Anti-Endometriotic Effects of Pueraria Flower Extract in Human Endometriotic Cells and Mice

Pueraria flowers have been used as a vegetable and an ingredient for tea and jelly. In this study, we investigated the effects of Pueraria flower extract (PFE) on endometriosis, a common gynaecological disease characterised by local sterile inflammation of peritoneal cavity. PFE suppressed the adhesion of human endometriotic cells 11Z and 12Z to human mesothelial Met5A cells. In addition, PFE significantly inhibited the migration of 11Z and 12Z cells as shown by wound-healing and transwell migration assays. PFE reduced the protein and mRNA levels of matrix metalloproteinase (MMP)-2 and MMP-9 in endometriotic cells. Moreover, extracellular signal-regulated kinase (ERK)1/2 was activated by PFE treatment, and an ERK1/2 inhibitor, PD98059, significantly inhibited PFE-inhibited cell migration in endometriotic cells. Furthermore, PFE significantly suppressed endometriotic lesion formation in a mouse model. These data suggest that Pueraria flower is a potential anti-endometriotic agent for the inhibition of endometriotic cell adhesion, migration, and MMP expression.


Introduction
Endometriosis is a common gynaecological disease that affect approximately 1 in 7 women and 30%-50% of infertile women [1]. The common symptoms of the disorder are dysmenorrhoea, chronic pelvic pain, and infertility. Endometriosis is characterised by the ectopic implantation and growth of endometrial tissue and local sterile inflammation of peritoneal cavity. Proliferation, adhesion, and migration of ectopic endometrial tissue are required to establish endometriotic lesions in the peritoneal cavity [2,3]. In addition, the expression of matrix metalloproteinase (MMP)-2 and MMP-9 have been shown to be higher in women with endometriosis compared to healthy controls [4][5][6].
Treatment options for endometriosis include surgery and medication. Current medical treatments for endometriosis fall into pain medication such as non-steroidal anti-inflammatory agents or hormone therapy such as gonadotropin-releasing hormone (GnRH) agonists, and androgens. Although the treatments can improve some symptoms, there is no cure for endometriosis. The treatments do not reduce the high recurrence rate of endometriosis and can have several adverse effects such as genital atrophy and hot flashes [7]. Therefore, the establishment of novel and effective therapeutic agents is urgently needed to improve clinical management of endometriosis patients.
Pueraria lobata Ohwi (Leguminosae), also known as "Kudzu" and "Japanese arrowroot" in the Western world, is a perennial leguminous vine endemic to eastern Asia [8]. In the United States,

Adhesion Assay
Met5A cells were grown to confluence on 96-well plates. Endometriotic cells (11Z and 12Z) were probed with CellTracker™ (10 mM) for 1 h at 37 • C and added (2 × 10 4 cells/well) to the mesothelial Met5A cells on 96-well plates. Nonadherent cells were washed out with medium following incubation at 37 • C for 1 h. The fluorescence at a wavelength of 490 nm in each well was analysed.

Wound-Healing Assay
Scratch wound-healing assays were performed in 12-well tissue culture plates with minor modifications [21]. Endometriotic cells (11Z and 12Z) (5 × 10 5 cells per well) were seeded in 12-well plates and grown to 80%-90% confluence. In order to create a denuded zone (gap) of constant width, the centres of the endometriotic cell monolayers were scraped with a sterile micropipette tip. After washing out the cellular debris with phosphate-buffered saline (PBS), cells were treated with PFE in the medium with 2% FBS. After 24 h treatment, the wound closure was photographed and analysed. The pictures of the initial wounded monolayers were compared with corresponding pictures of cells at the end of incubation. Artificial lines fitting the cut edges were drawn on pictures of the original wounds and overlaid on the pictures of cultures after incubation. Gap distance of the wound was measured using Photoshop software, and the data were normalised to the average of the control. Cells that migrated across the lines were counted in six random fields from each triplicate treatment, and data are presented as the mean ± SD.

Transwell-Migration Assay
A 24-well transwell unit (8 µm pore size) with polyvinylpyrrolidone-free polycarbonate (PVPF) filters was used for the in vitro transwell-migration assay. Endometriotic cells (3 × 10 4 ) were added in the upper part of the insert and incubated with the extract for 24 h at 37 • C. The cells that migrated to the lower surface of the membrane were stained with 0.05% crystal violet for 20 min after fixation with methanol. Migrative phenotypes were evaluated by counting the cells that migrated to the lower side of the filter under microscopy at 200×. All experiments were done in triplicate, and a minimum of five fields per filter was counted.

