Xiaoyaosan ameliorates CUMS-induced depressive-like and anorexia behaviors in mice via necroptosis related cellular senescence in hypothalamus

Ethnopharmacological relevance: Depression and anorexia often co-occur and share symptoms such as low mood, lack of energy, and weight loss. Xiaoyaosan is a classic formula comprising of a combination of eight herbs, possessing definitive therapeutic effects, minimal side effects, and economical benefits. It has been extensively employed in clinical treatment of ailments and symptoms such as depression, anxiety, and appetite problems. Nonetheless, its exact pharmacological mechanism with necroptosis remains incompletely explicit. Aim of the study: The aim of this study is to explore the potential mechanisms of anti-depressive and appetiteregulating effects of the active ingredients in Xiaoyaosan, and to investigate whether there is a correlation with necroptosis. Materials and methods: The network pharmacology method was conducted to identify active ingredients, which were used to predict the possible targets of Xiaoyaosan and explore the potential targets in treating depression and anorexia by overlapping with differentially expressed genes (DEGs) screened from GEO datasets (GSE125441, GSE198597, and GSE69151). Afterwards, the protein-protein interaction (PPI) network, enrichment analyses, hub gene identification, co-expression study and molecular docking were used to study the potential mechanism of Xiaoyaosan. Then, a mice model of depression was established by chronic unpredictable mild stress (CUMS) and the incidence of necroptosis in the hypothalamus of CUMS mice was investigated, while verifying the key therapeutic target of Xiaoyaosan. Results: Through network pharmacology research, it had been discovered that the 145 active ingredients of the 8 herbs in the Xiaoyaosan could regulate 198 disease targets. Through PPI network analysis and functional enrichment analysis, it had been found that the pharmacological mechanism of Xiaoyaosan mainly involved biological processes such as oxidative stress, kinase activity, and DNA metabolism. It is related to various pathways such as cellular senescence, immune inflammation, and the cell cycle, and 9 hub targets had been identified. Further analysis of the 9 hub targets and the key PPI network clusters clarified the key mechanisms by which Xiaoyaosan exerts anti-depressant and appetite regulating effects, possibly related to necroptosis-mediated cellular senescence. Molecular docking of the key indicators of cellular senescence screened by bioinformatics, SIRT1, ABL1, and MYC, revealed that the key component regulating SIRT1 is 2-[3,4-dihydroxyphenyl]-5,7-dihydroxy-6-[3-methylbut-2-enyl]chromone in licorice root, Glabridin in licorice root regulates ABL1, and & beta;-sitosterol found in Chinese angelica, debark peony root, and fresh ginger regulates MYC. Finally, through in vivo experiments, the expression of necroptosis in the hypothalamus of CUMS mice was verified. The regulatory effects of Xiaoyaosan on key substances RIPK1, RIPK3, MLKL, and p-MLKL were determined, while regulating effects on SIRT1, ABL1, and MYC were also observed. Conclusion: The present study have revealed the common mechanism of Xiaoyaosan in treating depression and anorexia, indicating that the active ingredients of Xiaoyaosan may alleviate the symptoms of depression and anorexia by intervening in the pathways related to necroptosis and cellular senescence. The hub genes and