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

Chaihu Shugan San produces rapid antidepressant-like effects within 2 h in female CRS-exposed mice. • The rapid effect is comparable in onset to ketamine in female chronically stressed mice. • Hippocampal TC2N is a negative regulator of the rapid antidepressant-like action of CSS. • TC2N down-regulation is associated with hippocampal ERβ–eNOS–CaMKII–BDNF signaling. • The estrogen receptor β-linked mechanism was demonstrated in female mice and requires validation in males. 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. Download: Download high-res image (318KB) Download: Download full-size image