Animal Study
Female mice (5 weeks of age, BALB/c) were obtained from Korea Orient Bio, Inc. (Seoul, Korea). Animals were maintained with free access to food and water in pathogen-free conditions in an air-conditioned room (humidity of 55% ± 5% and temperature of 24 • C ± 1 • C) under controlled lighting (12 h light/dark cycle). All animal-handling protocols and surgical procedures were approved by the Committee for the Care and Use of Laboratory Animals (KHP-2014-05-1) in College of Pharmacy, Kyung Hee University in compliance with institutional guidelines for experimental animal care. The endometriosis was induced in mice using a previously established method with modifications [22,23]. Briefly, the uterine horns of the donor mice were removed and put into a dish containing PBS. The endometrium-rich fragments (1 cm) from the middle-third of the uterine horn were finely and uniformly chopped. The fragments (~20 pieces) suspended in PBS were injected into the peritoneal cavity of recipient mice with a micropipette to induce the formation of endometriosis-like lesions. The 15 mice with induced endometriosis were randomly divided into three groups (5 mice/group): control, PFE 150, and PFE 300. Mice were orally administered either vehicle (200 µL of PBS) alone or PFE (150 and 300 mg/kg/day) for 5 weeks from a week before the inoculation with endometrial fragments. Mice body weight changes were measured once per week during the treatment period. Additionally, we investigated whether the administration of PFE induced kidney and liver toxicity in the mouse models. After induction of endometriosis for 4 weeks, the mice were sacrificed by cervical dislocation and the peritoneum and visceral organs were examined visually to measure the number of endometriotic lesions (1 > mm). The serum creatinine and blood urea nitrogen (BUN) levels were measured as indicators of the kidney function, while the alanine transaminase (ALT) and aspartate transaminase (AST) levels were measured to evaluate the liver function. The levels of serum creatinine, BUN, and ALT/AST activity in the PFE-treated mice were not different from those observed in the vehicle-treated control mice.

Statistical Analysis
For statistical analysis, GraphPad Prism version 5 (GraphPad, San Diego, CA, USA) was used. Statistical comparisons between two groups were made using Student's t-test. For comparing more than two groups, one-way analysis of variance (ANOVA) was used followed by the Tukey test with a significance of p < 0.05. All errors are shown as standard deviation (SD).

PFE Treatment Inhibits Endometriotic Cell Adhesion to Mesothelial Cells
The initial step of endometriotic lesion formation is the attachment of endometriotic cells to the layer of mesothelial cells that covers the peritoneal cavity. Using human endometriotic 11Z and 12Z cells and mesothelial Met5A cells, adhesion assay was performed to elucidate the effect of PFE on the adhesion ability of endometriotic cells to the mesothelial layer. Preincubation of 11Z and 12Z cells with PFE (25, 50, and 100 µg/mL) suppressed their adhesion to Met5A cells ( Figure 1A,B). The antiadhesive effect of PFE was not attributable to any cytotoxic effect, since the extract, used at concentrations of up to 200 µg/mL, did not affect the viability of the endometriotic or mesothelial cells over a period of 48 h ( Figure 1C).

PFE Treatment Inhibits Endometriotic Cell Adhesion to Mesothelial Cells
The initial step of endometriotic lesion formation is the attachment of endometriotic cells to the layer of mesothelial cells that covers the peritoneal cavity. Using human endometriotic 11Z and 12Z cells and mesothelial Met5A cells, adhesion assay was performed to elucidate the effect of PFE on the adhesion ability of endometriotic cells to the mesothelial layer. Preincubation of 11Z and 12Z cells with PFE (25, 50, and 100 μg/mL) suppressed their adhesion to Met5A cells ( Figure 1A,B). The antiadhesive effect of PFE was not attributable to any cytotoxic effect, since the extract, used at concentrations of up to 200 μg/mL, did not affect the viability of the endometriotic or mesothelial cells over a period of 48 h ( Figure 1C).

