Paeonol alleviates migration and invasion of endometrial stromal cells by reducing HIF-1α-regulated autophagy in endometriosis

Background: Dysregulated migration and invasion of endometrial stromal cells is implicated in the pathogenesis of endometriosis. Hypoxia functions as critical microenvironmental factor that results in promotion of endometrial stromal cells migration and invasion through upregulation of autophagy. Paeonol functioned as a tumor suppressor in human ovarian cancer and promoted cytoprotective autophagy. However, the role of paeonol in hypoxia-induced autophagy in endometriosis remains unknown. Methods: Stromal cells were isolated from endometriotic patients by enzymatic digestion of ectopic endometrial tissues, and then characterized by immunohistochemical analysis of cytoskeleton 19 (CK19) and vimentin. Cellular morphology was evaluated under microscope. Cell viability, proliferation and apoptosis of stromal cells were assessed by Cell Counting Kit-8, EdU labeling and flow cytometry, respectively. Wound healing and transwell assays were performed to detect metastasis of the stromal cells. Hypoxia-induced autophagy was evaluated through immunohistochemistry and western blot. Results: Paeonol treatment dosage dependently decreased cell proliferation and metastasis of the ectopic endometrial stromal cells (ecESCs), while promoted the cell apoptosis. Hypoxia-induced autophagy in the ecESCs was repressed by paeonol through down-regulation of LC3-II/LC3-I and Beclin-1, while up-regulation of p62. Hypoxia-inducible factor-1α (HIF-1α) was reduced post paeonol treatment, and paeonol-induced increase of p62 and decrease of LC3II/LC3-I and Beclin-1 were reversed by over-expression of HIF-1α. Over-expression of HIF-1α also attenuated the suppressive effect of paeonol on cell growth of ecESCs. Conclusions: Paeonol attenuated HIF-1α-induced promotion of ecESCsmigration and invasion through reducing autophagy, and reduced HIF-1α-induced endometriotic lesion in rats, providing potential therapeutic strategy for the treatment of endometriosis.


Abstract
Background: Dysregulated migration and invasion of endometrial stromal cells is implicated in the pathogenesis of endometriosis. Hypoxia functions as critical microenvironmental factor that results in promotion of endometrial stromal cells migration and invasion through upregulation of autophagy. Paeonol functioned as a tumor suppressor in human ovarian cancer and promoted cytoprotective autophagy. However, the role of paeonol in hypoxia-induced autophagy in endometriosis remains unknown. Methods: Stromal cells were isolated from endometriotic patients by enzymatic digestion of ectopic endometrial tissues, and then characterized by immunohistochemical analysis of cytoskeleton 19 (CK19) and vimentin. Cellular morphology was evaluated under microscope. Cell viability, proliferation and apoptosis of stromal cells were assessed by Cell Counting Kit-8, EdU labeling and flow cytometry, respectively. Wound healing and transwell assays were performed to detect metastasis of the stromal cells. Hypoxia-induced autophagy was evaluated through immunohistochemistry and western blot. Results: Paeonol treatment dosage dependently decreased cell proliferation and metastasis of the ectopic endometrial stromal cells (ecESCs), while promoted the cell apoptosis. Hypoxia-induced autophagy in the ecESCs was repressed by paeonol through down-regulation of LC3-II/LC3-I and Beclin-1, while up-regulation of p62. Hypoxia-inducible factor-1α (HIF-1α) was reduced post paeonol treatment, and paeonol-induced increase of p62 and decrease of LC3-II/LC3-I and Beclin-1 were reversed by over-expression of HIF-1α. Over-expression of HIF-1α also attenuated the suppressive effect of paeonol on cell growth of ecESCs. Conclusions: Paeonol attenuated HIF-1α-induced promotion of ecESCs migration and invasion through reducing autophagy, and reduced HIF-1α-induced endometriotic lesion in rats, providing potential therapeutic strategy for the treatment of endometriosis.

