Depression remains one of the most burdensome psychiatric illnesses worldwide, marked by persistent low mood, loss of pleasure, anxiety, cognitive difficulties, and impaired social functioning, and in severe cases by suicidal ideation. Current first-line drugs, including selective serotonin reuptake inhibitors and serotonin–norepinephrine reuptake inhibitors, work mainly by raising the availability of serotonin and norepinephrine in the brain. Yet these medications are far from perfect. They often take days or even weeks to produce noticeable benefit, and a considerable share of patients relapse after remission. This therapeutic gap has pushed researchers to look beyond the classic monoamine hypothesis and to explore alternative strategies, including compounds derived from traditional medicine systems that have been used clinically for centuries.

One such candidate is Chaihu Shugan San, a classical Chinese herbal formula composed of Bupleuri Radix (Chaihu), Paeoniae Radix Alba (Shaoyao), Cyperi Rhizoma (Xiangfu), Aurantii Fructus (Zhiqiao), Citri Reticulatae Pericarpium (Chenpi), Chuanxiong Rhizoma (Chuanxiong), and Glycyrrhizae Radix et Rhizoma (Gancao). The formula has long been prescribed for mood disorders, and recent clinical meta-analyses suggest it is effective and generally safe for improving depressive symptoms. Earlier studies hint that its mechanisms may involve modulation of monoaminergic neurotransmission, neuroinflammation, hypothalamic–pituitary–adrenal axis dysfunction, and neurotrophic signaling. However, a complete picture of how the formula acts in the brain has remained elusive, and one emerging line of evidence points to a less obvious suspect: the excitatory neurotransmitter glutamate.

Glutamate is the brain’s principal excitatory messenger, but when it accumulates outside cells it becomes toxic to neurons. Astrocytes, the star-shaped support cells of the central nervous system, keep extracellular glutamate in check largely through a transporter called excitatory amino acid transporter 2, also known as GLT-1, which handles the majority of synaptic glutamate uptake. Chronic stress has been shown to downregulate GLT-1 expression, potentially allowing glutamate to build up and injure neurons. Intriguingly, several bioactive constituents of Chaihu Shugan San have previously been reported to modulate GLT-1 and to protect neurons from injury. That convergence made GLT-1-mediated glutamate transport a compelling mechanistic lead for the research team behind the new study.

To pursue it, the investigators deployed an integrated strategy that has become a mainstay of modern herbal pharmacology research: ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) to map the chemical composition of the formula, network pharmacology to connect its constituents to disease targets, molecular docking to probe binding interactions, and in vivo experiments to test the predictions in living animals. The herbal materials, quality-certified and prepared in proportions of Chaihu 6 g, Chenpi 6 g, Chuanxiong 4.5 g, Xiangfu 4.5 g, Zhiqiao 4.5 g, Shaoyao 4.5 g, and Gancao 1.5 g, were extracted and analyzed on a high-resolution Q-Exactive HFX mass spectrometer in both positive and negative electrospray ionization modes, with chromatographic separation on an HSS T3 column using a carefully graded acetonitrile-formic acid elution program.

The chemical profiling identified thirty representative constituents spanning multiple classes, with flavonoids dominating the spectrum, followed by coumarins, triterpenes, monoterpenes, phenolic acids, and peptides. Prominent compounds included nobiletin, hesperetin, naringenin, glycyrrhizinate, paeoniflorin, and albiflorin, alongside tryptophan, gallic acid, and a series of polymethoxylated citrus flavones. In parallel, the network pharmacology pipeline screened candidate compounds from the Traditional Chinese Medicine Systems Pharmacology database using oral bioavailability of at least 30 percent and drug-likeness of at least 0.18 as thresholds, yielding 908 formula-associated protein targets after standardization to human gene symbols. Crossing these with 1,986 depression-related targets harvested from OMIM and GeneCards produced 330 overlapping targets considered central to the formula’s potential antidepressant-like effects.

Functional enrichment analysis of those shared targets painted a rich biological picture. Gene Ontology terms highlighted signal transduction, chemical synaptic transmission, responses to exogenous stimuli, and the upregulation of ERK1/ERK2 and PI3K/Akt signaling, while cellular component analysis concentrated on the plasma membrane, dendrites, synapses, and postsynaptic membranes. KEGG pathway mapping flagged circadian rhythm, serotonin synthesis, cAMP, HIF-1, Rap1, and, notably, the AGE–RAGE signaling pathway, which prior work has linked to glutamate homeostasis and stress-related neuroinflammation. Molecular docking then tested five flavonoids detected in the extract—kaempferol, hesperetin, isorhamnetin, naringenin, and nobiletin—against four AGE–RAGE pathway proteins: AKT1, MAPK10, MAPK3, and IL1B. The compounds showed favorable predicted binding affinities, with several flavonoid–MAPK3 and flavonoid–MAPK10 pairs displaying particularly strong scores, supporting the plausibility of the computationally predicted pathway.