PFE Inhibits Endometriotic Cell Migration
Wound-healing assays and transwell migration assays were performed to evaluate the effect of PFE on endometriotic cell migration. First, the wound-healing assay showed that the wound in endometriotic 11Z and 12Z cells was about 60% healed after 24 h in the absence of PFE treatment. In contrast, PFE significantly inhibited endometriotic cell migration compared with the control (Figure 2). Treatment of 11Z and 12Z cells with PFE at 100 μg/mL for 24 h resulted in only 30% migration. In addition, the migration assays demonstrated that PFE (25, 50, and 100 μg/mL) markedly suppressed the migration of human endometriotic cells ( Figure 3).

PFE Inhibits Endometriotic Cell Migration
Wound-healing assays and transwell migration assays were performed to evaluate the effect of PFE on endometriotic cell migration. First, the wound-healing assay showed that the wound in endometriotic 11Z and 12Z cells was about 60% healed after 24 h in the absence of PFE treatment. In contrast, PFE significantly inhibited endometriotic cell migration compared with the control (Figure 2). Treatment of 11Z and 12Z cells with PFE at 100 µg/mL for 24 h resulted in only 30% migration. In addition, the migration assays demonstrated that PFE (25, 50, and 100 µg/mL) markedly suppressed the migration of human endometriotic cells (Figure 3).

PFE Inhibits the Expression of MMP-2 and MMP-9 in Human Endometriotic Cells
MMP-2 and MMP-9 are well known to be involved in ectopic adhesion, invasion, implantation, and neovascularisation of the endometrium [24,25]. In this study, a Western blot assay was performed to measure the levels of MMP-2 and MMP-9 expression after 24 h of treatment with PFE (25, 50, and 100 μg/mL). PFE treatment markedly suppressed the levels of MMP-2 and MMP-9 protein in 11Z and 12Z cells (Figure 4). To investigate whether PFE regulates MMP-2 and MMP-9 expression at the transcriptional level, real-time RT-PCR was performed. As shown in Figure 5, PFE significantly inhibited the expression of MMP-2 and MMP-9 mRNA in human endometriotic 11Z and 12Z cells.

PFE Inhibits the Expression of MMP-2 and MMP-9 in Human Endometriotic Cells
MMP-2 and MMP-9 are well known to be involved in ectopic adhesion, invasion, implantation, and neovascularisation of the endometrium [24,25]. In this study, a Western blot assay was performed to measure the levels of MMP-2 and MMP-9 expression after 24 h of treatment with PFE (25, 50, and 100 μg/mL). PFE treatment markedly suppressed the levels of MMP-2 and MMP-9 protein in 11Z and 12Z cells (Figure 4). To investigate whether PFE regulates MMP-2 and MMP-9 expression at the transcriptional level, real-time RT-PCR was performed. As shown in Figure 5, PFE significantly inhibited the expression of MMP-2 and MMP-9 mRNA in human endometriotic 11Z and 12Z cells.

PFE Inhibits the Expression of MMP-2 and MMP-9 in Human Endometriotic Cells
MMP-2 and MMP-9 are well known to be involved in ectopic adhesion, invasion, implantation, and neovascularisation of the endometrium [24,25]. In this study, a Western blot assay was performed to measure the levels of MMP-2 and MMP-9 expression after 24 h of treatment with PFE (25, 50, and 100 µg/mL). PFE treatment markedly suppressed the levels of MMP-2 and MMP-9 protein in 11Z and 12Z cells (Figure 4). To investigate whether PFE regulates MMP-2 and MMP-9 expression at the transcriptional level, real-time RT-PCR was performed. As shown in Figure 5, PFE significantly inhibited the expression of MMP-2 and MMP-9 mRNA in human endometriotic 11Z and 12Z cells.

