Chaihu-Shugan-San alleviates functional dyspepsia by inhibiting central sensitization and regulating glutamate metabolism via NMDAR/CaMKII signaling pathway

ETHNOPHARMACOLOGICAL RELEVANCE: Functional dyspepsia (FD) is a common functional gastrointestinal disease. Chaihu-Shugan-San (CSS), a classical traditional Chinese medicine (TCM) formula from the Ming Dynasty "Jingyue Quanshu". Clinical evidence suggests that CSS is a highly effective therapeutic approach for the treatment of FD, however, its therapeutic mechanism for FD has not been fully clarified. AIM OF THE STUDY: The present study aimed to explore the potential mechanism of CSS in treating FD from the perspective of disorders of gut-brain interaction (DGBI). MATERIALS AND METHODS: The FD rat model was established via modified tail-clamp stimulation combined with gavage, followed by 4 weeks of CSS decoction intervention. An open-field test (OFT) was used to assess anxiety-like behaviors, gastric emptying rate was measured to evaluate gastrointestinal motility function, and spinal cord c-fos expression was detected to reflect visceral hypersensitivity. Hematoxylin and eosin (HE) staining was employed to clarify gastric tissue pathological changes, while transmission electron microscopy was performed to examine the microstructural integrity of nerve cells, synaptic clefts, and synaptic vesicles. Quantitative targeted metabolomics was used to screen potential metabolites involved in gut-brain interaction. C-fos was evaluated through immunohistochemistry. Ionized calcium-binding adapter molecule 1 (Iba1) and NADPH oxidase 2 (NOX2), Excitatory amino acid transporters (EAAT2) and Vesicular Glutamate Transporter 1 (VGLUT1) in the amygdala were evaluated through multiple immunofluorescences, quantitative real-time polymerase chain reaction (qPCR), and Western blot were applied to analyze the expression levels of proteins and mRNA in the NMDAR/CaMKII pathway in the gastric antrum and spinal cords. RESULTS: We identified 607 bioactive components, with 129 detected in serum and 134 in the cerebral cortex, confirming systemic exposure and cerebral penetration of key constituents. FD rats exhibited lower gastric motility, and immunohistochemistry showed strong activation of c-fos, a marker of hypersensitivity. CSS improved the gastric emptying rate and alleviated central sensitization. Fecal metabolomics revealed that CSS treats FD by regulating Linoleic acid metabolism, Cyanoamino acid metabolism, and Glycine, serine, and threonine metabolism. Serum Metabolomics revealed that CSS treats FD by regulating alanine, aspartate, and glutamate metabolism. After CSS treatement, Iba1 and NOX2 in the amygdala decreased compared to the FD rats, indicating the inhibition of central sensitization. CSS increased the levels of postsynaptic density protein 95 (PSD95), EAAT2 and VGLUT1 in the amygdala and spinal cord, and reducing the protein levels of Calcium-calmodulin-dependent protein kinase II (CaMKII), N-methyl-d-aspartate receptors (NMDAR), and Interleukin-1β (IL-1β) in the spinal cord. Correlation analysis also showed that CSS treatment significantly modulated the glutamate metabolic pathway in FD rats. CONCLUSION: These findings demonstrate that CSS ameliorates FD by inhibiting central sensitization - related activity and modulating glutamate metabolism, potentially mediated by the NMDAR/CaMKII signaling pathway.