Ginsenoside Rg3 inhibits angiogenesis in a rat model of endometriosis through the VEGFR-2-mediated PI3K/Akt/mTOR signaling pathway

Abstract

Objective: This study aimed to investigate the link between the inhibitory effect of ginsenoside Rg3 on the ectopic endometrium growth and the VEGFR-2-mediated PI3K/Akt/mTOR signaling pathway, a mechanism known to inhibit angiogenesis and induce ectopic endometrial cell apoptosis.

Materials and methods: A model of endometriosis was established by allotransplantation in rats. The rats were randomly divided into 5 groups: the ginsenoside Rg3 low-dose group (group A,5mg/kgBW/d of ginsenoside Rg3), the ginsenoside Rg3 high-dose group (group B, 10mg/kgBW/d of ginsenoside Rg3), the gestrinone group (group C, 0.5mg/kgBW/d of gestrinone), the control group (groupD, 10ml/kg BW/d of 0.5%CMC-Na) and the ovariectomized group (group E, 10ml/kgBW/d of 0.5%CMC-Na). Rats were executed after 21 days of continuous administration. The ectopic endometrium volume was measured and the inhibitory rate was calculated. The levels of serum estradiol (E2) and progesterone (P) were detected by Electro-Chemiluminescence Immunoassay (ECLI). The protein expressionof VEGF, VEGFR-2, p-Akt, and p-mTOR inthe ectopic endometrium wastested by immunohistochemistry(IHC) and Western Blotting. The mRNA expression levels of VEGF, VEGFR-2, Akt, and mTOR were tested by Real-Time Polymerase Chain Reaction (PCR). The apoptosis rate of the ectopic endometrial cells was detected by Terminal Deoxynucleotidyl Transferase-mediated Digoxigenin-dUTP Nick-End Labeling Assay(TUNEL).

Main results: Tissue measurements revealed a dose-dependent inhibition effect of ginsenoside Rg3 on the growth of the ectopic endometrium in treated rats compared to controls. Immunohistochemical and Western Blotting assays confirmed that the expression of VEGF, p-Akt, and p-mTOR was down-regulated in ginsenoside Rg3 -treated lesions. Real-time PCR results also showed that the mRNA expression levels of VEGF, Akt, and mTOR in the ectopic endometrium were reduced.

Conclusions: The present study demonstrates, for the first time, that ginsenoside Rg3 suppresses angiogenesis in developing endometrial lesions. The ginsenoside Rg3 inhibitory effect on the growth of the ectopic endometrium in EMs rats might occur through the blocking of the VEGFR-2-mediated PI3K/Akt/mTOR signaling pathway, thus halting angiogenesis and promoting the apoptosis of ectopic endometrial cells.

Fig 1. Molecular structure of the ginsenoside Rg3.

Fig 2. Weekly weight change in rats in before and after the treatment (n = 12).

Weight is represented as mean ± SD. (A: Ginsenoside Rg3 low-dosage group; B: Ginsenoside Rg3 high-dosage group; C: Gestrinone group; D: Model control group; E: Ovariectomized group).

Fig 3. Inhibitory effect of ginsenosideRg3 on ectopic endometrial growth.

(a) The volume of ectopic endometrial before treatment (n = 12); (b) The volume of ectopic endometrial after treatment (n = 12); (c) The inhibition rate of ectopic endometrialafter treatment (n = 12); **: P<0.01, *:P<0.05 (by Mann-Whitney U test). Volume is presented in median range (d) The epithelial height of ectopic endometrialafter treatment (n = 6). **: P<0.01 (by LSDt-test). Height is presented as means ± SDs. (A: Ginsenoside Rg3 low-dosage group; B: Ginsenoside Rg3 high-dosage group; C: Gestrinone group; D: Model control group; E: Ovariectomized group).

Fig 4. Effect of ginsenoside Rg3 on the levels of E₂ (left panel) and P (right panel) in serum as assessed by the ECLI assay (n = 12).

**: P<0.01, *:P<0.05 (by LSDt-test). Data are represented in means ± SDs. (A: Ginsenoside Rg3 low-dosage group; B: GinsenosideRg3 high-dosage group; C: Gestrinone group; D: Model control group; E: Ovariectomized group).

Fig 5. Effect of ginsenoside Rg3 on the protein expression levels and localization of VEGF, VEGFR-2, p-Akt, p-mTOR in the ectopic endometriaas measured by immunohistochemically.

(a) Protein expression and localization of VEGF, VEGFR-2, p-Akt, p-mTOR in the ectopic endometria by DAB staining, as assessed via electron microscopy. All magnifications were ×400. (b) Effect of ginsenoside Rg3 on VEGF, VEGFR-2, p-Akt, p-mTOR protein expression levels in the ectopic endometria (n = 6) as measured by the average gray values,**: P<0.01, *:P<0.05 (by LSD t-test). Average gray values are presented as mean ± SD. (A: Ginsenoside Rg3 low-dosage group; B: Ginsenoside Rg3 high-dosage group; C: Gestrinone group; D: Model control group; E: Ovariectomized group).

Fig 6. Effect of ginsenoside Rg3 on the protein expression levels of VEGF, VEGFR-2, p-Akt, p-mTOR in the ectopic endometria as assessed by Western blotting.

(a) Representative Western blotting results for VEGF, VEGFR-2, p-Akt, p-mTOR protein expression. (b) Relative fold change in protein expression levels of VEGF, VEGFR-2, p-Akt, p-mTOR in the ectopic endometria (n = 6). **: P<0.01, *:P<0.05 (by LSD t-test). Data are represented in mean ± SD. (A: GinsenosideRg3 low-dosage group; B: Ginsenoside Rg3 high-dosage group; C: Gestrinone group; D: Model control group; E: Ovariectomized group).

Fig 7. Effect of ginsenoside Rg3 on gene expression levels of VEGF, VEGFR-2, Akt, mTOR against reference gene in the ectopic endometria (n = 6).

(A: Ginsenoside Rg3 low-dosage group; B: Ginsenoside Rg3 high-dosage group; C: Gestrinone group; D: Model control group; E: Ovariectomized group).

Fig 8. Effect of ginsenoside Rg3 on the apoptotic morphological features of ectopic endometria.

(a) Apoptosis in ectopic endometria was observed by TUNEL assay. Green color represents TUNEL-positive cells as apoptosis. (b) The apoptotic index of ectopic endometrial cells after treatment (n = 6). *:P<0.05 (by LSD t-test). Data are represented in means ± SDs. (A: Ginsenoside Rg3 low-dosage group; B: Ginsenoside Rg3 high-dosage group; C: Gestrinone group; D: Model control group; E: Ovariectomized group).