ERK Signalling Is Involved in PFE-Induced Anti-Endometriotic Activity
Several studies have demonstrated that the extracellular signal-regulated kinase (ERK) pathway is involved in cell migration and adhesion [26]. Western blot analysis revealed that PFE markedly increased the phosphorylation of ERK1/2 in endometriotic cells, as shown in Figure 6A,B. To investigate the involvement of ERK1/2 signalling in the PFE-inhibited cell migration of endometriotic cells, we performed a transwell migration assay in the presence of a specific ERK1/2 inhibitor,

ERK Signalling Is Involved in PFE-Induced Anti-Endometriotic Activity
Several studies have demonstrated that the extracellular signal-regulated kinase (ERK) pathway is involved in cell migration and adhesion [26]. Western blot analysis revealed that PFE markedly increased the phosphorylation of ERK1/2 in endometriotic cells, as shown in Figure 6A,B. To investigate the involvement of ERK1/2 signalling in the PFE-inhibited cell migration of endometriotic cells, we performed a transwell migration assay in the presence of a specific ERK1/2 inhibitor,

ERK Signalling Is Involved in PFE-Induced Anti-Endometriotic Activity
Several studies have demonstrated that the extracellular signal-regulated kinase (ERK) pathway is involved in cell migration and adhesion [26]. Western blot analysis revealed that PFE markedly increased the phosphorylation of ERK1/2 in endometriotic cells, as shown in Figure 6A,B.
To investigate the involvement of ERK1/2 signalling in the PFE-inhibited cell migration of endometriotic cells, we performed a transwell migration assay in the presence of a specific ERK1/2 inhibitor, PD98059. As shown in Figure 6C, pre-treatment with PD98059 significantly reversed PFE-inhibited cell migration. These results indicate that, in endometriotic cells, PFE-inhibited migration may involve ERK1/2 signalling.
Four weeks after induction, mice were sacrificed to investigate the effect of PFE on the establishment of endometriosis-like lesions in vivo. As shown in Figure 7A, PFE-treated mice had a reduced number of total endometriotic lesions compared with vehicle-treated controls. Thus, PFE inhibits the formation of endometriosis-like lesions in mice. No significant weight loss or abnormal physiological change in mice treated with either vehicle or PFE was observed during the 5 weeks ( Figure 7B).

PFE Inhibits the Formation of Endometriosis-Like Lesions in Mice
Four weeks after induction, mice were sacrificed to investigate the effect of PFE on the establishment of endometriosis-like lesions in vivo. As shown in Figure 7A, PFE-treated mice had a reduced number of total endometriotic lesions compared with vehicle-treated controls. Thus, PFE inhibits the formation of endometriosis-like lesions in mice. No significant weight loss or abnormal physiological change in mice treated with either vehicle or PFE was observed during the 5 weeks ( Figure 7B). PD98059. As shown in Figure 6C, pre-treatment with PD98059 significantly reversed PFE-inhibited cell migration. These results indicate that, in endometriotic cells, PFE-inhibited migration may involve ERK1/2 signalling.

PFE Inhibits the Formation of Endometriosis-Like Lesions in Mice
Four weeks after induction, mice were sacrificed to investigate the effect of PFE on the establishment of endometriosis-like lesions in vivo. As shown in Figure 7A, PFE-treated mice had a reduced number of total endometriotic lesions compared with vehicle-treated controls. Thus, PFE inhibits the formation of endometriosis-like lesions in mice. No significant weight loss or abnormal physiological change in mice treated with either vehicle or PFE was observed during the 5 weeks ( Figure 7B).

Identification of Major Compounds of PFE and Anti-Endometriotic Effect of Tectorigenin
Three major isoflavones 1-3 ( Figure 8A) were isolated from the PFE by using repeated chromatography. These compounds were identified as tectorigenin-7-O-β-D-xylosylglucoside (1), tectoridin (2), and tectorigenin (3) on the basis of spectroscopic method ( 1 H-and 13 C-NMR) and by comparison with the published data. A mixed solution of three isoflavones and PFE extract were used for the optimisation of the UPLC condition. As shown Figure 8B,C, three isoflavones were separated within 7 min. Three isoflavones (peaks 1, 2, and 3) were identified by comparison of the retention time with those obtained from plant extract. The retention times of the three isoflavones 1-3 were 2.09 min, 2.47 min, and 4.64 min, respectively. Considering that tectorigenin-7-O-β-D-xylosylglucoside and tectoridin are generally metabolised into tectorigenin in the digestive tract, we investigated the effect of tectorigenin on the endometriotic cell adhesion and invasion. As shown in Figure 8D,E, tectorigenin significantly inhibited the cell adhesion and migration in endometriotic cells.

