Enhancement of Endometrial Receptivity by Cnidium officinale through Expressing LIF and Integrins

Improvement of endometrial receptivity is necessary for successful embryo implantation, and its impairment is associated with female infertility. In this study, we investigated the effect of the roots of Cnidium officinale Makino (CoM) on endometrial receptivity in both in vitro and in vivo model of embryo implantation. We found that CoM enhanced the adhesion of JAr cells to Ishikawa cells by stimulating expression of leukemia inhibitory factor (LIF) and integrins. In addition, blocking of LIFR using hLA or neutralization of integrins αV, β3, and β5 using antibodies significantly reduced the enhanced adhesion between JAr cell and CoM-treated Ishikawa cells, indicating that LIF and integrin play an important role in trophoblast-endometrium adhesion for embryo implantation. Furthermore, we identified that CoM significantly improved the implantation rate of blastocysts in the mouse model of RU-induced implantation failure. By collecting these results, here, we suggest that CoM has a therapeutic potential against female infertility associated with decreased endometrial receptivity.


Introduction
Embryonic implantation for a successful pregnancy is indispensable, which involves a complex process that requires molecular interactions of the endometrium and blastocyst [1]. During the window of implantation, the optimal environment of endometrium for adhering the embryo implies ultimately enhanced endometrial receptivity. is also affects embryo development and placenta formation [2]. e receptive endometrium is regulated by the interaction between hormones, growth factors, and cytokines [3]. Among cytokines in the endometrium, leukemia inhibitory factor (LIF) plays an important role in embryo implantation and is attributed to endometrial receptivity. LIF binds with its receptor (LIFR) and the coreceptor glycoprotein 130 (gp 130), resulting in activation of downstream signaling through signal transducer and activator of transcription factor 3 (STAT3) [4,5]. Interestingly, the expression of STAT3 is only possible during a certain period of endometrial receptivity [6]. Among the adhesion molecules that are regulated by cytokines such as secreted LIF, integrins are essential proteins to establish the required physical interaction between endometrium and blastocyst at the time of implantation. Like a LIF, the expression of integrins is used as a marker of endometrial receptivity [7,8].
e extract of Cnidium officinale Makino (CoM), which belongs to the Umbelliferae family, has been used as traditional herbal medicines to treat pain, inflammation, and menstrual disturbance in Asia [9]. Recent studies demonstrated that CoM has several pharmacological activities, such as anti-inflammation, antitumor, antimetastasis, and antiangiogenesis [9][10][11][12][13]. In a field of female disorders, several papers demonstrated that herbal formulae including C. officinale showed the estrogenic potential and antileukorrhea effect [14,15]. However, there are still no previous studies reporting the effect of CoM on the enhancement of the endometrial receptivity.
In this study, we showed a potential effect of CoM on the enhancement of the endometrial receptivity. e molecular regulation underlying CoM-increased enhanced endometrial receptivity was related to the expression of LIF and integrin molecules. From these results, we suggest that CoM might be a good candidate for a novel drug for ameliorating female infertility related to decreased endometrial receptivity.

Preparation of Herbal Extract.
Dried roots of C. officinale were purchased from Omni Herb (Daegu, Korea) in 2012.
is material was confirmed by Dr. Goya Choi of Korea Institute of Oriental Medicine (KIOM) (Daejeon, Korea). A voucher specimen (2-17-36) was deposited at the Clinical Medicine Division, KIOM. e extraction method is shown in Figure 1(a). Briefly, the extract was prepared in KIOM's laboratory from a mixture of chopped crude herbs that was extracted using distilled water at 100 ± 2°C for 3 h by reflux extraction (COSMOS, Kyungseo Machine Co., Incheon, Korea). e solution was filtered through filter paper. e filtered solution was concentrated using a vacuum evaporator (Ev-1020; Daihan Scientific Co., Ltd, Gangwon-do, Korea). e extract was freeze-dried to create a powder (Genesis 25L; SP Scientific, Stone Ridge, NY). e prepared powder (CoM) was stored at − 70°C.
e lyophilized extract (10 mg) was dissolved in 70% methanol (5 mL) and then filtered through a 0.2 μm membrane filter (Woongki Science Co., Ltd., Seoul, Korea) before being injected into HPLC for component analysis. e ferulic acid (Sigma-Aldrich; purity over 98.0%) was used as a standard indicator of CoM in this study [17]. e HPLCgrade solvents, methanol, acetonitrile, and water were obtained from J. T. Baker (Phillipsburg, NJ). Trifluoroacetic acid (analytical reagent grade) and the standards were procured from Sigma-Aldrich. e HPLC system consisted of a Waters Alliance 2695 system coupled with a 2998 photodiode array detector (Waters Co., Milford, MA). Data processing was performed with Empower software (version 3; Waters Co.). e ferulic acid in CoM was separated using a Luna 5 μm C 18 100 A column (4.6 × 250 mm, 5 μm particle size; Phenomenex Inc., Torrance, CA). e monitoring was performed at 240 nm for ferulic acid. e mobile phases consisted of water with 0.1% (v/v) distilled water (solvent A) and trifluoroacetic acid (solvent B) at a flow rate of 1.0 mL/ min. e gradient conditions changed as presented in Table 1. e injection volume was 10 μL.

