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. 2022 Sep 27:16:3297-3314.
doi: 10.2147/DDDT.S367871. eCollection 2022.

Network and Experimental Pharmacology to Decode the Action of Wendan Decoction Against Generalized Anxiety Disorder

Qi Jin #  1 , Jie Li #  2 , Guang-Yao Chen #  1 , Zi-Yu Wu  3 , Xiao-Yu Liu  2 , Yi Liu  4 , Lin Chen  5 , Xin-Yi Wu  5 , Yan Liu  2 , Xin Zhao  2 , Yue-Han Song  2
Affiliations

Network and Experimental Pharmacology to Decode the Action of Wendan Decoction Against Generalized Anxiety Disorder

Qi Jin et al. Drug Des Devel Ther. .

Abstract

Objective: The mechanism of Wendan Decoction (WDD) against Generalized Anxiety Disorder (GAD) was predicted by network pharmacology and validated by in vivo and in vitro experiments.

Methods: The targets of WDD for the treatment of GAD were obtained by a search of online databases. Further, PPI network and KEGG enrichment were used to identify the key targets and pathways. Ultimately, these key targets and pathways were validated by in vivo experiments on GAD mice modeled by repeated restraint stress (RRS) and in vitro experiments on inflammatory factor stimulated BV-2 cells.

Results: Through searching the databases, the 137 ingredients of WDD that correspond to 938 targets and 4794 targets related to GAD were identified. Among them, 569 overlapping targets were considered as the therapeutic targets of WDD for GAD. PPI analysis showed that the inflammation-related proteins IL-6, TNF, SRC and AKT1 were the key targets, and KEGG enrichment suggested that PI3K/AKT and MAPK signaling pathways were key pathways of WDD in the treatment of GAD. In vivo experiments, RRS mice exhibited abnormality in behavioristics in open field test (OFT) and elevated plus maze (EPM) and increases in serum corticosterone and the percentage of lymphocytes positive for IL-6 in peripheral blood. These abnormal changes can be reversed by WDD and the positive control drug paroxetine. In vitro experiments, WDD can inhibit IL-6 induced activation of PI3K/AKT and MAPK signaling pathways in BV2 cells, and suppress the ensuing release of inflammatory factors TNF-α, IL-1β and PGE2, and showed a dose-dependent effect.

Conclusion: WDD is able to resist GAD by relieving inflammatory response in peripheral and central system.

Keywords: Generalized Anxiety Disorder; Interleukin-6; MAPK signaling pathway; PI3K/AKT signaling pathway; Wendan Decoction; network pharmacology.

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Conflict of interest statement

All authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The herb-target network of Wendan Decoction (WDD) treating Generalized Anxiety Disorder (GAD). Yellow nodes represent all herbs of WDD. Blue nodes represent the intersection targets of WDD and GAD. Common targets of multiple herbs are located in the center of the network.
Figure 2
Figure 2
PPI topological analysis of Wendan Decoction treating Generalized Anxiety Disorder. (A)PPI network of 569 targets. (B) The subnet of PPI with 142 hub targets screened based on twice medium degree. (C) According to the Betweenness Centrality, Closeness Centrality, Degree Centrality and Eigenvector Centrality separately, TOP 10 targets in subnet from each criterion were labeled yellow. (D) Venn diagram of 4 topological analysis data. TNF, SRC, IL-6 and AKT1 were common targets.
Figure 3
Figure 3
(A) KEGG enrichment analysis of 569 intersection targets of Wendan Decoction and Generalized Anxiety Disorder. The redder bubble is, the less P value is. (B) Network of top 5 KEGG pathways and their corresponding targets. Red nodes represent pathways. Yellow nodes represent related targets.
Figure 4
Figure 4
Identification of active ingredients of Wendan Decoction. (A) Total ion chromatography (TIC) on positive of Wendan Decoction. (B) TIC on negative of Wendan Decoction. (C-F) Extraction ion chromatography of Wendan Decoction. (C) Identification of naringenin. (D) Identification of glabridin. (E) Identification of luteolin and baicalein. (F) Identification of coniferyl aldehyde, syringaldehyde, quercetin, beta-sitosterol and hederagenin.
Figure 5
Figure 5
Behavioristic changes of mice in each group. (A) Representative trajectory diagram of each group in open field test. (B) Representative heat maps of the movement of each group in open field test. (C) Representative trajectory diagram of each group in elevated plus maze. (D) Representative heat maps of the movement of each group in elevated plus maze. (E) Total time spent in the central zone of open field. (F) Percentage of number of entries in the open arms. (a) blank control group; (b) repeated restraint stress (RRS) model group; (c) Wendan Decoction group; (d) paroxetine group. *P < 0.05, compared with blank control group; #P < 0.05, compared with RRS model group.
Figure 6
Figure 6
Plasma corticosterone concentration of mice in each group. (A) blank control group; (B) repeated restraint stress (RRS) model group; (C) Wendan Decoction group; (D) paroxetine group. *P < 0.05, compared with blank control group; #P < 0.05, compared with RRS model group.
Figure 7
Figure 7
Percent of IL-6+ T lymphocytes in blood of mice in each group. (A) Representative flow cytometric analysis. (I)Sorting cell fragments and cells; (II)Sorting cells and T lymphocytes; (III)sorting T lymphocytes and CD3+ T lymphocytes; (IV)Sorting CD3+ T lymphocytes and CD3+ CD4+ T lymphocytes; (V)Sorting CD3+ CD4+ T lymphocytes and IL-6+ CD4+ T lymphocytes. (B) The proportion of CD3+ lymphocytes. (C) The proportion of CD4+ lymphocytes. (D) The proportion of IL-6+ CD4+T lymphocytes. (a) blank control group; (b) repeated restraint stress (RRS) model group; (c) Wendan Decoction group; (d) paroxetine group. *P < 0.05, compared with blank control group; #P < 0.05, compared with RRS model group.
Figure 8
Figure 8
Effects of different concentrations of Wendan Decoction (WDD) on BV2 microglial cell proliferation. *P
Figure 9
Figure 9
Effect of different concentrations of Wendan Decoction (WDD) and 100μmol/L Dexamethasone (DEX) on pro-inflammatory factors of IL-6 induced BV-2 microglia cells, respectively. (A) Concentrations of TNF-α in the cell supernatants. (B) Concentrations of PEG2 in the cell supernatants. (C) Concentrations of IL-1β in the cell supernatants. *P < 0.05, compared with blank control group; #P < 0.05, compared with IL-6-induced group.
Figure 10
Figure 10
Effect of different concentrations of Wendan Decoction (WDD) and 100μmol/L Dexamethasone (DEX) on PI3K/AKT and MAPK signaling pathways of IL-6 induced BV-2 microglia cells. (A) Western blots of PI3K, p-PI3K, AKT, p-AKT, MAPK p38 and p-MAPK p38. (B) Quantification analysis of p-PI3K/PI3K. (C) Quantification analysis of p-AKT/AKT. (D) Quantification analysis of p-p38/p38. *P < 0.05, compared with blank control group; #P < 0.05, compared with IL-6-induced group; ΔP < 0.05, compared with 700μg/mL WDD intervention group.
Figure 11
Figure 11
The hypothesis of Wendan Decoction treating Generalized Anxiety Disorder.
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