The crucial question, of course, was whether the formula would actually work in a living nervous system. To find out, the team turned to a chronic restraint stress model in which male BALB/c mice were confined in 50 mL centrifuge tubes for six hours daily over 21 consecutive days. The animals were divided into five groups of six: unstressed controls receiving saline, stressed mice receiving saline, and stressed mice treated with low-dose Chaihu Shugan San (6.5 g/kg), high-dose Chaihu Shugan San (13 g/kg), or the reference antidepressant fluoxetine (4 mg/kg), all given by daily oral gavage. Behavioral assessment followed, using the sucrose preference test to gauge anhedonia, the open field test to measure exploration and anxiety-like behavior, and the tail suspension test to quantify behavioral despair.

The results were strikingly consistent across tests. Chronic restraint stress significantly reduced sucrose preference, shortened total distance traveled and time spent in the center of the open field arena, and lengthened immobility in the tail suspension test. Both doses of the herbal formula reversed much of this damage: high-dose treatment significantly increased central-zone time and total distance in the open field, low-dose treatment significantly raised sucrose preference, and both doses cut tail suspension immobility time. Fluoxetine improved sucrose preference and open field performance but did not significantly alter immobility time. Beyond behavior, Nissl staining revealed that stressed mice lost Nissl body density and staining intensity in the hippocampal CA1 and CA3 regions, hallmarks of neuronal stress damage, while both doses of the formula restored neuronal morphology, with high-dose treatment reaching significance in CA3 as well as CA1.

The biochemical story tied these threads together. Hippocampal glutamate levels were significantly elevated in stressed mice, and both doses of the formula, like fluoxetine, brought them back down. Immunohistochemistry showed that chronic stress reduced expression of GLT-1, the astrocytic glutamate transporter, and immunofluorescence revealed reduced GFAP, a marker of astrocytes; the herbal treatment significantly increased GLT-1 at both doses and raised GFAP at the low dose, partially repairing the astrocytic damage. Western blotting then validated the network pharmacology prediction in vivo: stressed mice showed elevated hippocampal RAGE expression and increased phosphorylation of NF-κB p65, and the formula significantly suppressed both, indicating inhibition of stress-induced RAGE/NF-κB pathway activation.

The authors are careful about causality. NF-κB’s relationship with GLT-1 is context dependent—the GLT-1 promoter contains multiple NF-κB-binding sites, and under pathological conditions such as chronic stress, sustained NF-κB activation can suppress GLT-1 transcription, potentially through recruited corepressors. The concurrent changes in RAGE/NF-κB signaling, GLT-1 expression, and glutamate levels observed here suggest an association consistent with restored glutamate homeostasis, but the researchers acknowledge that selective pathway inhibitors will be needed to establish whether these molecular shifts cause the behavioral improvements, and that further functional studies must clarify the role of astrocytic GLT-1 in glutamate regulation. Flavonoids such as kaempferol, hesperetin, isorhamnetin, naringenin, and nobiletin have independently been reported to exert anti-inflammatory, antioxidant, and neuroprotective effects, including reduction of excessive glutamate accumulation, lending plausibility to the multicomponent, multitarget interpretation. For now, the study stands as an experimental proof of concept: a centuries-old herbal prescription may ease stress-induced depressive-like behavior in mice, in part by coaxing the brain’s glutamate-clearing machinery back into balance—a mechanism that could inspire new avenues for treating a disorder that current medications still leave many patients behind.

Subject of Research: The antidepressant-like effects of the traditional Chinese medicine formula Chaihu Shugan San and their link to glutamate homeostasis, GLT-1 expression, and RAGE/NF-κB signaling in a chronic stress mouse model.

Article Title: Network Pharmacology and Experimental Validation Reveal the Potential Antidepressant‐Like Effects of Chaihu Shugan San Associated With Glutamate Homeostasis

Article References: Xu, Y., Xia, L., Zhou, K., Liu, C., Tian, X., & Yang, Z. (2026). Network Pharmacology and Experimental Validation Reveal the Potential Antidepressant‐Like Effects of Chaihu Shugan San Associated With Glutamate Homeostasis. Food Science & Nutrition, 14(9), Article e72384. https://doi.org/10.1002/fsn3.72384

Image Credits: AI Generated

DOI: 10.1002/fsn3.72384

Keywords: Chaihu Shugan San, depression, glutamate homeostasis, GLT-1, RAGE/NF-κB signaling, network pharmacology, molecular docking, chronic restraint stress, traditional Chinese medicine, flavonoids, astrocytes, hippocampus

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Tags: alternative depression therapies from Chinese medicineAncient Chinese herbal medicine for depressionastrocytesChaihu Shugan SanChaihu Shugan San antidepressant effectschronic restraint stressclinical meta-analysis of herbal antidepressantsDepressionflavonoidsGLT-1glutamate homeostasisherbal approaches to treatment-resistant depressionherbal formula neuroinflammation reductionherbal impact on hypothalamic-pituitary-adrenal axisherbal modulation of brain glutamate balanceherbal treatment for depressive symptomshippocampusmolecular dockingnetwork pharmacologyplant-based neurotrophic signalingRAGE/NF-κB signalingtraditional Chinese medicinetraditional Chinese medicine for mood disorderstraditional medicine and neurotransmitter regulation