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

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.


Introduction
Implantation is a complex biological process, requiring communication between the appropriately developed trophoblast and the receptive endometrium [1]. This process is established and maintained by diverse biological factors, including cytokines, growth factors, and receptors [2].
Embryo implantation is adversely affected by abnormal expression of the genes related to the establishment of uterine receptivity [3]. Among these factors, leukemia inhibitory factor (LIF) plays a key role in determining the outcome of implantation [4][5][6]. Genetic mutations and aberrant expression of LIF are known to contribute to implantation failure in mice and humans [7,8]. In addition, inhibition of LIF activity by addition of an anti-LIF antibody or LIF antagonist effectively prevents embryo implantation in mice, monkeys, and humans [9][10][11][12].
In recent years significant advances have been made in assisted reproductive technologies (ART). However, pregnancy rates remain low [13]. High doses of exogenous gonadotropins used for ovarian stimulation during in vitro fertilization (IVF) procedures are known to impair endometrial receptivity [14]. Traditional herbal remedies and acupuncture, therefore are being proposed as alternative treatments for improving endometrial receptivity [15][16][17]. Several groups have reported that LIF is an important molecular target of herbal remedies and acupuncture [18][19][20].
The roots of P. lactiflora Pall., belonging to the Paeoniaceae family, have been used as a therapeutic drug for treating fever, rheumatoid arthritis, systemic lupus erythematosus, hepatitis, and gynecological disorders in Korea, Japan, and China [21,22]. Especially, P. lactiflora has been used for several years to treat various gynecological problems such as dysmenorrhea, cramps and spasms during pregnancy, and infertility [23,24]. Different components extracted from the roots of P. lactiflora are reported to have anti-inflammatory, immunomodulatory, anti-allergic, anti-arthritic, and hepatoprotective activities [21,[25][26][27]. However, the biological effects of P. lactiflora extract on endometrial receptivity have not been explored.
Therefore, in this study, we evaluated the effects of P. lactiflora extract on the LIF expression in endometrial Ishikawa cells and adhesion of trophoblastic JAr spheroids to Ishikawa cells. In addition, the effect of P. lactiflora extract on embryo implantation was examined in an implantation-failure mouse model.

Extract preparation
The roots of P. lactiflora were purchased from Omniherb Co. (Daegu, Korea). The herb was authenticated by a botanist at Omniherb Co. A voucher specimen (DC-H21) in the form of a frozen rhizome was deposited in the Korean Medicine Research Center for Healthy Aging, Pusan National University (Yangsan, Korea). The extraction method is described in S1 Fig. PL-WE and PL-PP were freshly dissolved in distilled water before performing subsequent experiments.
Fingerprinting high-performance liquid chromatography (HPLC) analysis HPLC analysis was performed using a Shimadzu HPLC system (Shimadzu Co., Kyoto, Japan), consisting of a solvent delivery unit, an on-line degasser, a column oven, an autosampler, and an SPD-20A UV detector. For data analysis, LC solution software (Version 1.25) was used. The analytical column used was an ACE C18 column (4.6 × 250 mm i.d., 5 μm pore size). The mobile phases were solvent A (acetonitrile) and solvent B (0.1% phosphoric acid). The gradient flow was as follows:  (46-50 min). The column temperature was maintained at 25°C. The analysis was carried out at a flow rate of 1 mL/min with UV detection at 254 nm. The column injection volume was 20 μL. The standard solution and PL-PP were prepared by dissolution in dimethyl sulfoxide (DMSO; 1 mM and 50 mg/mL, respectively). The solutions were filtered through a 0.45-μm membrane filter (Millipore) and applied to HPLC.

Cell culture
Human endometrial Ishikawa cell line, derived from a human adenocarcinoma, was kindly provided by Dr. Jacques Simard (CHUL Research Center, Quebec, Canada). The cells were maintained as monolayers at 37°C in an atmosphere containing 5% CO 2 /air in Dulbecco's Modified Eagle Medium (DMEM; Welgene, Daegu, Korea) containing 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin/streptomycin (Gibco, Rockville, MD, USA). The human choriocarcinoma JAr cells were obtained from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI1640 (Welgene) containing 10% heat-inactivated FBS and 1% penicillin/streptomycin. AN3CA cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Cell viability Assay
The cytotoxic effects of PL-WE and PL-PP were examined using the methylthiazolyldiphenyltetrazolium bromide assay (MTT, Sigma-Aldrich). In brief, Ishikawa cells were cultured in 24-well plates with PL-WE and PL-PP (indicated concentrations) for 24 h. After removal of the culture medium, 300 μL of 1× MTT solution (0.5 mg/mL) was added to each well. After incubation for 4 h at 37°C in a CO 2 incubator, the supernatants were removed and formazan crystals formed in viable cells were dissolved in 300 μL of ethanol:DMSO (v/v, 1:1). To detect the cytotoxicity, the absorbance in each well was measured at 540 nm with a microplate reader. The percentage of living cells in treated cultures was calculated relative to that in untreated cultures.

Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated from Ishikawa cells using RiboEx™ (GeneAll, Seoul, Korea). Equal amounts of total RNA (1 μg) from each sample were then subjected to reverse transcription with oligo-dT primers by using M-MLV reverse transcriptase (Enzynomics, Daejeon, Korea). The cDNA was amplified by PCR using DiaStar™ Taq DNA Polymerase (Solgent Co., Daejeon, Korea). The PCR conditions, amplified size of each target gene, and primers used in this study are shown in Table 1.

LIF knockdown by shRNA
To knock down endogenous hLIF, lentiviral shRNA constructs were obtained from Open Biosystems (Thermo Scientific, Waltham, MA, USA). 293T cells (1.5 × 10 6 ) were subcultured in a 10 cm dish. Twenty-four hours after cell seeding, the lentiviral vector encoding hLIF shRNA (3 μg), packaging vector pCMV-VSVG (0.3 μg), and the envelop plasmid pHRCMV-8.2 ΔR (3 μg) were transfected in 293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Forty-eight hours after transfection, the supernatant harboring lentiviruses was collected and filtered using a sterilized 0.45 μm syringe filter. LIF shRNA lentiviral supernatant with polybrene (5 μg/mL) was added to the Ishikawa cells. To select cells with stable integration of LIF shRNA, the viral infected Ishikawa cells were treated with 3 μg/mL puromycin for 1 week. The knockdown efficiency of the LIF shRNA was verified by western blot analysis. The best hLIF shRNA #5, among 5 hLIF shRNA clones (#3-#7), was used for subsequent experiments. The sequence of LIF shRNA #5 used for knocking down LIF was 5'-TTACCCGAGGTGT CAGGGCCG-3'. JAr spheroid adhesion assay Ishikawa cells (3 × 10 5 cells) were seeded into 24 well plates and cultured for 24 h. The medium was replaced and the cells were incubated in serum-free -DMEM containing PL-PP for 48 h.
To make JAr spheroids, we used the GravityPLUS™ Kit (InSphero Co.) according to the manufacturer's manual. Briefly, after JAr cells were trypsinized and counted, the suspended JAr cells (1 × 10 3 /40 μl RPMI medium) were gently delivered into each well of the GravityPLUS™ plate and incubated in a humidified 5% CO 2 incubator at 37°C for 3 days. JAr spheroids (20/well) were gently delivered onto a confluent monolayer of Ishikawa cells grown in a 24 well culture plate. After incubation for 2 h at 37°C, the cells were washed with 10% FBS/RPMI medium three times to remove non-binding JAr spheroids. The attached JAr spheroids were visualized using a camera and counted.

Animals
Male and female C57BL/6 mice (7-8 weeks old, weight 20-22 g), inbred in a specific pathogenfree (SPF) facility, were purchased from Orient Bio, Co. (Seongnam, Korea). They were bred separately and had free access to water and a standard diet with a 12 h light / 12 h dark cycle. All experimental procedures were examined and approved by the Animal Research Ethics Committee at the Pusan University of Korea.

Animal models and treatment
All 33 female mice were randomly divided into 3 groups, including a control group, RU486 group, and RU486 with PL-PP group. To improve implantation, the female mice were treated with PL-PP every day for 17 days. Female mice belonging to the RU486 with PL-PP group received PL-PP orally (1.06 mg/20 g) every day, while the mice belonging to the control and RU486 groups received the same amount of PBS. After seven days of daily treatment with PL-PP, all females were caged with males (ratio 2:1) at 6 pm and the day when vaginal plugs were detected was designated as day 1 of pregnancy. The embryo implantation failure model was developed according to previous reports with some modifications [20,28]. Briefly, the mice belonging to the RU486 group and RU486 with PL-PP group were injected subcutaneously with 0.08 mg/0.1 mL of RU486 solution, while the mice belonging to the control group were injected with the same volume of corn oil on day 4 of pregnancy at 9 am. Seven days after RU486 injection, all mice were euthanized by inhalation of CO 2 gas and both uterine horns were excised to determine the number of implantation sites. The number of implanted embryos on each uterine horn was recorded.

