Jiawei Xiaoyao Pills improves depression-like behavior in rats by regulating neurotransmitters, inhibiting inflammation and oxidation and modulating intestinal flora

doi:10.12122/j.issn.1673-4254.2025.02.16

Abstract

Abstract:

Objective To explore the bioactive components in Jiawei Xiaoyao Pills (JWXYP) and their mechanisms for alleviating depression-like behaviors. Methods The active compounds, key targets, and pathways of JWXYP were identified using TCMSP and TCMIP databases. Thirty-six SD rats were randomized equally into 6 groups including a control group and 5 chronic unpredictable mild stress (CUMS)-induced depression groups. After modeling, the 5 model groups were treated with daily gavage of normal saline, 1.8 mg/kg fluoxetine hydrochloride (positive control drug), or JWXYP at 1.44, 2.88, and 4.32 g/kg. The depression-like behaviors of the rats were evaluated using behavioral tests, and pathological changes in the liver and hippocampus were examined with HE staining. The biochemical indicators in the serum and brain tissues were detected using ELISA. Serum metabolomics analysis was performed to identify the differential metabolites using OPLS-DA, and gut microbiota changes were analyzed using 16S rDNA sequencing. Results Network pharmacology revealed that menthone and paeonol in JWXYP were capable of penetrating the blood-brain barrier to regulate inflammatory pathways and protect the nervous system. In the rat models subjected to CUMS, treatment with JWXYP significantly improved body weight loss, sucrose preference and open field activities, reduced liver inflammation, alleviated structural changes in the hippocampal neurons, decreased serum levels of TNF‑α, IL-1β, IL-6 and LBP, and increased 5-HT and VIP concentrations in the serum and brain tissue, and these effects were the most pronounced in the high-dose group. Metabolomics analysis showed changes in such metabolites as indole-3-acetamide and acetyl-L-carnitine in JWXYP-treated rats, involving the pathways for bile acid biosynthesis and amino acid metabolism. 16S rDNA analysis demonstrated increased gut microbiota diversity and increased abundance of Lactobacillus species in JWXYP-treated rats. Conclusion JWXYP alleviates depression-like symptoms in rats by regulating the neurotransmitters, inhibiting inflammation and oxidation, and modulating gut microbiota.

Key words: Jiawei Xiaoyao Pills, network pharmacology, intestinal flora, serum metabolome, depression-like behavior

Ying LIU, Borui LI, Yongcai LI, Lubo CHANG, Jiao WANG, Lin YANG, Yonggang YAN, Kai QV, Jiping LIU, Gang ZHANG, Xia SHEN. Jiawei Xiaoyao Pills improves depression-like behavior in rats by regulating neurotransmitters, inhibiting inflammation and oxidation and modulating intestinal flora[J]. Journal of Southern Medical University, 2025, 45(2): 347-358.

Figures/Tables 8

Fig.1 Network pharmacology results. A: JWXYP and depression intersection genes. B: MCODE algorithm for screening the key targets. C: Chinese medicine-component-target network diagram. D: GO analysis of the key genes. E: Bubble map of KEGG analysis of the key genes.

Tab.1 Physicochemical properties of the bioactive compounds in Jiawei Xiaoyao Pills （JWXYP）

Compound	MW	OB(%)	BBB	DL
Quercetin	302.25	46.43	-0.77	0.28
Luteolin	286.25	36.16	-0.84	0.25
Kaempferol	286.25	41.88	-0.55	0.24
Thymol	150.24	41.47	1.68	0.03
Stigmasterol	412.77	43.83	1	0.76
Paeonol	166.19	28.79	0.84	0.04
Oleanolic acid	456.78	29.02	0.07	0.76
Saikosaponin A	781.1	32.39	-2.93	0.09
Pulegone	152.26	51.6	1.74	0.03
Isoliquiritigenin	256.27	85.32	-0.41	0.15
Paeoniflorin	480.51	53.87	-1.86	0.79
Saikogenin F	472.78	25.88	-0.71	0.63
Licochalcone A	338.43	40.79	-0.21	0.29

Fig.2 Establishment of depression rat models and behavioral test results of the rats. A: Body weight curves of rats in each group. B: Sugar water preference test. C: External hair of rats before modeling (1), after modeling (2) and after drug treatment (3). D: Open- field test of the rats (activity time, distance of the edge, total distance, and number of activities). E: Trajectory diagram of the rats in open field test. *P<0.05, **P<0.01, ***P<0.001 vs model group (CUMS); #P<0.05 vs control group.

Fig.3 ELISA results, liver and spleen index, liver and hippocampus HE staining results. A, D: Content of VIP and 5-HT in the brain tissues. B, C, E-H: Serum levels of IL-6, TNF-α, VIP, 5-HT, ILβ and LBP. J, K: Thymus index and spleen index. L: HE staining of the liver tissues in each group (Original magnification: ×100). M: HE staining of rat hippocampus (×100). N: HE staining of hippocampal CA1 region of the rats (×200) . *P<0.05, **P<0.01, ***P<0.001 vs model group (CUMS); #P<0.05, ##P<0.01 vs control group.

Fig.4 Identification of differential metabolites and multivariate statistical analysis. A: Principal component analysis. B: Partial least squares discriminant analysis (OPLS-DA). C: Differential metabolite volcano plot analysis. The threshold of the difference was VIP≥1 and T-test P<0.05 in the OPLS-DA model.

Fig.5 Effects of serum metabolites and metabolic pathway analysis. A: VIP (importance of variables in projection) diagram of OPLS-DA in POS mode. B: VIP diagram of OPLS-DA in NEG mode. C: KEGG enrichment circle diagram (CUMS vs JWJWXYP). D: KEGG enriched bubble diagram of the top 20 pathways with the smallest Q value. E: Metabolic pathway and classification between JWXYP and CUMS groups.

Fig.6 Intestinal flora species abundance and differential species. A: Distribution of samples in PC1 and PC2 dimensions. B: PCA analysis and PCoA analysis of the distribution of samples in PCo1 and PCo2 dimensions. C: β diversity box plot. D: Shaanon diversity index: ACE, Sob, Chao1 and Shannon index of Control, CIHMS and JWXYP groups. E: Distribution of Shannon index. F: Relative abundance of species at the phylum, class and species levels in control, CUMS and JWXYP groups.

Fig.7 Spearman analysis of depression-related components, differential metabolites and differential intestinal flora in JWXYP-treated rats with CUMS-induced depression. P value and color depth represent the degree of correlation.*P<0.05, **P<0.01,***P<0.001, JW vs CUMS group.

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