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

Abstract

Objectives: 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.

Conclusions: JWXYP alleviates depression-like symptoms in rats by regulating the neurotransmitters, inhibiting inflammation and oxidation, and modulating gut microbiota.

Keywords: Jiawei Xiaoyao Pills; depression-like behavior; intestinal flora; network pharmacology; serum metabolome.

图1

网络药理学结果 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.

图2

大鼠模型建立及行为学测试结果测定 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.

图3

ELISA结果、肝脾指数、肝脏照片及HE染色结果 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.

图4

差异代谢物的鉴定及多元统计分析 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.

图5

血清代谢物的影响及代谢通路分析 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.

图6

肠道菌群物种丰度及差异物种 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.

图7

组内分析采用Spearman分析对加味逍遥丸干预CUMS诱导的抑郁相关成分、大鼠体内差异代谢物、差异肠道菌群 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.