The Mechanism of Xiaoyao San in the Treatment of Ovarian Cancer by Network Pharmacology and the Effect of Stigmasterol on the PI3K/Akt Pathway

Abstract

Purpose: This study was aimed at exploring the regulatory mechanism of Xiaoyao San (XYS) and its main compound, Stigmasterol, in the biological network and signaling pathway of ovarian cancer (OC) through network pharmacology-based analyses and experimental validation.

Methods: The active compounds and targets of XYS were studied by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). The GeneCards and OMIM databases were used to screen common targets of XYS in the treatment of OC. Combined with the STRING database and Cytoscape 3.6.0, the core compounds and targets of XYS were obtained. GO and KEGG pathway enrichment analyses of core target genes were carried out by using the Metascape and DAVID databases. Molecular docking has been achieved by using the AutoDock Vina program to discuss the interaction of the core targets and compounds of XYS in the treatment of OC. The effect of Stigmasterol on proliferation and migration were assessed by CCK8 and wound healing assay. Western blot and qRT-PCR were used to analyze the protein and mRNA expressions of PI3K, Akt, and PTEN after treatment of Stigmasterol.

Results: A total of 113 common targets of XYS for the treatment of OC were obtained from 975 targets related to OC and 239 targets of XYS's effect. The main compounds of XYS include Quercetin, Naringenin, Isorhamnetin, and Stigmasterol, which mainly regulate the targets such as TP53, Akt1, and MYC and PI3K/Akt, p53, and cell cycle signal pathways. At the same time, molecular docking showed that Stigmasterol and Akt1 had good docking conformation. Stigmasterol inhibited OC cell proliferation and migration in vitro and reduced the protein and mRNA expressions of the PI3K/Akt signaling pathway.

Conclusion: Stigmasterol as the one of the main compounds of XYS suppresses OC cell activities through the PI3K-Akt signaling pathway.

Figure 1

(a) The targets of XYS in treatment of OC. Venn diagram showing 975 targets associated with OC and 239 targets of XYS. Overlap represents 113 common target genes. (b) The high expression of Akt1 and TP53 and low expression of MYC in OC. ^∗∗P < 0.01, ^∗∗∗P < 0.001. (c) Interaction target network of XYS treatment of OC. The top 3 targets in terms of degree value are TP53, Akt1, and MYC.

Figure 2

(a) GO enrichment in targets of XYS treatment of OC. (b) Top 20 of KEGG pathway enrichment in targets of XYS treatment of OC. (c) Results of molecular docking. Diagram of interaction between Akt1 protein and Stigmasterol (3D). (d) Relationship between the mRNA levels of Akt1 and the survival outcomes of patients with OC. A is the ROC curve of Akt1 in OC. B is the KM curve of Akt1 in OC.

Figure 3

(a) Stigmasterol inhibited the migration in A2780 cells. (b) The effect of Stigmasterol on expressions of the PI3K-Akt signaling pathway by qRT-PCR in A2780 cells. (c, d) Western blot assay showed that Stigmasterol downregulated the expression of p-PI3K and p-Akt protein levels and upregulated the expression of PI3K, Akt, and PTEN protein levels in A2780 cells. (e) Stigmasterol inhibited the migration in SKOV3 cells. (f) The effect of Stigmasterol on expressions of the PI3K-Akt signaling pathway by qRT-PCR in SKOV3 cells. The effect of Stigmasterol on expression of the PI3K-Akt signaling pathway in SKOV3 cells. Stigmasterol increased the mRNA expression level of PTEN and decreased the mRNA expression level of PI3K and Akt. (g, h) Western blot assay showed that Stigmasterol downregulated the expression of p-PI3K and p-Akt protein levels and upregulated the expression of PI3K, Akt, and PTEN protein levels in SKOV3 cells. ^∗P < 0.05, ^∗∗P < 0.01 vs. control group. The dosage of Stigmasterol was 25 μM and 50 μM in A2780 and SKOV3 cells, respectively.