Introduction
Endometriosis is an estrogen-dependent benign chronic disease [1], and affects 2-10% of premenopausal women as a major cause of infertility and chronic pelvic pain [2]. Endometriosis is generally characterized by the presence of endometrial glands and extracellular matrix, and defined as extra-growth of the endometrial tissues [3]. The pathogenesis of endometriosis is still unclear, and the most widely accepted theory is the implantation theory in which endometrial tissue is implanted outside the uterus through an open fallopian tube during the menstrual cycle [4]. Meanwhile, the enhanced proliferation, adhesion, migration and invasion of endometrial cells, including stromal and epithelial cells, facilitated the ectopic implantation of the endometrial tissues and development of endometriotic lesions [5]. The suppression of endometrial stromal cells metastasis contributed to amelioration of endometriosis [5].
Autophagy is an evolutionary conservative mechanism that eliminates and circulates non-essential cytoplasmic components to maintain homeostasis [6]. Dysregulated autophagy in the eutopic or ectopic endometrium retarded cell apoptosis and promoted hyperplasia of stromal cells and endometriotic tissues, thus contributing to the pathogenesis of endometriosis [7]. The level of autophagy is relatively high in the ectopic endometrium of patients with ovarian endometriosis [8], and autophagy could promote the migration and invasion of endometrial stromal cells under the condition of hypoxia [9]. Inhibition of autophagy inhibited the angiogenesis of endometriosis and attenuated the ectopic endometriotic lesions [10]. Therefore, regulation of autophagy might be helpful for the suppression of endometrial stromal cells metastasis.
Paeonol (4-methoxy-2-hydroxyacetophenone), an active compound extracted from traditional chinese medicine, demonstrated antioxidant, anti-tumor, antiinflammatory and immunomodulatory effects [11]. For example, paeonol promoted colorectal cancer cell apoptosis [12], and retarded non-small-cell lung cancer motility [13]. Moreover, paeonol decreased vascular endothelial growth factor to enhance radiotherapy-induced apoptosis of ovarian cancer [14]. However, whether paeonol has an effect on endometriosis has not been studied yet. Paeonol activated autophagy flux in ovarian cancer [15], and promoted autophagy to demonstrate anti-atherosclerotic in vascular smooth muscle cell [16]. Additionally, oxidized low-density lipoprotein-induced vascular endothelial cell autophagy was suppressed by paeonol [17]. The detailed role of paeonol on autophagy in endometriosis needs further research.
This study aimed to investigate roles of paeonol on migration and invasion of isolated ectopic endometrial stromal cells (ecESCs), as well as endometriotic lesion in rats. Specifically, whether autophagy was implicated in paeonol-mediated progression of endometriosis was also determined.

Isolation and culture of ecESCs
Patients (N = 7; age of 18 to 50) with ovarian endometriosis were recruited at The Hospital Affiliated to Shandong University of Traditional Chinese Medicine. The presence of endometriosis was confirmed by pathological examination and laparoscopy. The pathological examination was performed by an experienced fertility specialist based on the criteria of Noyes. The study was approved by the Center of Reproduction and Genetics, The Hospital Affiliated to Shandong University of Traditional Chinese Medicine and the patients signed written informed consents. Laparoscopy was performed to isolate the ectopic endometrial tissues, and the normal endometrial tissues were also isolated from regularly cycling women without endometriosis. The tissues were digested with collagenase A and DNase (Sigma Aldrich, St. Louis, MO, USA) for 2 hours. The cells were then cultured in DMEM medium (Lonza, Basel, Switzerland) containing 10% fetal bovine serum (Lonza) at 37 • C incubator. The adherent cells were grown to 80% confluence before the evaluation under microscope (Olympus Inc., Tokyo, Japan) and immunohistochemical analysis of CK19 and vimentin.

Cell Counting Kit-8 and EdU staining
The ecESCs with indicated transfection and treatment, as well as normal endometrial stromal cells and IOSE80 cells, were plated and incubated with Cell Counting Kit-8 solution (Dojindo, Tokyo, Japan) for 2 hours. Absorbance at 450 nm was determined by Microplate Autoreader (Thermo Fisher Scientific). For EdU staining, cells were incubated with diluted EdU solution (1:1000, Sigma Aldrich, St. Louis, MO, USA) for 2 hours, and then fixed in 4% paraformaldehyde. Cells were incubated with 100 µL 1 × Apollo® dyeing reaction solution (Sigma Aldrich, St. Louis, MO, USA) and DAPI solution (Sigma Aldrich, St. Louis, MO, USA) for 1 hour. The fluorescence was measured under fluorescence microscope (Olympus, Tokyo, Japan).

Flow cytometry
The ecESCs with indicated transfection and treatment were stained using an Annexin V-fluorescein isothiocyanate apoptosis detection kit (Beyotime, Beijing China). The proportion of apoptotic cells was determined via flow cytometer (Attune, Life Technologies, Darmstadt, Germany).

