Modified Chaihu Guizhi Decoction alleviates anxiety- and depression-like behaviors in mice with chronic unpredictable mild stress by inhibiting the JAK2/STAT3 signaling pathway

doi:10.12122/j.issn.1673-4254.2025.10.11

Abstract

Abstract:

Objective To investigate the mechanisms of Modified Chaihu Guizhi Decoction (MCGD) for ameliorating anxiety- and depression-like behaviors in a mouse model of chronic unpredictable mild stress (CUMS). Methods The main chemical constituents of MCGD were identified through literature review, and network pharmacology analysis was performed to predict the potential pharmacological mechanisms of MCGD. For in vivo validation, male C57BL/6J mice were randomized into control group, CUMS model group, fluoxetine (FLX) treatment group, and low- and high-dose MCGD treatment groups (n=15), and in all but the control group, CUMS models were established by daily exposure to two randomized stressors for 28 consecutive days. Starting from 3 days prior to modeling, MCGD and fluoxetine treatments were administered daily via gavage and intraperitoneal injection, respectively. Depression- and anxiety-like behaviors of the mice were assessed using sucrose preference test, forced swim test, open field test and elevated plus maze test. The changes in mRNA expressions of the clock genes and inflammatory markers and expressions of the JAK2/STAT3 signaling proteins were detected using RT-qPCR and Western blotting, and immunofluorescence staining was used to detect microglia activation in the mice. Results The key active compounds in MCGD identified by network pharmacology analysis included quercetin, acacetin, formononetin, nobiletin, and baicalein. GO analysis identified 607 enriched pathways, and KEGG pathway enrichment revealed significant involvement of the JAK2/STAT3 and NF-κB signaling pathways. In the mouse models of CUMS, treatment with both fluoxetine and MCGD significantly alleviated anxiety- and depression-like behaviors. MCGD treatment significantly reduced Iba1 expression, improved the inflammatory markers, reversed the decrease in clock gene circadian rhythm amplitude, and obviously downregulated the expressions of JAK2, p-STAT3, p-NF-κB, IL-1β, and IL-6 proteins. Conclusion MCGD effectively alleviates anxiety- and depression-like behaviors in CUMS mice by modulating the inflammatory pathways and inhibiting the JAK2/STAT3 signaling pathway.

Key words: Modified Chaihu Guizhi Decoction, chronic unpredictable mild stress model, JAK2/STAT3, network pharmacology, anxiety, depression

Xiaotao LIANG, Xiaoshan LIANG, Yifan XIONG, Shiru XIE, Xiaoyu ZHU, Wei XIE. Modified Chaihu Guizhi Decoction alleviates anxiety- and depression-like behaviors in mice with chronic unpredictable mild stress by inhibiting the JAK2/STAT3 signaling pathway[J]. Journal of Southern Medical University, 2025, 45(10): 2146-2159.

Figures/Tables 12

Tab.1 Primer sequences used for RT-qPCR in this study

Primer name	Sequence
IFN-γ Forward	AGCAACAACATAAGCGTCATTGA
IFN-γ Reverse	TGACCTCAAACTTGGCAATACTC
TNF-α Forward	GCCTCCCTCTCATCAGTTCTATG
TNF-α Reverse	ACCTGGGAGTAGACAAGGTACAA
IL-1β Forward	GCAACTGTTCCTGAACTCAACT
IL-1β Reverse	ATCTTTTGGGGTCCGTCAACT
IL-4 Forward	TCCTCACAGCAACGAAGAACA
IL-4 Reverse	AGGCATCGAAAAGCCCGAAA
IL-6 Forward	ACAACCACGGCCTTCCCTACTT
IL-6 Reverse	CACGATTTCCCAGAGAACATGTG
IL-10 Forward	GGTTGCCAAGCCTTATCGGA
IL-10 Reverse	AGACACCTTGGTCTTGGAGCTTA
Bmal1 Forward	CTCCAGGAGGCAAGAAGATTC
Bmal1 Reverse	ATAGTCCAGTGGAAGGAATG
Clock Forward	TTCCTTCCTTAGAGACGAGACT
Clock Reverse	CTAAATGCTACCCTGAGGATAGAG
Cry1 Forward	GATCCACCATTTAGCCAGACAC
Cry1 Reverse	ACAGCCACATCCAACTTCCA
Cry2 Forward	CAAGCACTTGGAACGGAAGG
Cry2 Reverse	GAAGAGGCGGCAGGAGAG
Per1 Forward	CAGGCTAACCAGGAATATTACCAGC
Per1 Reverse	CACAGCCACAGAGAAGGTGTCCTGG
Per2 Forward	CCACACTTGCCTCCGAAATA
Per2 Reverse	ACTGCCTCTGGACTGGAAGA
GAPDH Forward	CAACTACATGGTCTACATGTTC
GAPDH Reverse	CTCGCTCCTGGAAGATG

Fig.1 Network pharmacology analysis of the mechanisms by which Modified Chaihu Guizhi Decoction (MCGD) alleviates anxiety- and depression-like behavior in CUMS. A: Venn diagram of shared targets between MCGD and depression. B: Component-target-disease map. C: Core intersection targets. D: Gene Ontology (GO) analysis of the shared targets, categorized into Biological Processes (BP), Molecular Functions (MF), and Cellular Components (CC). E: Bubble chart of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for the shared targets.

