia. However, ZSS and FZSS improve the quality of sleeping by regulating different metabolic pathways and regulating gut microbiota structure. The results of this study provide an experimental basis for illustrating the action mechanism of ZSS and FZSS in treating insomnia and provide a scientific basis for clinical rational medication guidance.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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