Mechanisms underlying the therapeutic effects of Semen cuscutae in treating recurrent spontaneous abortion based on network pharmacology and molecular docking

Abstract

Background: This paper aims to analyse the active components of Semen cuscutae (SC) by network pharmacology and screen the most stable compounds with tumour necrosis factor-alpha (TNF-α) by molecular docking to explore the mechanisms of SC treatment of recurrent spontaneous abortion (RSA) and provide a theoretical basis for drug development. Methods: The active compounds of SC and the potential inflammatory targets of RSA were obtained from the Traditional Chinese Medicine Systems Pharmacology database and GeneCards, respectively. The RSA-SC target gene interaction network was obtained and visualized using the STRING database and Cytoscape software. GO and KEGG pathway enrichment analyses were obtained from DAVID to further explore the RSA mechanism and therapeutic effects of SC. Interactions between TNF-α and drugs were analysed by molecular docking. Treatment of human trophoblast cells with sesamin and TNF-α was carried out to detect their proliferative and apoptotic abilities, and WB assay was carried out to detect EGFR, PTGS2, and CASP3 protein expression. Results: Ten compounds and 128 target genes were screened from SC, of which 79 overlapped with RSA target inflammatory genes, which were considered potential therapeutic targets. Network pharmacological analysis showed that sesamin, matrine, matrol, and other SC compounds had a good correlation with the inflammatory target genes of RSA. Related genes included PGR, PTGS1, PTGS2, TGFB1, and CHRNA7. Several signalling pathways are involved in the pathogenesis of RSA, such as the TNF-α signalling pathway, HIF-1 signalling pathway, oestrogen signalling pathway, proteoglycans in cancer cells, and FoxO signalling pathway. Molecular docking results suggested that sesamin was the most suitable natural tumour necrosis factor inhibitor (TNFi). Sesamin can promote proliferation and inhibit apoptosis in human trophoblasts by downregulating EGFR and CASP3 expression and upregulating PTGS2 expression. Conclusion: Our findings play an important role and basis for further research into the molecular mechanism of SC treatment of RSA and drug development of TNFi.

Keywords: Semen cuscutae; immune-inflammatory; molecular docking; network pharmacology; recurrent spontaneous abortion; tumor necrosis factor-alpha inhibitor.

FIGURE 1

Venn diagram of 79 target genes: potential targets for Semen cuscutae (SC) treatment of recurrent spontaneous abortion (RSA).

FIGURE 2

Potential target genes and protein–protein interaction (PPI) network map of SC therapy for RSA. (A) PPI network map of 79 target genes, and (B) list of the top 10 genes of the PPI network map.

FIGURE 3

SC-RSA–compound–potential target gene network map. The yellow triangle represents SC, red v represents RSA, green diamonds denote the active ingredients of SC, and pink circles indicate 79 overlapping target genes.

FIGURE 4

Validation of gene expression in GEO data (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001). (A): GSE165004 was used to verify the critical gene levels (B): GSE43256 was used to verify the critical gene levels.

FIGURE 5

Top 20 biological functions.

FIGURE 6

Top 15 signalling pathways for KEGG enrichment of core targets.

FIGURE 7

Core target of the tumour necrosis factor (TNF) signalling pathway. Red represents SC action targets in the network, and arrows indicate upstream and downstream relationships between genes.

FIGURE 8

Three-dimensional structure of TNF-α and the site of binding.

FIGURE 9

Three-dimensional map of the binding of compounds. (A) Quercetin, (B) NSC63551, (C) isorhamnetin, (D) beta-sitosterol, (E) kaempferol, (F) and CLR, (G) campest-5-en-3beta-ol, (H) Isofucosterol, (I) matrine, (J) sesamin with TNF-α.

FIGURE 10

Sesamin can promote proliferation and inhibit apoptosis in human trophoblasts by downregulating EGFR and CASP3 expression and upregulating PTGS2 expression (A). CCK-8 assay was used to detect cell proliferation (B). Flow cytometry was performed to assess the cell apoptotic rate (C). Western blot analysis was used to detect EGFR, CASP3, and PTGS2 protein levels at 24 h after treating the cells with sesamin and TNF-α. GAPDH was used as an internal control. All data are presented as mean ± SDs. *p < 0.05; **p < 0.005; and ***p < 0.001.