Adhesion Assay.
Adhesion assay between JAr and Ishikawa cells were prepared as described previously [18]. To make cell monolayers, Ishikawa cells (1.5 × 10 6 cells) were seeded into 6-well plates and incubated with CoM treatment for 48 h. e JAr cells (2 × 10 6 cells) were labeled with 5chloromethylfluorescein diacetate (CMFDA) fluorescence dye (CellTracker Green; Life Technologies, Carlsbad, CA) for 15 min. To check adhesion between Ishikawa and JAr cells, labeled JAr cells were added to the Ishikawa cell monolayer. After shaking gently for 30 min, the cells were washed to remove unbound JAr cells. e attached cells were visualized with a fluorescence microscope (Axio Imager M1; Zeiss, Oberkochen, Germany) and counted.

Western Blot Analysis.
Total protein from each sample was extracted using 1% NP-40 lysis buffer ( ermo Fisher Scientific), separated by SDS-PAGE, and electrotransferred to nitrocellulose membranes (0.45 μm; ermo Fisher Scientific). e membranes were incubated with 1X blocking solution and then with primary antibodies, respectively. After the reaction with appropriate HRP-conjugated secondary antibodies, the bands were visualized using the ECL chemiluminescence system (GE Healthcare, Chicago, IL).

Animals.
Male and female C57BL/6 mice (7-8 weeks old, weight 20-22 g) were purchased from Orient Bio, Co. (Seongnam, Korea). ey were bred in a specific pathogenfree facility with free access to water and a standard diet under a 12 h light/12 h dark cycle. All experimental procedures were approved by the Animal Research Ethics Committee at the Pusan University of Korea (no. PNU-2018-1943).

Animal Models and Treatment.
e embryo implantation failure model was performed as described previously [19]. A schematic diagram of an in vivo experimental schedule is shown ( Figure S1). Briefly, forty female mice were randomly divided into four groups: control (Con), RU486 (RU), CoM + RU, and CoM. Female mice of CoM + RU and CoM or Con and RU groups were dosed daily with CoM (67.75 mg/ kg/day) or normal saline solution for 15 days, respectively. e doses of CoM were determined by daily intake criteria for humans (16 g intake for an average human 60 kg) in the clinic, which is about 67.75 mg/kg after considering the yield of CoM extraction (25.48%). Forty-eight hours before mating to synchronize the estrous cycle of female mice, 5 IU PMSG was pretreated and then 5 IU hCG was intraperitoneally injected. All female mice of groups were then mated with males (ratio 1 : 1). e next day, vaginal plug was observed and it was designated as day 1. To induce embryo implantation failure model, RU486 (4 mg/kg/day) or corn oil was orally administered to female mice of RU and CoM + RU or Con and CoM groups daily from day 4 to day 7. All mice were sacrificed the next day after separating from males, and both uterine horns  were excised to examine the number of implantation sites. e number of implanted embryos on each uterine horn was counted.

Statistical Analysis.
e statistical significance from results was performed by a Student's t-test or one-way analysis of variance with Tukey's post hoc test using GraphPad Prism (GraphPad Software, San Diego, CA) and expressed as the mean ± SEM. e minimum significance level was set at a p value of 0.05. All experiments were independently performed at least 3 times, except for animal study.

