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. 2022 Aug 8:13:917544.
doi: 10.3389/fphar.2022.917544. eCollection 2022.

Wenshenyang recipe treats infertility through hormonal regulation and inflammatory responses revealed by transcriptome analysis and network pharmacology

Lan Xie  1   2 , Shuai Zhao  2 , Xiaoling Zhang  2 , Wenting Huang  1   2 , Liansheng Qiao  1   2 , Delin Zhan  1 , Chengmei Ma  2 , Wei Gong  2 , Honglei Dang  2 , Hua Lu  3
Affiliations

Wenshenyang recipe treats infertility through hormonal regulation and inflammatory responses revealed by transcriptome analysis and network pharmacology

Lan Xie et al. Front Pharmacol. .

Abstract

The Wenshenyang recipe (WSYR) has the effect of treating infertility, but the mechanisms underlying this activity have not been fully elucidated. In this study, network pharmacology and RNA sequencing were combined, with database-based "dry" experiments and transcriptome analysis-based "wet" experiments used conjointly to analyse the mechanism of WSYR in the treatment of infertility. In the dry analysis, 43 active compounds in WSYR and 44 therapeutic targets were obtained through a database search, 15 infertility pathways were significantly enriched, and key targets, such as ESR1, TP53, AKT1, IL-6, and IL-10 were identified. Then the wet experiments were performed to detect the expression changes of the 412 genes from 15 infertility pathways identified by dry analysis. HK-2 cells were treated with the three herbs of WSYR and subjected to targeted RNA sequencing. Based on the results, 92 of the 412 genes in 15 infertility pathways were identified as DEGs. Additionally, key targets, such as ESR2, STAT1, STAT3, and IL6, were also identified in the wet experiments. RT-qPCR experiments further verified that WSYR played an anti-inflammatory role by upregulating IL-4 and IL-10 and Epimedium brevicornu Maxim (Yinyanghuo) showed broader effect than Drynaria fortunei (Kunze) J. Sm (Gusuibu) and Cistanche deserticola Y.C.Ma (Roucongrong). By screening compounds of WSYR using molecular docking models of ESR1 and ESR2, it was further found that xanthogalenol in Gusuibu, arachidonate in Roucongrong, and anhydroicaritin in Yinyanghuo had good affinity for estrogen receptors. These findings provide evidence for an estrogen-regulating role of the three herbs in WSYR.

Keywords: GO enrichment analysis; infertility; molecular docking; network pharmacology; pathway enrichment analysis; transcriptome analysis; wenshenyang recipe.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Experimental technical roadmap.
FIGURE 2
FIGURE 2
Functional analysis of infertility-related targets of WSYR identified in dry experiments (A) Venn diagram of the potential targets in WSYR and the therapeutic targets for infertility (B) Bubble chart of GO biological process enrichment analysis of 44 infertility treatment targets in WSYR (C) Bubble chart of pathway enrichment analysis of 44 infertility treatment targets in WSYR (p value ≤ 10−3) (D) Pathway enrichment analysis of infertility treatment-related targets of Roucongrong, Yinyanghuo and Gusuibu (p value ≤ 10−3) (E) Comparison of pathways regulated by Roucongrong, Yinyanghuo and Gusuibu.
FIGURE 3
FIGURE 3
Interactive networks of infertility treatment targets based on databases (A)“Herbs-Compounds-Targets-Pathways” network. Orange diamond: TCM; dark yellow triangle: active compound; green circle: gene; blue hexagon: pathway (B) Protein–protein interaction network (PPIN) of 44 WSYR infertility therapeutic targets. Dark pink circle: key targets; blue circle: other targets (C) Hormone-related PPIN based on database targets. Dark pink circle: key targets; blue circle: other targets (D) Inflammatory immune-regulated PPIN based on database targets. Dark pink circle: key targets; blue circle: other targets.
FIGURE 4
FIGURE 4
Functional analysis of WSYR DEGs based on transcriptome analysis (A) Volcano map of DEGs of Roucongrong, Yinyanghuo and Gusuibu. Red dots: upregulated genes; green dots: downregulated genes; black dots: unchanged genes (B) Bubble chart of GO biological processes enriched among the 92 DEGs related to WSYR (top 15) (C) Bubble chart of pathways enriched among the 92 DEGs related to WSYR (top 10) (D) Pathway enrichment analysis of DEGs in Roucongrong, Yinyanghuo and Gusuibu (p value ≤ 10−5) (E) Comparison of pathways regulated by Roucongrong, Yinyanghuo and Gusuibu.
FIGURE 5
FIGURE 5
Interactive networks of WSYR based on DEGs (A) “Herbs-DEGs-Pathways” network. Orange diamond: TCM; green circle: gene; blue circle: pathway (B) Protein—protein interaction network (PPIN) of all DEGs from transcriptome sequencing. Dark pink circle: key targets; blue circle: other targets (C) Hormone-related PPIN based on DEGs.Dark pink circle: key targets; blue circle: other targets (D) Inflammatory immune-regulated PPIN based on DEGs.Dark pink circle: key targets; blue circle: other targets.
FIGURE 6
FIGURE 6
Action of WSYR and its herbs Gusuibu, Roucongrong, Yinyanghuo on SK-OV-3 cells. SK-OV-3 cells were treated with WSYR (100, 200, 300 μg/ml), Gusuibu (200 μg/ml), Roucongrong (200 μg/ml), Yinyanghuo (200 μg/ml) for 24 h. The mRNA expression of IL-4 (A,B), IL-10 (C,D) was analyzed by qPCR. *p < 0.05 and **p < 0.01 indicate statistical significance compared to control.
FIGURE 7
FIGURE 7
The 3D interaction of the original ligand GQD (A), marckine (B), xanthogalenol (C), arachidonate (D) and yinyanghuo A (E), linoleyl acetate (F), suchilactone (G) and davallioside A_qt (H) with ESR1.
FIGURE 8
FIGURE 8
The 3D interaction of the original ligand THC (A), anhydroicaritin (B), 8-isopentenyl-kaempferol (C), davallioside A_qt (D), xanthogalenol (E) and linoleyl acetate (F) with ESR2.
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