Identification of Major Compounds of PFE and Anti-Endometriotic Effect of Tectorigenin
Three major isoflavones 1-3 ( Figure 8A) were isolated from the PFE by using repeated chromatography. These compounds were identified as tectorigenin-7-O-β-D-xylosylglucoside (1), tectoridin (2), and tectorigenin (3) on the basis of spectroscopic method ( 1 H-and 13 C-NMR) and by comparison with the published data. A mixed solution of three isoflavones and PFE extract were used for the optimisation of the UPLC condition. As shown Figure 8B,C, three isoflavones were separated within 7 min. Three isoflavones (peaks 1, 2, and 3) were identified by comparison of the retention time with those obtained from plant extract. The retention times of the three isoflavones 1-3 were 2.09 min, 2.47 min, and 4.64 min, respectively. Considering that tectorigenin-7-O-β-D-xylosylglucoside and tectoridin are generally metabolised into tectorigenin in the digestive tract, we investigated the effect of tectorigenin on the endometriotic cell adhesion and invasion. As shown in Figure 8D,E, tectorigenin significantly inhibited the cell adhesion and migration in endometriotic cells.

Discussion
Recent pharmacological studies have suggested that Pueraria flower has a wide range of biological activities, including hepatoprotective, antioxidant, hypoglycaemic, hypolipidaemic, antimutagenic, and hormone-regulating effects [14][15][16][17][18]. For example, Pueraria flower extract (PFE) was shown to have a protective effect on acute alcohol intoxication via modulation of alcohol metabolising and antioxidant enzymes in mice [27]. Liu et al. reported that PFE improves cognitive impairment in diabetic mice through the normalisation of acetylcholinesterase (AChE) activity and metabolic abnormalities in the brain [28]. Kamiya et al. demonstrated that PFE has anti-fatty liver and

