TJZYF Improves Endometrial Receptivity through Regulating VEGF and PI3K/AKT Signaling Pathway

Abstract

The endometrium receptivity was impaired by controlled ovarian hyperstimulation (COH), which would then lead to fertility issues and increased abortion clinically. In the present study, to explore the effectiveness of Tiaojing Zhuyun Formula (TJZYF) in improving endometrial receptivity of COH rats and the possible active ingredients and mechanisms, an approach of network pharmacology was performed and a COH animal model was established. As analyzed, stigmasterol and quercetin may be the active ingredients of TJZYF on improving endometrial receptivity and positive regulation of ion transport, the cytokine-mediated signaling pathway, and endocrine process, and vascular endothelial growth factor receptor signaling pathway may be involved. Eighty female rats were divided into four groups randomly: control, model, TJZYF, and TJZYF+si-VEGFA. COH rat models were constructed by injecting with human menopausal gonadotropin (HMG) and human chorionic gonadotropin (HCG). We found that both endometrial thickness and number of embryo implantations in model were substantially reduced vs. control. The gene and protein expressions of VEGF, PI3K, and p-Akt in the uterus were significantly reduced. TJZYF could increase the endometrial thickness and number of embryo implantations and enhance the expressions of VEGF, PI3K, and p-Akt in the uterus. In the TJZYF+si-VEGFA group, the effect of TJZYF was impaired. Generally, TJZYF could improve the endometrium receptivity and facilitate embryo implantation of COH rats by upregulating VEGF and enhancing the PI3K/Akt signaling pathway.

Figure 1

The network pharmacology analysis between TJZYF and decreased endometrial receptivity. (a) Venn diagram of overlapped genes between TJZYF and reducing endometrial receptivity. (b) Upset map of the intersection gene of TJZYF and decreased endometrial receptivity. The upset map is divided into upper (bar graph) and bottom part (bar graph on the left, set name in the middle and dot matrix on the right). The horizontal bar chart on the bottom left shows the number of elements in each set (the number of targets corresponding to each single herb), the set name is obtained from the ingredients of TJZYF, and the dot matrix on the bottom right should be interpreted together with the bar chart above, and the dots represent related sets. (c) Protein-protein interaction (PPI) network diagram. (d) Drug-active ingredient-target network diagram of TJZYF and decreased endometrial receptivity. White rectangle represents the targets of TJZYF and decreased endometrial receptivity.

Figure 2

Enrichment of GO and KEGG pathways for candidate targets of TJZYF against decreased endometrial receptivity. (a) Intersection gene GO biological process, (b) cellular component, and (c) molecular function bubble plot. The y-axis on the left indicates GO pathway terms, and the x-axis indicates P values. The circle indicates the number of genes aligned in the pathways, a larger circle with a large number. The color from green to red indicates a greater ratio of genes aligned to pathways. (d) KEGG circle map. The outer right circle indicates terms of signaling pathways, and the left indicates genes. The inner left circle represents P values of genes with significance corresponding to the pathways.

Figure 3

Molecular docking analysis between two key components of TJZYF and AKT1, VEGFA. (a) Docking studies of human AKT1 with quercetin and (b) stigmasterol. (c) Docking studies of human VEGFA with quercetin and (d) stigmasterol. The protein structures of AKT1 and VEGFA are shown as cyan and rosy cartoon, respectively. The green color indicates molecules. The residues involved in hydrogen bonding interaction (yellow dash lines) have been shown in sticks.

Figure 4

Effect of TJZYF on estrogen and progesterone in rats. (a) HE staining detection of rat vaginal secretion. (b) Serum estrogen and (c) progesterone in rats were detected by ELISA assay. ^∗P < 0.05, ^∗∗P < 0.01 vs. model; ^#P < 0.05, ^##P < 0.01 vs. TJZYF based on one-way ANOVA and Tukey's tests.

Figure 5

Effect of TJZYF on rats' endometrial receptivity. (a) Histopathological HE staining of endometrial tissue of rats in each group. Scale bar = 50 μm (magnification, ×200). (b) Treatment effects on endometrium thickness (×40) and (c) (×100), and (d) mean endometrium thickness statistics (n = 10). (e) Embryo implantation statistics of rats in each group (n = 10). (f) The pinocytosis of endometrial epithelium under an electron microscope. ^∗P < 0.05, ^∗∗P < 0.01 vs. model; ^#P < 0.05, ^##P < 0.01 vs. TJZYF based on one-way ANOVA and Tukey's tests.

Figure 6

Endometrial VEGF, AKT, PI3K, and p-AKT protein expression of rats among each group. (a) The bands chart and semiquantification of protein VEGF, AKT, PI3K, and p-AKT via Western blot. (b) VEGF, PI3K, and AKT expression in endometrium. GAPDH was used as a reference. ^∗P < 0.05, ^∗∗P < 0.01 vs. model; ^#P < 0.05, ^##P < 0.01 vs. TJZYF as per one-way ANOVA and Tukey's tests.

Figure 7

Immunohistochemical detection of VEGF, PI3K, AKT, and p-AKT expression in endometrial of each group. Scale bar = 50 μm (magnification, ×400). Brown color indicates positive immunostaining.