Chaihu Shugan San inhibits C3/C3aR-mediated microglial M1 polarization to improve hippocampal synaptic plasticity in mice with menopausal syndrome with liver qi stagnation

doi:10.12122/j.issn.1673-4254.2026.08.10

Abstract

Abstract:

Objective To investigate the therapeutic mechanism of Chaihu Shugan San (CSS) for treatment of menopausal syndrome (MPS) with liver qi stagnation. Methods Fifty-six female C57BL/6 mice were randomized into sham-operated group (n=13) and bilateral ovariectomy (OVX) groups (n=43), and the latter group was further randomized equally into menopausal group, menopausal liver qi stagnation (MPS+CUMS) group subjected to chronic unpredictable mild stress (CUMS), and CSS treatment group (with CSS gavage at daily dose of 6.47 g/kg for 28 days). MPS model establishment was validated by vaginal smears and behavior tests. The levels of sex hormones, neurotransmitters, and inflammatory cytokines in the serum and hippocampus were measured by ELISA. Hippocampal morphology was observed using HE and Golgi staining, and immunofluorescence staining and Western blotting were used to detect the expressions of microglial markers, synaptic plasticity proteins and C3/C3aR pathway proteins in the hippocampal CA1 region. Results The OVX mice showed disrupted estrous cycles. The MPS+CUMS mice exhibited obvious depression-like behaviors, abnormal sex hormones and neurotransmitters, elevated inflammatory factors, hippocampal CA1 neuronal loss with disordered arrangement and pyknosis, reduced dendritic spines, increased microglial markers, decreased synaptic plasticity proteins, and upregulated C3/C3aR pathway. All these changes were significantly improved or corrected after CSS treatment in MPS+CUMS mice. Conclusion CSS alleviates MPS with liver qi stagnation possibly by inhibiting C3/C3aR-mediated M1 microglial polarization and improving synaptic plasticity.

Key words: menopausal syndrome, liver qi stagnation, Chaihu Shugan San, microglia, C3/C3aR pathway

Yao ZHONG, Xiaoshi CAI, Xinyuan LI, Suying WANG, You WU, Zhaoxi CHEN, Yue WANG, Bingrong SHEN, Lin GONG, Jianying SHEN, Li MIN, Wenna LIANG. Chaihu Shugan San inhibits C3/C3aR-mediated microglial M1 polarization to improve hippocampal synaptic plasticity in mice with menopausal syndrome with liver qi stagnation[J]. Journal of Southern Medical University, 2026, 46(8): 1823-1834.

Figures/Tables 11

Fig.1 Papanicolaou staining results of mouse vaginal exfoliated cells (Original magnification: ×100). A: Proestrus. B: Estrus. C: Late estrus. D-H: Interestrous. Blue arrows indicate nucleated cells, green arrows indicate keratinized cells, and red arrows indicate leukocytes.

Tab.1 Comparison of serum E2, FSH and LH levels in the mice among the 4 groups (Mean±SD)

Group	E₂(pg/mL)	FSH (ng/mL)	LH (mU/mL)
Sham	10.57±1.31	7.62±0.97	18.97±0.76
MPS	5.77±0.74**	9.38±0.26**	24.58±0.72**
MPS+CUMS	4.89±0.71**	11.20±0.92**	33.13±1.48**
CSS	6.98±1.31^##	9.68±0.89^#	22.88±1.42^##

Tab.2 Comparison of baseline general condition and behavioral test results of the mice among the 4 groups (Mean±SD)

Group	Weight (g)	OFT		SPT Sucrose solution preference rate (%)	FST Immobile time (s)	TST Immobile time (s)
Group	Weight (g)	Total distance (m)	Time in the center (s)	SPT Sucrose solution preference rate (%)	FST Immobile time (s)	TST Immobile time (s)
Sham	18.88±0.87	14.26±2.61	32.21±11.78	62.21±8.58	33.00±7.07	139.09±5.05
MPS	18.52±0.57	14.05±2.76	32.67±13.02	62.37±6.70	32.43±8.06	138.83±8.97
MPS+CUMS	19.52±0.98	12.80±2.29	32.67±10.91	61.70±2.58	33.69±4.16	139.56±11.17
CSS	18.86±0.95	12.90±1.54	32.41±14.05	65.82±4.58	31.44±7.46	136.19±5.83

Tab.3 Comparison of behavioral tests after modeling and intervention among the 4 groups (Mean±SD)