Wound healing and transwell assays
The ecESCs with indicated transfection and treatment were plated and scratched by a plastic tips. Fortyeight hours later, the wound was calculated under microscope (Olympus). For transwell assay, cells in serum-free medium were seeded in the upper Matrigel-coated Transwell chamber, and medium with 20% fetal bovine serum was added into the bottom chamber. Forty-eight hours later, the invasive cells in the bottom chamber were fixed with paraformaldehyde and stained with 0.1% crystal violet before measurement under microscope (Olympus).

Immunofluorescence
The ecESCs with indicated transfection and treatment were fixed with 4% paraformaldehyde before incubation with 5% bovine serum albumin for 1 hour. Specific antibody against LC3B (1:300; Abcam, Cambridge, MA, USA) was applied to incubated the cells overnight. Following incubation with FITC-conjugated secondary antibody (1:100; Abcam, Cambridge, MA, USA), DAPI was used to stain cell nucleus before measurement under fluorescence microscope (Olympus).

qRT-PCR
Total RNAs extracted from ecESCs by TRIzol Reagent (Sigma Aldrich) were reversed into cDNAs under cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). SYBR Green Master (Roche, Mannheim, Germany) was applied to determined mRNA expression of HIF-1α and GAPDH by 2 −∆∆Ct method. GAPDH was used as internal reference. The primers were listed below: HIF-1α: F 5 ′ -AACATAAAGTCTGCAACATGGAAG-

Animal model
Experiments were performed according to National Institutes of Health Laboratory Animal Care and Use Guidelines. and approved by The Hospital Affiliated to Shandong University of Traditional Chinese Medicine. Female SD rats (6-8 weeks old, 200-220 g) were purchased from Envigo (Indianapolis, IN, USA), and housed in cages with 12:12 h light and dark cycles and controlled environment (24 ± 2 • C ). Rats were divided into sham and endometriosis groups. To establish endometriotic rats, rats were anaesthetized by halothane in oxygen, and the bowels were exposed by mid-ventral incision. The right uterine horn was removed, opened longitudinally via scissors in sterile PBS, and cut into four square pieces (4 mm 2 ). The pieces were sutured to the intestine mesentery with single stitch. The abdominal wall was closed, and the ras were kept for endometriosis induction for 4 weeks. Laparotomy was performed to confirm the endometriotic lesions. Rats in the sham group (N = 6) were conducted with surgery without transplantation of uterine tissue towards intestine mesentry. Rats in the endometriosis group were randomly divided into 3 groups: endometriosis (EMs) group (N = 6), EMs with 20 mg/kg paeonol group (N = 6) and EMs with 50 mg/kg paeonol group (N = 6). Rats were orally treated with paeonol for 21 days before the functional analyses.

ELISA
Rats were sacrificed by carbon dioxide inhalation, the serum samples were collected, and then performed with commercial ELISA kits (Krishgen Biosystem, Mumbai, India) to determine levels of IL-1β, IL-6 and TNF-α.

Histological examination
The endometriotic lesions were surgically removed from the rats, and fixed in formalin. The paraffinembedded tissues were cut into 5 µm thick sections. The sections were stained with hematoxylin and eosin (Sigma-Aldrich), and examined under microscope.

Western blot
Proteins extracted from ecESCs or endometriotic lesions were separated by SDS-PAGE, and then transferred to PVDF membranes (Sigma Aldrich). The membrane was blocked in 5% bovine serum albumin before incubation with primary antibodies against HIF-1α

Statistical analysis
Data with at least three times repeat were presented as mean ± standard deviations. Student's t test or one-way analysis of variance were used to perform statistical comparison under Graphpad Prism® statistical analysis software. A p values < 0.05 was considered statistically significant.

Paeonol repressed cell proliferation of ecESCs
To explore the effects of paeonol on endometriosis, ecESCs were isolated and characterized under microscope. The cells showed spindle shape (Fig. 1A). Moreover, immunohistochemical analysis demonstrated negative staining of CK19 and positive staining of vimentin (Fig. 1A), indicating that endometrial stromal cells were successful isolated. The ecESCs under hypoxia treatment were applied to paeonol condition. Results showed that paeonol condition dosage dependently reduced cell viability of ecESCs (Fig. 1B), while dosage dependently promoted cell apoptosis of ecESCs (Fig. 1C). However, paeonol 100 µm had no significant effect on the cell viability of normal endometrial stromal cells and IOSE80 cells (Supplementary Fig. 1A). Lower EdU staining of ecESCs under paeonol condition (Fig. 1D) further demonstrated the anti-proliferative role of paeonol on ecESCs.

