Rapid antidepressant-like effects of Chaihu Shugan San in female restraint-stressed mice: involvement of hippocampal TC2N and ERβ-eNOS-CaMKII-BDNF signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Chaihu Shugan San (CSS), a classical seven-herb formula from the Ming-dynasty text Jing-Yue Quanshu, is widely prescribed for depressive disorders in China. Previous preclinical studies reported antidepressant-like effects only after weeks of repeated administration, mostly in male animals. Whether a single dose of CSS can produce rapid antidepressant-like effects in female animals, and through what mechanism, remains unclear. AIM OF THE STUDY: To determine whether a single administration of CSS produces rapid antidepressant-like effects in female mice exposed to chronic restraint stress (CRS), and to explore the underlying hippocampal mechanism. METHODS AND MATERIALS: CSS quality was controlled by HPLC quantification of four index components. Female CRS mice received a single dose of CSS (1, 2 or 4 g/kg, i.g.) or ketamine (10 mg/kg, i.p.), followed by behavioral testing (tail suspension, forced swimming, sucrose preference and open field tests) within 2-24 h. Hippocampal RNA sequencing, RT-qPCR, Western blotting and ELISA were used to identify and validate candidate targets; hippocampal AAV-mediated TC2N overexpression and pretreatment with the ERβ antagonist acolbifene or the CaMKII inhibitor KN-93 were used for interventional validation. RESULTS: Batch-to-batch variation of the four index components was below 10% (RSDs 3.51-5.66%), with naringin (313.07 ± 11.16 μg/mL) and hesperidin (196.40 ± 11.11 μg/mL) as the major constituents. Acute administration of CSS (2 and 4 g/kg), like ketamine, shortened immobility time in the tail suspension test at 2 h and in the forced swimming test at 12 h, and restored sucrose preference at 24 h, without affecting locomotor activity. Hippocampal transcriptomics identified Tc2n as up-regulated by CRS and down-regulated by CSS, with Esr2, Nos3 and Camk2a showing the opposite pattern; these changes were confirmed at the mRNA and protein levels within 2 h. Hippocampal TC2N overexpression or pretreatment with acolbifene or KN-93 abolished the rapid behavioral effects of CSS and the associated changes in ERβ-eNOS-CaMKII-BDNF signaling. CONCLUSION: CSS produces rapid, ketamine-comparable antidepressant-like behavioral effects in female CRS-exposed mice, accompanied by down-regulation of hippocampal TC2N and involvement of ERβ, eNOS, CaMKII and BDNF. Because only female mice were studied, these findings are specific to females and support an estrogen-linked mechanism that warrants confirmation in male animals and further translational study.