Tab.2 Key active components in Modified Chaihu Guizhi Decoction (MCGD) for treatment of depression

Code	ID	Name	Relevance	Soucre
CH9	MOL000098	Quercetin	128	Chinese thorawax root
HQ1	MOL001689	Acacetin	62	Baikal skullcap root
BS4	MOL000422	Kaempferol	55	White peony root
GC9	MOL003896	7-Methoxy-2-methyl Isoflavone	40	Liquorice root
GC10	MOL000392	Formononetin	36	Liquorice root
ZK4	MOL005828	Nobiletin	33	Submature bitter orange
CH10	MOL000354	Isorhamnetin	32	Chinese thorawax root
GC6	MOL002565	Medicarpin	32	Liquorice root
BX3	MOL002714	Baicalein	31	Ternate pinellia
GC63	MOL000497	Licochalcone a	30	Liquorice root

Tab.3 Binding energies of the core active components of MCGD for depression treatment for docking with the core target molecules JAK2 and STAT3

Target	Sample	Estimated ΔG (kcal/mol)
JAK2	CH9	-6.45
JAK2	HQ1	-6.48
JAK2	BS4	-6.40
JAK2	GC9	-5.53
JAK2	GC10	-6.27
JAK2	ZK4	-6.27
JAK2	CH10	-6.7
JAK2	GC6	-5.73
JAK2	BX3	-5.43
JAK2	GC63	-6.13
STAT3	CH9	-5.48
STAT3	HQ1	-5.16
STAT3	BS4	-5.08
STAT3	GC9	-5.9
STAT3	GC10	-5.09
STAT3	ZK4	-7.44
STAT3	CH10	-5.64
STAT3	GC6	-5.02
STAT3	BX3	-4.78
STAT3	GC63	-5.8

Fig.2 Visualization of the results of molecular docking analysis. A: Docked molecular conformation of JAK2 with CH9. B: Molecular conformation of JAK2 docked with CH10. C: Molecular conformation of JAK2 docked with BS4. D: Molecular conformation of JAK2 docked with ZK4. E: Molecular conformation of JAK2 docked with HQ1.

Fig.3 Visualization of the results of molecular docking analysis. A: Docked molecular conformation of STAT3 with CH9. B: Molecular conformation of STAT3 docked with CH10. C: Molecular conformation of STAT3 docked with ZK4. D: Molecular conformation of STAT3 docked with GC9. E: Molecular conformation of STAT3 docked with GC63.

Fig.4 MCGD significantly improves depression-like behavior in CUMS mice. A: Experimental design. B: Sucrose preference percentage in the sucrose preference test (SPT). C: Immobility time in the forced swim test (FST). D: Swimming times in the FST. Data are presented as Mean±SD (n=15). Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. ##P<0.01 vs Control group; *P<0.05, **P<0.01 vs CUMS group.

Fig.5 MCGD significantly improves anxiety-like behavior in CUMS mice. A: Trajectory map in the open field test (OFT). B: Total distance in OFT. C: Time percentage spent in center of OFT. D: Trajectory map in the elevated plus maze test (EPM). E: Time percentage spent in open arms of EPM. F: Number of entries spent into open arms of EPM. Data are presented as Mean±SD (n=15). Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. ##P<0.01 vs Control group; **P<0.01 vs CUMS group.

Fig.6 Effects of MCGD on mRNA expression of inflammatory factors in the hippocampus of CUMS mice. A: Relative mRNA level of IL-1β. B: Relative mRNA level of IL-6. C: Relative mRNA level of TNF-α. D: Relative mRNA level of IFN-γ. E: Relative mRNA level of IL-4. F: Relative mRNA level of IL-10. Data are presented as Mean±SD (n=6). Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. #P<0.05, ##P<0.01 vs Control group; *P<0.05, **P<0.01 vs CUMS group.

Fig.7 Effects of MCGD on the JAK2/STAT3 signaling pathway in the hippocampus of CUMS mice. A: Protein bands of JAK2, p-STAT3, STAT3, p-NF-κB, NF-κB, IL-1β, and IL-6 detected by Western blotting. B-F: Quantitative analysis of JAK2, p-STAT3, STAT3, p-NF-κB, NF-κB, IL-6, and IL-1β protein expression levels. Data are presented as Mean±SD (n=3). Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. ##P<0.01 vs Control group; *P<0.05, **P<0.01 vs CUMS group.

Fig.8 Regulatory effect of MCGD on expression levels of clock protein genes in the hippocampus of CUMS mice. A: Normalized circadian rhythm amplitude of Bmal1. B: Normalized circadian rhythm amplitude of Clock1. C: Normalized circadian rhythm amplitude of Cry1. D: Normalized circadian rhythm amplitude of Cry2. E: Normalized circadian rhythm amplitude of Per1. F: Normalized circadian rhythm amplitude of Per2. Data are presented as Mean±SD (n=3). Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. #P<0.05, ##P<0.01 vs Control group; *P<0.05, **P<0.01 vs CUMS group.

Fig.9 Effects of MCGD on microglia activation level in the hippocampal CA1 region of CUMS mice. A: Immunofluorescence images of Iba1 expression (Original magnification: ×200). B: Statistics of Iba1+ cells in the hippocampal CA1 region. C: Statistics of microglia total length of process in the hippocampal CA1 region. Each group included 3 mice, and 4 slices were analyzed per mouse. Data are presented as Mean±SD. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. ##P<0.01 vs Control group; **P<0.01 vs CUMS group.

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