Effects Of Xiaoyaosan On The CAMP Signaling Pathway in The Cerebral Cortex Of Depressed Rats
Nov 26, 2025
Abstract
Objective: To evaluate changes in serum prostaglandin E2 (PGE2) and substance P (SP) and the mRNA expression of cyclic adenosine monophosphate (cAMP) and cAMP response element-binding protein 1 (CREB1) in the cerebral cortex of depressed rats, and to explore the molecular mechanism by which Xiaoyaosan exerts antidepressant effects.
Methods: Forty-eight male SD rats were randomly assigned to four groups (n = 12 each): normal, model, Xiaoyaosan, and fluoxetine. A depression model was established using chronic immobilization stress (CIS). Xiaoyaosan was administered by gavage 30 minutes before daily modeling for 21 days. Antidepressant-like effects were assessed by macroscopic behavioral observation, sucrose preference test, open-field test, and forced swim test. Cortical cAMP and CREB1 mRNA levels were quantified by real-time fluorescence qPCR. Serum PGE2 and SP were measured by enzyme-linked immunosorbent assay (ELISA).
Results: Compared with the model group, both Xiaoyaosan and fluoxetine significantly attenuated depression-like behavior; increased sucrose consumption, vertical rearing counts, horizontal crossings, and grooming in the open-field test; and reduced cumulative immobility time in the forced swim test. Both treatments improved cortical histopathology, elevated cAMP and CREB1 mRNA expression in the cerebral cortex, and reduced serum PGE2 and SP levels (P < 0.01).
Conclusion: Xiaoyaosan exerts clear antidepressant effects in depressed rats, potentially via regulation of the cortical cAMP signaling pathway.
Keywords: Xiaoyaosan; Depression; cAMP; CREB1; Prostaglandin E2 (PGE2); Substance P (SP)
Introduction
Depression is a leading cause of morbidity and disability worldwide, profoundly impairing quality of life. Xiaoyaosan, a classical multi-herb traditional Chinese medicine (TCM) formula, has demonstrated clinical utility across common depressive presentations, supporting its continued development as a botanical therapy.
Mechanistically, the cAMP–protein kinase A (PKA)–CREB signaling cascade is central to the regulation of mood and affective behavior. Downregulation of this pathway can precipitate depressive phenotypes. CREB is implicated in the pathophysiology of depression, and activation by cAMP enhances transcription of downstream genes; polymorphisms and functional reductions in this axis have been considered depression susceptibility factors.
Inflammatory mediators also contribute to depression. Prostaglandin E2 (PGE2) mediates inflammatory responses, and elevated PGE2 can induce depressive behaviors. Substance P (SP), a neuropeptide linked to stress and affect, is often elevated in the serum of patients with depression. Thus, altered cortical cAMP and CREB1 mRNA expression together with elevated serum PGE2 and SP may represent a molecular mechanism underlying cortical injury in depression.
Although numerous TCM compound formulas have been investigated for antidepressant mechanisms, the influence of most formulas on the cAMP pathway remains unclear. Using a chronic immobilization stress (CIS) paradigm to induce depression-like behavior in rats, this study further investigates the antidepressant mechanism of Xiaoyaosan and compares it with fluoxetine, aiming to inform future development of botanical therapeutics and provide actionable insights for people seeking help for depression.
HOW TCM HERB SUPPLEMENTS RELIEVE PROSTATITIS SYMPTOMS
Materials
Animals
Forty-eight SPF-grade healthy adult male Sprague–Dawley rats (8 weeks old, 200–220 g) were purchased from Beijing Viton Lihua Experimental Animal Technology Co., Ltd. [License No.: SCXK (Beijing) 2016-0006]. Rats were housed in the barrier animal facility of Guizhou University of Traditional Chinese Medicine at 22 ± 2°C, relative humidity 35%–55%, 12 h light/12 h dark (7:00–19:00), four rats per cage, with 7 days of acclimatization. Ethics approval: Guizhou University of TCM (No. 20210142).
HOW TCM HERB SUPPLEMENTS RELIEVE PROSTATITIS SYMPTOMS
TCM HERB SUPPLEMENTS FOR SALE
Drugs
The Xiaoyaosan formula followed the Taiping Huimin Heji Ju Fang proportions. Crude herbs were purchased from Beijing Tongrentang (Bozhou) Decoction Pieces Co., Ltd.: Bupleurum (Chaihu), White Peony (Baishao), Angelica sinensis (Danggui), Stir-fried Atractylodes (Baizhu), Poria (Fuling), Honey-fried Licorice (Zhigancao), Mentha (Bohe), and Fresh Ginger (Shengjiang), combined in a ratio of 5:5:5:5:5:4:1:5. The decoction and concentration were prepared in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China by Hunan Jiuzhitang, serving as the TCM intervention. Doses were converted for rats, dissolved in deionized water, and prepared as a suspension.
Fluoxetine hydrochloride capsules (Patheon France; packaged by Eli Lilly Suzhou Pharmaceutical Co., Ltd.; batch No. 6663B; 20 mg/capsule) were used as the reference antidepressant.
Reagents
ExTaq (TAKARA, Cat. DRR100A); SYBR Green/Fluorescein qPCR Master Mix (2×) (Fermentas, Cat. #K0242); primers (Qingke Bio); Rat PGE2 and SP ELISA kits (Shanghai Yuanju Biotechnology Center, Cat. CK-E30697 and CK-E30122).
Instruments
7900 real-time fluorescence qPCR system (Applied Biosystems, USA); micropipettes (Eppendorf); paraffin microtome (HY1508, Jinhua Huiyou); Aquapro ultrapure water system (AJY-0501, Aikepu).