CoM Increases the Adhesion of JAr Cells to Ishikawa Cells.
To evaluate the phytochemical property of CoM, the waterextracted CoM was analyzed by HPLC. e yield of CoM was 25.48% and the retention time of ferulic acid was 13.98 min on absorbance of 240 nm (Figure 1(b)). To evaluate the effect of CoM on the cell viability, MTT assay was performed.
e results showed CoM did not induce significant cytotoxicity up to 500 μg/ml (Figure 2(a)). In subsequent experiments, we used CoM concentration at 50 μg/mL. To investigate the effect of CoM on embryo attachment for successful embryo implantation, we first performed an in vitro adhesion assay using human trophoblastic JAr cells and endometrial Ishikawa cells. As shown in Figure 2

e LIF and Integrins Mediate the CoM-Induced Endometrial Receptivity.
To determine the effect of CoM on the expression of LIF and integrins for endometrial receptivity, qRT-PCR analysis was performed using CoM-treated Ishikawa cells. As shown in Figure 3(a), CoM treatment elevated the mRNA level of LIF, ITGAV, ITGB3, and ITGB5 in Ishikawa cells in a concentration-dependent manner. In addition, western blot analysis also showed that the expression of LIF, ITGAV, ITGB3, and ITGB5 was enhanced by CoM stimulation (Figure 3(b)). To further investigate LIFdependent adhesion between trophoblast and endometrium, we performed the adhesion assay on Ishikawa cells by hLA pretreatment. As shown in Figure 4(a), CoM increased the adhesion of JAr cells to Ishikawa cells, whereas pretreatment with hLA markedly alleviated the adhesion by inhibition of the LIF/LIFR signaling pathway. Similarly, we next investigated whether integrins play a role in CoM-enhanced endometrial receptivity toward trophoblast. e treatment of integrin αV, β3, and β5 antibodies for neutralization significantly reduced the enhanced adhesion between JAr cell and CoM-treated Ishikawa cells (Figure 4(b)).

CoM Improves In Vivo Embryo Implantation in Mice.
To determine the effect of CoM on blastocyst attachment for embryo implantation, we performed CoM administration on RU486-induced implantation failure mouse model in vivo. A simple schematic of the experiment is shown in Figure S1. As shown in Figure 5, the number of implantation sites in the RU group (0.9 ± 2.51) was markedly depleted compared to the Con group (7.8 ± 2.2), whereas CoM + RU group (6 ± 3.33) was significantly higher than the RU (9 ± 0.94) group.

