摘要：

Fuzi-Lizhong pill (FLP) is a well-validated traditional Chinese medicine (TCM) formula that has long been used in China for gastrointestinal disease and adjunctive therapy for depression. In our previous study, we reported that the principal herb of FLP, Aconitum carmichaelii Debx. (Fuzi), exhibits antidepressant-like effects. However, there have been no reports on whether FLP produces antidepressant-like effects and its potential molecular mechanisms. We aim to demonstrate the antidepressant-like effects of FLP in chronic restraint stress (CRS) mice and to explore the associated molecular mechanisms. The active components and probable molecular targets of FLP, as well as the targets related to depression, were identified through network pharmacology. A protein-protein interaction (PPI) network was generated using the overlapping targets, followed by the visualization as well as identification of the core targets associated with the antidepressant-like action of FLP. Subsequently, KEGG and GO enrichment analyses were conducted. UHPLC-MS/MS was employed to further detect the active compounds in FLP. Molecular docking was applied to assess the connections between the active components as well as the core targets. The efficacy of FLP in treating depression and its molecular mechanisms were examined using western blotting, ELISA, 16S rRNA sequencing, HE staining, Nissl staining, and Golgi-Cox staining in a CRS-induced mouse model. Network pharmacology and UHPLC-MS/MS analyses indicated that the active compounds of FLP comprised taraxerol, songorine, neokadsuranic acid B, ginkgetin, hispaglabridin B, quercetin, benzoylmesaconine and liquiritin. KEGG pathway analysis implicated that the PI3K/Akt/mTOR as well as MAPK signaling pathways are closely related to the therapeutic effects of FLP on depression. Molecular docking analysis demonstrated that the main components of FLP bind to PI3K, AKT, mTOR, BDNF and MAPK. FLP significantly decreased immobility in mice that were elevated by CRS in the FST and the TST. FLP also significantly increased sucrose preference in mice after CRS in the SPT. FLP upregulated proteins associated with BDNF-TrkB and PI3K/Akt/mTOR signaling and downregulated proteins associated with MAPK signaling. Serum levels of CORT, IL-6, IL-1β, and TNF-α in CRS mice were significantly decreased following treatment with FLP. In addition, FLP ameliorated CRS-induced gut microbiota dysbiosis as demonstrated by 16S rRNA sequencing analysis. FLP ameliorated CRS-induced intestinal inflammation and neuronal damage. Finally, antidepressant-like effects and concomitant increases in dendritic spine density induced by FLP administration were also reduced after rapamycin treatment. These results demonstrate that FLP has antidepressant-like effects in mice exposed to CRS that involve activation of the PI3K/Akt/mTOR signaling pathway, increase in spinogenesis, inhibition of the MAPK signaling pathway, decrease in inflammation, and amelioration of gut microbiota dysbiosis. These findings provide novel evidence for the clinical application of FLP on depression.

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