Methods
Grouping and modeling
Rats were randomly assigned to normal, model, Xiaoyaosan, and fluoxetine groups (n = 12 each). Except for the normal group, all groups underwent CIS for 3 h/day at randomized times for 21 days. Normal rats were not restrained but were food- and water-deprived during the same time windows to match handling.
Drug administration
Based on prior studies, Xiaoyaosan was administered by gavage at 2.22 g crude drug·kg−1·day−1; fluoxetine at 2.0 mg·kg−1·day−1. Drugs were dissolved in deionized water. Doses were calculated by body surface area conversion; gavage volume was 10 mL/kg, given 30 minutes before each daily restraint session. Normal and model groups received equal volumes of deionized water.
Outcome measures and procedures
3.1 Macroscopic observations
Daily observations recorded mental status, locomotion, and general appearance.
3.2 Sucrose preference test
Rats were trained for 3 days. Day 1: two bottles of 1% sucrose per cage. Day 2: one bottle replaced with equal-volume double-distilled water. After 24 h food and water deprivation, each rat received one bottle of 1% sucrose and one of double-distilled water for 1 h. Bottles were weighed to calculate sucrose preference (%): sucrose intake / (sucrose + water intake) × 100%.
3.3 Open-field test
A black-walled open field (125 cm × 125 cm × 50 cm) was used in a quiet room. Rats were placed at the center; over 5 minutes, vertical rearing counts, horizontal grid crossings, and grooming events were recorded. The arena was cleaned with 75% ethanol and water between trials.
3.4 Forced swim test
Rats were individually placed in glass cylinders with 35 cm water at 20 ± 2°C. From immersion, behavior was recorded for 5 min with a high-resolution camera. Cumulative immobility time over 5 min was measured (immobility defined as minimal movement to keep nose above water, slight body flexion for balance). Water was changed between animals.
3.5 Histopathology
Rats were anesthetized with 3% sodium pentobarbital (0.15 mL/100 g). Six rats per group underwent transcardial perfusion with 4% paraformaldehyde followed by 0.9% saline for fixation, decapitation, and brain extraction. Brains were immersed in 4% paraformaldehyde (4°C) for 72 h, paraffin-embedded, trimmed, and sectioned at ~5 μm. Sections were floated at 48°C, mounted, baked at 60°C for 4 h, deparaffinized and rehydrated, stained with hematoxylin, differentiated, rinsed, counterstained with eosin, dehydrated, cleared, and coverslipped for microscopic examination. The remaining six rats per group were euthanized; cerebral cortex was rapidly dissected on ice and stored at −80°C for RNA extraction.
3.6 mRNA quantification
Target sequences were retrieved from NCBI and primers designed with Primer 6.0.
cAMP: Forward 5′-CCGTGCTGTGGATGACTTCAACC-3′; Reverse 5′-AGTCTCCTTCACTCGGAACCTCA-3′; 133 bp.
CREB1: Forward 5′-GGAGCAGACAACCAGCAGAGTG-3′; Reverse 5′-GGATACCTGGGCTAATGTGGCA-3′; 105 bp.
GAPDH: Forward 5′-AAGGGCTCATGACCACAGTC-3′; Reverse 5′-GGATGCAGGGATGATGTTCT-3′; 116 bp.
qPCR conditions: pre-denaturation 90°C for 30 s; 45 cycles of 95°C 10 s, 60°C 30 s, 72°C 30 s. Melt-curve analysis: 95°C 1 min, 55°C 1 min, 95°C 1 min.
3.7 Serum inflammatory factors
For ELISA: (i) set standards and samples; add 50 μL standards at graded concentrations to standard wells; (ii) add 40 μL sample diluent to blank and sample wells, then 10 μL sample and mix; (iii) add 100 μL enzyme conjugate to all but blanks; (iv) seal and incubate at 37°C for 1 h; (v) dilute concentrated wash buffer 1:20; (vi) wash 5 times and pat dry; (vii) add 50 μL Substrate A then 50 μL Substrate B, incubate at 37°C protected from light for 15 min; (viii) add stop solution; (ix) read absorbance at 450 nm with blanks set to zero.
Statistical analysis
Data were analyzed in SPSS 26.0 and expressed as mean ± SD. After confirming normality and homogeneity of variance, one-way ANOVA was used. P < 0.05 was considered statistically significant.
Reader's note for practitioners and people seeking help
For developers of botanical/TCM antidepressants: The study links a classical formula to a specific neurobiological pathway (cAMP–CREB) and inflammatory mediators (PGE2, SP). Such dual modulation-neuroplasticity signaling plus inflammation-aligns with contemporary depression biology and may guide formula optimization, biomarker selection, and combination strategies with SSRIs like fluoxetine.
For individuals exploring help for depression: Preclinical results cannot replace medical care, but they suggest Xiaoyaosan may improve depressive behaviors by normalizing brain signaling and reducing inflammatory signals. If you consider herbal options, consult a qualified clinician, especially if you are on antidepressants, to avoid interactions and ensure dosing and quality control.
Transitional takeaway
Xiaoyaosan improved behavior, brain histology, and molecular markers in CIS-induced depressed rats, likely via upregulating cortical cAMP/CREB signaling while lowering peripheral PGE2 and SP. Building on this, the next study will dissect upstream regulators of cAMP (e.g., adenylyl cyclase isoforms and phosphodiesterases) and determine whether Xiaoyaosan directly modulates these targets or acts primarily through anti-inflammatory pathways converging on CREB.