Paeonol repressed cell migration and invasion of ecESCs
The role of paeonol on metastasis of endometriosis was then investigated. Data from wound healing ( Fig. 2A) and transwell (Fig. 2B) assays demonstrated that cell migration and invasion of ecESCs were dosage dependently repressed by paeonol treatment, suggesting the anti-invasive role of paeonol on ecESCs.

Paeonol repressed autophagy of ecESCs
To explore role of paeonol in autophagy of ecESCs, indicators of autophagy, including LC3B, Beclin and p62, were examined. As shown in Fig. 3A, paeonol treatment dosage dependently induced decrease in LC3-II and Beclin, and increase in p62, suggesting that paeonol repressed autophagy of ecESCs. Moreover, there was a gradual decrease in the immunofluorescence of LC3 in ecESCs (Fig. 3B), further confirming that paeonol could induce suppression of autophagic flux in ecESCs.

Paeonol repressed HIF-1α in ecESCs
Hypoxia-induced HIF-1α was shown to be positively correlated with autophagy in ectopic endometrium. Expression level of HIF-1α was determined in ecESCs post paeonol treatment. There was a gradual decrease in the mRNA (Fig. 4A) and protein (Fig. 4B) expression of HIF-1α in ecESCs post paeonol treatment, further confirming the suppressive role of paeonol on HIF-1α expression in ecESCs.

Paeonol repressed autophagy of ecESCs through down-regulation of HIF-1α
The ecESCs transfected with pcDNA-mediated HIF-1α over-expression were treated with 50 µM paeonol to examine the effect of HIF-1α on paeonol-mediated autophagy in ecESCs. The transfection efficiency of pcDNAmediated HIF-1α over-expression was shown in Supplementary Fig. 1B. Over-expression of HIF-1α attenuated paeonol-induced decrease in LC3-II and Beclin-1, and the increase in p62 (Fig. 5A), suggesting that paeonol repressed autophagy of ecESCs through down-regulation of HIF-1α. Moreover, the decrease in the immunofluorescence of LC3 was reversed by in pcDNA-mediated HIF-1α over-expression in ecESCs (Fig. 5B), further confirming that paeonol could induce suppression of autophagic flux in ecESCs through down-regulation of HIF-1α.

Paeonol repressed migration and invasion of ecESCs through suppression of autophagy
The ecESCs transfected with pcDNA-mediated HIF-1α over-expression were treated with 50 µM paeonol to examine the effect of HIF-1α on paeonol-mediated migration and invasion of ecESCs. Over-expression of HIF-1α counteracted with the suppressive effect of paeonol on cell viability of ecESCs (Fig. 6A). The promoted cell apoptosis of ecESCs by paeonol was also reversed by pcDNAmediated HIF-1α over-expression (Fig. 6B). Moreover, over-expression of HIF-1α also counteracted with the suppressive effect of paeonol on cell migration (Fig. 6C) and invasion (Fig. 6D) of ecESCs. These results showed that paeonol repressed migration and invasion of ecESCs through suppression of autophagy.

Paeonol ameliorated endometriotic lesions of rats in a HIF-1α dependent manner
To investigate role of paeonol in in vivo endometriosis, endometriotic rats were established through transplantation of uterine tissue towards intestine mesentry. Histopathological analysis by H&E staining showed hallmarks of endometriosis in the endometriotic rats, as demonstrated by perforation of stroma, increased number of glands and thickening walls of endometrial glands compared with the sham group (Fig. 7A). In addition, the number and thickened walls of glands were reduced by paeonol (Fig. 7A). Serum levels of IL-1β, IL-6 and TNF-α in endometriotic rats were down-regulated by paeonol (Fig. 7B). Paeonol attenuated endometriosis-induced increase in HIF-1α, Beclin-1 and p62 in the rats (Fig. 7C), suggesting that paeonol ameliorated endometriotic lesions of rats in a HIF-1α dependent manner.