Discussion
Recent pharmacological studies have suggested that Pueraria flower has a wide range of biological activities, including hepatoprotective, antioxidant, hypoglycaemic, hypolipidaemic, antimutagenic, and hormone-regulating effects [14][15][16][17][18]. For example, Pueraria flower extract (PFE) was shown to have a protective effect on acute alcohol intoxication via modulation of alcohol metabolising and antioxidant enzymes in mice [27]. Liu et al. reported that PFE improves cognitive impairment in diabetic mice through the normalisation of acetylcholinesterase (AChE) activity and metabolic abnormalities in the brain [28]. Kamiya et al. demonstrated that PFE has anti-fatty liver and antiobesity effects in high-fat diet-induced obese mice by suppressing lipogenesis in the liver, stimulating lipolysis in white adipose tissue, and promoting thermogenesis in brown adipose tissue [29]. However, to the best of our knowledge, no report has demonstrated the effect of PFE on endometriosis. In this study, we first investigated the effect of PFE on endometriosis using human immortalised endometriotic 11Z and 12Z cells. The 11Z and 12Z cells were established from active endometriotic lesions and have been validated to maintain the phenotypic characteristics and in vivo properties of active phase endometriosis [20,30]. Endometriotic cells are characterised by enhanced proliferation, invasion, adhesion, and migration [30]. In this study, we demonstrated that PFE significantly inhibited the adhesion and migration of endometriotic cells with no significant change in cell viability. In contrast, any change in cell invasion was not observed following PFE treatment (data not shown), possibly due to the low invasiveness of 11Z and 12Z cells used in this study. Due to our promising in vitro results, the anti-endometriotic effect of PFE was examined in vivo using endometriosis-induced mice. PFE administration significantly disrupted the establishment of endometriosis-like lesions, suggesting that PFE should be considered as a potential preventive and therapeutic agent for endometriosis in humans.
MMPs, a family of zinc-dependent endopeptidases, are known to regulate the migration, invasion, and proliferation of various cell types [31]. Recently, accumulating data suggest that MMPs are associated with the establishment and progression of endometriosis. For example, MMP levels were shown to be enhanced in ectopic endometriotic tissues [32]. Styer et al. demonstrated that MMP levels in endometrial tissue are related to the ability of the tissue to progress into ectopic endometriotic lesions in a mouse endometriosis model [33]. Among the various MMPs, gelatinases MMP-2 and MMP-9, which degrade the principal component of basement membranes, collagen IV, have been intensively investigated in the context of endometriosis. MMP-2 and MMP-9 are elevated in the peritoneal fluid of patients with endometriosis compared to healthy people [5,34,35]. Similarly, induction of endometriosis enhanced the levels of MMP-2 and MMP-9 in peritoneal fluid and cells in an in vivo study using BALB/c mice [36]. In addition, human endometriotic tissues were shown to have higher levels and activities of MMP-2 and MMP-9 [4,37]. In this study, we showed that PFE significantly suppressed the mRNA and protein levels of MMP-2 and MMP-9 in both 11Z and 12Z cells, suggesting that PFE modulated MMP-2/-9 expression at the transcriptional level. Downregulation of MMP-2 and MMP-9 by PFE may involve the inhibition of PFE-induced migration in human endometriotic cells, considering that MMPs have been implicated in the endometriotic cell migration.
The mitogen-activated protein kinase (MAPK) pathways are well known to regulate a wide range of cellular events in eukaryotic cells. Activated MAPK coordinates diverse cellular activities including cellular metabolism, differentiation, cell cycle, motility, apoptosis, and survival. One of the MAPKs, ERK1/2, has been suggested as a key regulator of the majority of the signalling pathways and cascades associated with the pathogenesis of endometriosis [38]. For example, some factors in the endometriotic milieu such as tumour necrosis factor α (TNFα) and monocyte chemotactic protein-1 (MCP-1) have been shown to regulate ERK signalling in endometriotic cells [39]. In addition, Gentilini et al. have showed that ERK1/2 signalling pathway is associated with 17beta-estradiol and growth factor-induced cell migration of endometriotic stromal cells [40]. Interestingly, ERK1/2 has been implicated in the regulation of MMP expression [41][42][43]. In this study, we showed that treatment with PFE markedly increased the activation of ERK1/2 in endometriotic cells and that PFE-suppressed migration was significantly reversed in the presence of PD98059, a specific ERK1/2 inhibitor. The ERK1/2 activity remained elevated throughout the experiment (up to 24 h) (Supplementary Figure 1). These data suggest that ERK1/2 signalling is involved in PFE-inhibited endometriotic cell migration.
Pueraria flowers are rich source of isoflavones, such as genistein, daidzein, kakkalide, puerarin, and tectoridin [44]. In fact, most of biological activities of the flowers have been attributed to the isoflavones. We found that tectoridin, tectorigenin-7-O-β-D-xylosylglucoside, and tectorigenin were the major components of PFE used in this study. This result is consistent with a previous finding that tectoridin is the major isoflavone component of Pueraria flowers during the first 5 years of storage [45]. Tectoridin and its major metabolite tectorigenin have been suggested to have a wide range of biological activities (e.g., antioxidant, anti-inflammatory, anti-angiogenic, anti-tumour, hepatoprotective, and endocrine-modulating effects) [12,[46][47][48][49][50]. To the best of our knowledge, there have been no reports on the anti-endometriotic effects of the isoflavones of PFE. It is of note that tectorigenin-7-O-β-D-xylosylglucoside, and tectoridin are known to be metabolised into tectorigenin in the digestive tract [51], suggesting that the major isoflavones in the PFE are likely to be absorbed in to the bloodstream in the forms of tectorigenin. Like other isoflavones, tectorigenin, a type of O-methylated isoflavone, has been suggested to possess estrogenic activities [17]. Interestingly, tectorigenin has been shown to inhibit cell migration through downregulation of MMPs, including MMP-2 and MMP-9, in osteosarcoma cells [52]. In addition, tectorigenin has been demonstrated to exert some of their biological actions via the ERK1/2 pathway [53,54]. Additionally, we found that tectorigenin significantly inhibits the endometriotic adhesion and migration in this study. Thus, it is reasonable to speculate that the anti-endometriotic effects of PFE are mainly attributed to the major isoflavones. However, in vivo efficacy of the active constituents and its more exact molecular mechanisms of action still need to be further examined in future.

Conclusions
In summary, we demonstrate that PFE inhibits the adhesion and migration of endometriotic cells and the establishment of endometriosis-like lesions. The anti-endometriotic activity of PFE is thought to be associated with the downregulation of MMP-2 and MMP-9 and the regulation of ERK1/2 signalling. Taken together, these results suggest that PFE has potential adjuvant intervention for treating and preventing endometriosis.