Discussion
In recent years, many research studies have focused on the control of endometrial receptivity [20]. Even in traditional medicine in Eastern Asia, Zhong et al. [21] reported that acupuncture improves endometrial receptivity. e reason is because new approaches are needed for treating female infertility, which has not yet been fully resolved, despite the technological advances on assisted reproductive technologies [22]. us, our research has explored from traditional herbs to improve endometrial receptivity as another option of infertility treatment. e components of CoM have abundant polyphenol including ferulic acid, caffeic acid, cnidilide, senkyunolide, and ligustilide [23]. ese polyphenol components of CoM have pharmacological properties as an anticancer agent through induction of cell death by the expression of Bax/p53 gene and antiangiogenic activity through inhibition of retinal neovascularization [9,10]. It also has anti-inflammatory activity in LPS-induced murine macrophages via suppression of JNK, ERK, and STAT signaling pathways [24]. However, several major components, such as ferulic acid and caffeic acid, were not effective to improve the endometrial receptivity nor LIF expression (data not shown). To elucidate which compounds of CoM are effective in endometrial receptivity, further extensive experiments are needed.
Many researchers have shown that diverse molecules, such as LIF, interleukin-1 (IL-1), and colony-stimulating factor-1 (CSF-1), play an important role in endometrial receptivity for blastocyst implantation [25][26][27]. Among them, LIF is the main cytokine regulating the endometrial receptivity [28]. In addition, the embryo implantation process for successful pregnancy involves the adhesion of trophoblast to the receptive endometrium [29,30]. Significant changes in the expression of adhesion molecules in endometrium have effects on endometrial receptivity. omas et al. [31] have reported the increased expression of integrin αVβ3, as an important marker of endometrial receptivity, in the luminal epithelium during the implantation window. Another group has shown that various integrin subunits α1, α4, β1, and β5 were known to be expressed in endometrial epithelium during the implantation window [7]. Recently, Kumar et al. [32] showed that integrin β8 activates VAV-RAC1 signaling axis via FAK to facilitate the endometrial epithelial cell receptivity for blastocyst implantation. Previously, we also reported that LIF regulates endometrial receptivity via integrins αVβ3 and αVβ5 [18]. e binding of secreted LIF and its receptor in the endometrial epithelium increases expression of integrins αVβ3 and αVβ5 on the plasma membrane surface of endometrial cells, which is involved in the adhesion of the trophoblastic cells to the endometrial cells for blastocyst implantation.  were examined by qRT-PCR (a) and western blot (b), respectively. β-actin was used as an internal control. Band intensity was calculated by fold of control and is shown as mean ± SEM. * p < 0.05, * * p < 0.01, and * * * p < 0.001 in comparison with control groups.
Based on these articles, our results showed that CoM increases the expression of LIF, ITGAV, ITGB3, and ITGB5 in Ishikawa cells, indicating the promotion of endometrial receptivity for embryo implantation (Figure 3). In addition, the enhanced endometrial receptivity by CoM may affect adhesion of JAr cells to Ishikawa cells (Figure 4). Blocking of LIFR using hLA diminished the adhesion of JAr cells to LIFstimulated Ishikawa cells which is due to adhesion between JAr cells and CoM-stimulated Ishikawa cell through LIFdependent pathway. Neutralization of αV, β3, and β5 expression on the endometrial cell surface by using integrin αV, β3, and β5 antibodies significantly reduced the adhesion between trophoblastic cells and CoM-stimulated endometrial cells, suggesting that all integrins αV, β3, and β5 play a critical role in cell-cell adhesion for embryo implantation. Furthermore, these results clearly showed improved implantation of blastocysts in the mouse model of RU-induced implantation failure ( Figure 5). Further clinical studies are needed to confirm the effect of CoM on infertile women with decreased endometrial receptivity. e safety properties of CoM were not validated in this study. Previous study reported that its LD 50 of i.p. injection in mouse is lower than 34.5 g/kg and LD 50 of p.o. administration is estimated to 23 g/kg [33]. e dose used in this study (67.75 mg/kg/day) was deduced from a general dose of clinical use of CoM, as previously mentioned in the Material section. In addition, the concentration is much lower compared with a previously reported lethal dose. us, we supposed that the dosage might be safe. However, to exactly assess the safety of CoM, further acute and chronic toxicological studies at good laboratory practice level should be performed.
In conclusion, as illustrated in Figure 6, CoM enhances the expression of LIF. e expression of ITGAV, ITGB3, and ITGB5 in CoM-treated endometrial cells also increases the adhesion of trophoblastic cells to endometrial cells for blastocyst implantation. Blocking of LIFR using hLA and neutralization of integrin subunits αV, β3, and β5 using antibodies efficiently inhibits the adhesion of the trophoblastic cells to the CoM-stimulated endometrial cells. CoM + RU CoM Con Figure 5: In vivo effect of CoM administration on embryo implantation. To confirm improvement of embryo implantation by CoM, female mice were orally administered with CoM (67.75 mg/kg/day) in 100 μL of normal saline solution for 15 days. On day 4 from pregnancy estimated by vaginal plugs, the female mice were injected with RU486 (4 mg/kg/day) for 4 days. All mice were sacrificed, and both uterine horns were excised to examine the number of implanted embryos. e number of implantation sites was calculated as means ± SEM. Uterine tissue sections were analyzed histologically after hematoxylin and eosin staining. * * p < 0.01 and * * * p < 0.0001 for comparisons between two groups. erefore, our results suggest that CoM has a therapeutic potential for ameliorating female infertility related to reduced endometrial receptivity.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request. is study provides evidence that CoM increased adhesion of trophoblast to endometrium through the enhanced expression of LIF, integrin αV, β3, and β5, indicating improved endometrial receptivity.