Paeonia lactiflora Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression

Abstract

In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins β3 and β5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells.

Fig 1. PL-PP HPL chromatogram.

HPLC was performed as described in the Materials and Methods. Retention times of the standard compounds were 4.8, 10.5, 11.5, 17.5, and 28 min for gallic acid, catechin, methyl gallate, paeoniflorin, and benzoic acid, respectively.

Fig 2. The effect of PL-PP on LIF expression and adhesion of JAr spheroids to Ishikawa cells.

(A, B) Total RNA and protein were isolated after treatment with the indicated concentrations of PL-PP in serum—free medium for 24 h. Relative levels of LIF mRNA and protein were examined by RT-PCR and western blot analysis, respectively. The intensity of the band of interest was estimated by densitometric analysis and calculated as the mean ± SD of three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001 compared to the control group). (C) Ishikawa cells were cultured in 24-well plates and treated with or without PL-PP (50 μg/mL) for 48 h. Twenty JAr spheroids were added onto the Ishikawa cell monolayer. The number of JAr spheroids bound to confluent Ishikawa cells was manually counted and calculated as the mean ± SD of three independent experiments (* P < 0.05 compared to each group).

Fig 3. Identification of signaling pathways involved in PL-PP-induced LIF expression.

Ishikawa cells were pre-treated with SP600125 (10 μM), LY294002 (15 μM), SB203580 (10 μM), and/or U0126 (0.5 μM) for 1 h. After treatment of each signaling inhibitor, the cells were stimulated with PL-PP (50 μg/mL) for 24 h. Total RNA and protein were isolated from each sample and LIF expression was evaluated by RT-PCR (A) and western blot analysis (B). The intensity of the band of interest was estimated by densitometric analysis and calculated as means ± SD of two independent experiments (^# P < 0.01 vs. the untreated control. * P < 0.05 and ** P < 0.01 vs. the PL-PP-treated control). Ishikawa cells were treated with PL-PP for the indicated times (0, 0.5, 1, 2, and 4 h). Total protein was isolated from each sample and p38 and ERk phosphorylations were evaluated by western blot analysis (C). The intensity of the band of interest was estimated by densitometric analysis and calculated as means ± SD of three independent experiments (* P < 0.05 compared to each control).

Fig 4. Adhesion of JAr spheroids to PL-PP-stimulated LIF-knockdown Ishikawa cells.

Ishikawa cells harboring pLKO.1 or shLIF vector were cultured in 24-well plates and treated with or without PL-PP (50 μg/mL) in serum-free medium for 48 h. Twenty JAr spheroids were added onto the Ishikawa cell monolayer. Representative pictures were taken and the number of adhered JAr spheroids to Ishikawa cells was counted and calculated as the mean ± SD of three independent experiments (** P < 0.01 and *** P < 0.001 compared to each group).

Fig 5. Expression of adhesion molecules in PL-PP treated Ishikawa cells.

(A) Ishikawa cells were treated with or without PL-PP (50 μg/mL) for 24 h, and total RNA was extracted. The expression levels of LIF, ITGAV, ITGB1, ITGB3, ITGB4, ITGB5, ICAM-1, L-selectin, E-cadherin, and CD44 mRNA were analysed by RT-PCR. β-actin was used as an internal control. (B) The pLKO.1 –or shLIF—transfected Ishikawa cells were treated with PL-PP (50 μg/mL) for 24 h. Total RNA was extracted and ITGB3 and ITGB5 mRNA expression levels were measured by RT-PCR. β-actin was used as an internal control.

Fig 6. In vitro and In vivo effect of PL-PP on implantation in the presence of a progesterone antagonist.

(A) Ishikawa cells in the absence or presence of RU486 (10 μM) were treated with or without PL-PP (50 μg/mL) in serum free medium for 48 h. Twenty JAr spheroids were added onto the Ishikawa cell monolayer. The number of adherent JAr spheroids to Ishikawa cells was counted in representative pictures and calculated as the mean ± SD of three independent experiments (* P < 0.05, *** P < 0.001 compared to each group and ns means no significance) (B) Female mice were treated with or without RU486 and PL-PP. After 7 days, all mice were euthanized and both uterine horns were excised to determine the number of implantation sites. Pictures of implanted embryos are shown. Arrows indicate implanted embryos. The number of implantation sites was counted and calculated as the mean ± SD of three independent experiments (** P < 0.05 and *** P < 0.001 compared to each group). (C) The uterin tissue sections were analyzed histologically after H&E staining. Embryo implantation sites are indicated by arrows.

Fig 7. Schematic representation of the mechanism underlying the P. lactiflora induced LIF expression in the endometrium and its relevance.

P. lactiflora induces LIF expression through the activation of p38 and MEK/ERK pathways, leading to an increase in the trophoblast adhesion to the endometrium. Inhibition of LIF expression blocks the expression of adhesion molecules, such as integrin β3 and β5, and adhesion of the trophoblast to the endometrium.