Densitometry and statistical analysis
The intensity of the bands obtained from RT-PCR and western blot analysis, and the attached spheroids from the adhesion assay were quantified with ImageJ software. Data from all experiments are indicated as the percentage or fold compared to the control and expressed as the mean ± SD. The differences between the mean values of 2 groups were determined using a standard t-test with the GraphPad Prism (GraphPad Software, CA, USA). Furthermore, for comparison among groups, one-way analysis of variance (ANOVA) tests with a Dunnet's post-hoc comparison were used with the assistance of GraphPad Prism. The minimum significance level was set at a P value of 0.05. All experiments were independently performed at least 3 times.

Results
Phytochemical validation of P. lactiflora extracts and effect on cell viability of Ishikawa cells As shown in Fig 1, PL-PP contained gallic acid, catechin, methyl gallate, paeoniflorin, and benzoic acid. PL-PP contains 151.14 μg/mg (>15%) paeoniflorin, which was the most abundant molecule among the five compounds tested (S1 Table). As shown in S2A Fig, PL-WE treatment inhibited cell growth in a dose-dependent manner. Treatment with 10, 50, 100, and 500 μg/mL PL-WE showed 8, 22, 31, and 63% inhibition, respectively, while PL-PP treatment at concentrations of 500 μg/mL inhibited cell proliferation by 21% (S2B Fig). PL-PP did not have significant cytotoxic activity up to 100 μg/mL; therefore, we used PL-PP concentrations up to 50 μg/mL for the subsequent experiments.  (Fig  2A and 2B). We next examined whether PL-PP enhances the adhesion of Jar spheroids to Ishikawa cells. As shown in Fig 2C,

PL-PP regulates LIF expression through the p38 and MEK/ERK signaling pathways
To determine the upstream signaling pathways responsible for PL-PP-induced LIF expression, we used various signaling inhibitors. The specific inhibitors of JNK (SP600125) and PI3K (LY294002) had no effect on the PL-PP-induced LIF expression (Fig 3A and 3B). However, p38MAPK (SB203580) and MEK/ERK inhibitors (U0126) did inhibit the PL-PP-induced LIF expression. As shown in Fig 3C, PL-PP markedly induced the phosphorylation of p38 and ERK. These results suggest that the activation of p38 and ERK, but not that of JNK or PI3K, is required for the PL-PP-induced expression of LIF. To further investigate whether the PL-PP induced increase in the expression of adhesion molecules is mediated via a LIF-dependent pathway, we assessed ITGB3 and ITGB5 transcript levels in PL-PP treated pLKO.1-and shLIF-transfected Ishikawa cells. The transcript levels of ITGB3 and ITGB5 were found to be lower in PL-PP treated LIF knock-down cells compared with PL-PP treated pLKO.1-transfected cells (Fig 5B). These results suggest that PL-PP induces an increase in the expression of ITGB3 and ITGB5 through a LIF-dependent pathway.

PL-PP administration restores implantation in an implantation-failure mouse model
We next investigated whether the administration of PL-PP could effectively promotes blastocyst implantation in vivo. First, we assessed the effect of RU486, a well-known contraceptive drug, on LIF expression in human endometrial Ishikawa cells. RU486 treatment did not have any significant effect on LIF expression (data not shown). We also examined the in vitro effect of PL-PP on the adhesion of JAr spheroids to RU486 treated-Ishikawa cells. As shown in Fig  6A, the number of JAr spheroids adhered to RU486-pretreated Ishikawa cells increased from 41% to 61.66% after treatment with PL-PP. Further, the total number of implanted embryos in the RU486 with PL-PP group (3.36 ± 3.07), though lower than that in the control group (6.70 ± 2.26), was significantly higher than that in the RU486 group (0.18 ± 0.60). This indicated that PL-PP restores the implantation defect induced by RU486 treatment. Taken together, the data suggest that PL-PP-induced LIF expression plays a crucial role in the adhesion of Jar spheroids to the endometrium (Fig 7).

