Liqi Yangyin Formula Ameliorates CUMS-Induced Depression and Comorbid Constipation via ACE/FFAR2 Modulation of the Microbiota-Gut-Brain Axis

Lianjie Xu^1*Shun Seng Ong¹Xiaoyue Deng¹Yunzhi Qian²Hai Lu³Yang Song³Yang Wang³Xu Tianshu³

• ¹Nanjing University of Chinese Medicine, Nanjing, China
• ²The University of North Carolina at Chapel Hill Libraries, Chapel Hill, United States
• ³Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital, Nanjing, China

Background: The gut-brain axis, involving bidirectional signaling between the gastrointestinal tract and the central nervous system. Clinical observations have shown that Liqi Yangyin (LQYY) can effectively relieve symptoms of depression accompanied by constipation. However, whether LQYY exerts its effects through gut-brain crosstalk remains to be elucidated. Methods: A chronic unpredictable mild stress (CUMS) protocol was employed to establish a mouse model. H&E and Nissl staining were used to examine pathological changes in the prefrontal cortex (PFC) and colon. The ultrastructure of the intestinal barrier was observed via transmission electron microscopy, while the expression of the blood-brain barrier tight junction proteins was quantified by Western blotting (WB). ELISA quantified inflammatory factors and serotonin (5-HT) levels. Immunohistochemistry, immunofluorescence, and WB analyzed IBA-1 and Free fatty acid receptor 2 (FFAR2) expression levels. Gut microbiota composition was analyzed via 16S rDNA sequencing, and SCFAs levels were quantified using UHPLC-TSQ Altis Plus. Additionally, in vitro studies using BV-2 cells involved treatments with acetic acid (ACE) and an FFAR2 antagonist, after which the expression of relevant indicators was assessed. Results: Our results demonstrated that LQYY significantly ameliorated CUMS-induced behavioral changes and improved intestinal motility. These effects were associated with the restoration of gut microbiota balance and an increase in ACE levels. LQYY increased FFAR2 expression, leading to reduced neuroinflammation and enhanced colonic 5-HT secretion. Furthermore, LQYY protected intestinal and blood-brain barrier integrity and improved neuronal morphology in the PFC. In vitro studies confirmed that ACE suppresses microglial inflammation through upregulating FFAR2 expression, an effect that was attenuated by the FFAR2 inhibitor GLPG0974. Conclusions: These findings suggest that LQYY modulates the gut-brain axis through ACE/FFAR2, offering a promising therapeutic approach for depression and constipation.