Group	Weight (g)	OFT		SPT Sucrose solution preference rate (%)	FST Immobilization time(s)	TST Immobilization time(s)
Group	Weight (g)	Total distance (m)	Time in the center(s)	SPT Sucrose solution preference rate (%)	FST Immobilization time(s)	TST Immobilization time(s)
Sham	24.27±1.03	12.22±0.55	30.05±7.77	74.34±14.33	27.38±4.31	117.29±10.06
MPS	25.20±1.21	10.44±1.17*	26.25±9.67	52.80±12.72**	45.72±8.14**	149.60±10.98**
MPS+CUMS	22.48±0.84**	7.71±1.16**	12.44±6.55**	49.91±5.78**	88.91±10.65**	188.66±12.86**
CSS	23.13±0.70	14.20±2.18^##	25.76±9.19^#	72.18±7.36^##	39.95±7.74^##	150.36±14.17^##

Tab.4 Serum levels of Glu and GABA in each group (Mean±SD)

Group	Glu (μmol/L)	GABA (ng/mL)
Sham	22.07±1.20	9.92±0.48
MPS	25.21±1.48**	9.26±0.37*
MPS+CUMS	29.53±1.83**	7.08±0.35**
CSS	25.47±1.45^##	9.89±0.55^##

Tab.5 Serum and hippocampal levels of IL-1β and TNF-α in each group (Mean±SD)

Group	Serum		Hippocampal
Group	IL-1β (pg/mL)	TNF-α (pg/mL)	IL-1β (pg/mg.pro)	TNF-α (pg/mg.pro)
Sham	1.02±0.53	0.33±0.20	12.45±0.78	87.04±3.23
MPS	2.96±0.45**	1.87±0.65**	22.76±1.21**	105.10±1.25**
MPS+CUMS	8.29±1.58**	5.00±0.74**	26.85±1.47**	194.16±9.43**
CSS	3.59±1.09^##	3.16±0.67^##	18.41±1.92^##	116.13±4.58^##

Fig.2 HE staining of the hippocampal CA1 region in each group (Original magnification: ×200). The red arrows in the figure indicate necrotic cells with karyopyknosis. MPS: Menopausal syndrome; CUMS: Chronic unpredictable mild stress; CSS: Chaihu Shugan San.

Fig.3 Results of Golgi staining in the hippocampal CA1 region of the mice in each group (Upper panel: ×10; Lower panel: ×1000).

Fig.4 Effect of Chaihu Shugan San on expressions of microglial activation related proteins Iba1, Iba1/iNOS, and Iba1/Arg1 in the hippocampal CA1 region of the mice. A, B: Immunofluorescence staining of Iba1 expression (×200) and Iba1+ cell counts in the hippocampal CA1 region. C, D: Immunofluorescence staining of Iba1/iNOS expression (×200) and Iba1+/iNOS+ cell counts in the hippocampal CA1 region. E, F: Immunofluorescence staining of Iba1/Arg1 expression (×200) and Iba1+/Arg1+ cell counts in the hippocampal CA1 region. Data are presented as Mean±SD (n=3). Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. Iba1: Ionized calcium-binding adapter molecule 1; iNOS: Inducible nitric oxide synthase; Arg1: Arginase 1. **P<0.01 vs Sham group, ##P<0.01 vs MPS+CUMS group.

Fig.5 Effect of Chaihu Shugan San on expressions of synapse related proteins PSD95 and SYT-1 in the hippocampal CA1 region of the mice. A, B: Immunofluorescence staining of PSD95 expression (×200) and relative fluorescence intensity in the CA1 region. C, D: Immunofluorescence staining of SYT-1 expression (×200) and relative fluorescence intensity in the CA1 region. Data are presented as Mean±SD (n=3). Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. PSD95: Postsynaptic density protein 95; SYT-1: Synaptotagmin 1. **P<0.01 vs Sham group, ##P<0.01 vs MPS+CUMS group.

Fig.6 Effect of Chaihu Shugan San on expression of classical complement pathway-related proteins C3, C3aR and pSTAT3/STAT3 in the hippocampus of the mice. A: Protein bands of C3, C3aR, p-STAT3 and STAT3 detected by Western blotting. B-D: Quantitative analysis of C3, C3aR, p-STAT3, and STAT3 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. C3: Component 3; C3aR: Complement C3a receptor; pSTAT3: Phosphorylated signal transducer and activator of transcription 3; STAT3: Signal transducer and activator of transcription 3; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase. *P<0.05, **P<0.01 vs Sham group; #P<0.05, ##P<0.01 vs MPS+CUMS group.

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