Discussion
Embryo quality and endometrial receptivity are crucial factors for successful implantation. In this study, we investigated the potential of medicinal herbs to improve uterine receptivity. Although P. lactiflora is one of the most well-known herbs for the treatment of diverse gynecological diseases [21,22], the effects of P. lactiflora on endometrial receptivity have not been investigated. PL-WE, which is extracted from the roots of P. lactiflora with boiling water, contains polysaccharides. It has been reported that polysaccharides cause an abnormal cellular immune response and interruption of cell-cell interactions [29,30]. In the present study, PL-WE did not induce LIF expression in the endometrium and was found to be cytotoxic. PL-PP was devoid of polysaccharides. Biochemical analysis suggests that paeoniflorin is one of the major compounds contributing to the effects of P. lactiflora.
Several factors such as integrins, interleukin-1 (IL-1), calcitonin, amphiregulin, epidermal growth factor (EGF), HB (heparin binding)-EGF, colony stimulating factor-1, LIF, mucins, trophinin/tastin, Hoxa-10, and cyclooxygenase (COX) contribute to endometrial receptivity [31]. LIF, a key factor for embryo implantation, is expressed in the glandular epithelial, luminal 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).
doi:10.1371/journal.pone.0148232.g003 epithelial and stromal cells of the endometrium [5,32]. Our investigations demonstrated that in vitro PL-PP treatment increases the LIF expression and the adhesion of Jar spheroids to Ishikawa cells. Further LIF knockdown inhibited the adhesion of JAr spheroids to PL-PP-treated Ishikawa cells. These results suggest that an increase in the LIF expression in response to PL-PP favors the adhesion of JAr spheroids to Ishikawa cells.
Interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) induce the phosphorylation of nuclear factor κB (NF-κB), which binds to the corresponding responsive elements in the LIF promoter region [33]. IL-1β stimulated LIF induction is mediated by MEK/ERK and NF-κBpathways in normal articular human chondrocytes [34]. TNF-α is known to induce LIF expression through p38MAPK signaling pathway in bone marrow stromal cells from pediatric patients with myelodysplastic syndrome and neuronal retinal cells [35,36]. Our results showed involvement of p38 and MEK/ERK signaling pathways in PL-PP-induced LIF expression. It has been reported that the inhibition of LIF-induced STAT3 activation in the endometrium suppresses implantation [37]. We also observed that the PL-PP induced increase in LIF expression involves STAT3 activation.
Implantation is associated with modulation in the endometrial expression of several adhesion molecules, including integrins [38][39][40]. Integrins α2β1, α3β1, and α6β4 are reported to be constitutively expressed in Ishikawa cells. Further the expression of α1β1 and αvβ3 integrins is reported to be under hormonal regulation [41]. The cell surface expression of adhesion molecules determines the adhesiveness of JAr spheroids to endometrial cells [42]. Human endometrial RL95-2 cells, known as highly adhesive cells, express integrin α6, β1, and β4 subunits. On the other hand, non-receptive human endometrial AN3-CA cells lack β4 integrin subunit and show reduced expression of α6 and β1 integrin subunits. This results in lesser adhesion of JAr spheroids to endometrial AN3-CA cells [43].
E-cadherin is located on the plasma membrane of the epithelial cells and is critical for the maintenance of adherent junctions [44][45][46]. It has been reported that E-cadherin expression is reduced in the uterine epithelium during implantation in rodents [47]. This results in the reorganization of the adherent junctions between epithelial cells to facilitate blastocyst implantation [47]. Our data also demonstrated a reduction in E-cadherin expression in endometrial cells, following PL-PP treatment. Further the study demonstrated that PL-PP induces the expression of integrins β3 and β5 in a LIF-dependent manner.
Danielsson et al. showed that RU486 treatment immediately post ovulation reduces endometrial glandular LIF expression at the expected time of implantation [48]. Lalitkumar et al. reported that RU486 inhibits human blastocyst attachment to an endometrial three-dimensional cell model in vitro [49]. A previous report showed that various cytokines such as EGF, TGF-α, PDGF, and IL-1β are potent inducers of LIF expression in endometrial stromal cells [48]. However, steroid hormones such as estradiol and progesterone do not exert a regulatory effect on LIF expression in human endometrial cells [50]. We also did not observe any adverse effect of RU486 on LIF expression in Ishikawa cells. Also the number of JAr spheroids attached to Ishikawa cells did not change due to pre-treatment with RU486. PL-PP treatment also augmented the number of implanted embryos in an in vivo implantation failure model, developed using RU486. It is likely that a PL-PP induced increase in LIF expression does not involve the progesterone receptor signaling pathway.  Conclusions PL-PP increases the LIF expression in Ishikawa cells through p38 MAPK and MEK/ERK signaling pathways in endometrial Ishikawa cells. The increase in LIF expression by PL-PP favors the adhesion of human trophoblastic JAr spheroids to Ishikawa cells. Also, the administration of PL-PP improves embryo implantation in RU486-treated mice. These results suggest that PL-PP may facilitate the adhesion of the blastocyst to the endometrium. PL-PP may be considered as a potential therapeutic herbal medicine for improving pregnancy rates. AN3-CA 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, Lselectin, E-cadherin and CD44 mRNA were examined by RT-PCR. β-actin was used as an internal control. (B) AN3-CA cells 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. The number of adherent JAr spheroids to Ishikawa cells was counted in representative pictures, and calculated as the mean ± SD of three independent experiments ( ÃÃ P < 0.01 compared to each group). (TIF) S1 Table. Quantitative phytochemical constituents of PL-PP. (DOCX)