Discussion
Previous study has shown that the dysregulation of endometrial stromal cells leads to endometriotic lesions during the development of endometriosis [5]. Endometriosis was found to be related to increased risk of clear-cell carcinoma and other malignancies [18]. Impaired autophagy was found to be closely associated with stage and prognosis of endometriosis, and mediated the cell proliferation, apoptosis, migration and invasion of endometrial cells [19]. Inhibition of autophagy contributed to suppression of endometrial cell migration and invasion [20]. Since paeonol promoted cytoprotective autophagy in ovarian cancer cells  [15], and suppressed the oxidized low-density lipoproteininduced vascular endothelial cell autophagy [17], the functional role of paeonol in autophagy, migration and invasion of endometrial stromal cells was investigated in this study.
The increase in endometrial stromal cell migration and invasion contributed to epithelial-to-mesenchymal transition in endometriosis [21]. Suppression of endometrial stromal cell migration and invasion could ameliorate endometriotic lesions [22]. Results in this study indicated that paeonol treatment decreased cell viability, proliferation, migration and invasion of ecESCs, and promoted the cell apoptosis. In vivo endometriosis showed that paeonol ameliorated hallmarks of endometriosis in the endometriotic rats, suggesting that paeonol might ameliorate endometriotic lesions during the development of endometrio-sis. Since epithelial-to-mesenchymal transition was considered as the original establishment of endometriotic lesions [23], the effects of paeonol on epithelial-to-mesenchymal transition of ecESCs should be investigated in the further study.
Autophagy was found to be promoted in ovarian endometriosis [8], and the induction of autophagy resulted in abnormal apoptosis during development of endometriosis [24]. Expression of HIF-1α and LC3 were reduced in eutopic endometrium in women with and without endometriosis compared with ectopic endometrial lesions [25], while hypoxia induced increase in HIF-1α, LC3 and Beclin-1, and decrease in p62 [9]. Our results demonstrated that HIF-1α expression was reduced in ecESCs under paeonol treatment, and hypoxia-induced autophagy was also ameliorated by paeonol treatment through downregulation of LC3 and Beclin-1, and up-regulation of p62, suggesting that paeonol suppressed autophagy during the development of endometriosis to ameliorate endometriotic lesions. However, previous research also showed that autophagy was reduced in ecESCs compared with the normal women [26], and the enhanced autophagy in ecESCs could retard the ectopic lesions through stimulation of cytotoxic activity of NK cells [27]. The protective effect of paeonol against autophagy in ecESCs might dependent on the immune microenvironment and individual characteristic of the patients.
HIF-1α was reported to promote the development of endometriosis through induction of epithelial-mesenchymal transition of endometrial epithelial cells [28]. Endometrial stromal cells migration and invasion [9], as well as autophagy and epithelial-mesenchymal transition [29], were also provoked to participate in the pathophysiology of endometriosis. Moreover, HIF-1α promoted autophagy to facilitate for the migration and invasion of endometrial stromal cells [9], and hypoxia-induced pro-survival autophagy was suppressed by knockdown of lncRNA MALAT1 to enhance the cell apoptosis [30]. pcDNA-mediated HIF-1α over-expression in ecESCs attenuated paeonol-induced increase in cell apoptosis, and decrease in cell viability, proliferation, migration and invasion, suggesting that paeonol repressed migration and invasion of ecESCs through suppression of autophagy. Paeonol attenuated endometriosis-induced increase in HIF-1α, Beclin-1 and p62 in the rats, suggesting that paeonol ameliorated endometriotic lesions of rats in a HIF-1αdependent manner. In addition, inflammatory response was reported to participate in the progression of endometriosis through migration and invasion of endometrial cells in the ectopic regions [31], and paeonol could attenuate the inflammation [32]. Serum levels of IL-1β, IL-6 and TNFα in endometriotic rats were down-regualted by paeonol in this study, suggesting that paeonol might exert antiinflammatory effect against endometriosis.

Conclusions
In general, our results showed that paeonol repressed migration and invasion of ecESCs through attenuation of hypoxia-triggered autophagy, and ameliorated histopathological changes and inflammatory response of endometriotic rats in a HIF-1α dependent manner, suggesting that paeonol might serve as a novel therapeutic strategy for the treatment of endometriosis. Moreover, according previous study [33], paeonol might be intraperitoneally introduced into the patients with endometriosis.

Author contributions
CP and ZW designed the study, supervised the data collection, XX analyzed the data, interpreted the data, WY, XW and YQ prepared the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript.

Ethics approval and consent to participate
Written informed consent was obtained from a legally authorized representative(s) for anonymized patient information to be published in this article. All animal care and treatments were done as approved by Animal Care Committee, the Hospital Affiliated to Shandong University of Traditional Chinese Medicine.

Acknowledgment
Thanks to all the peer reviewers for their opinions and suggestions.

Conflict of interest
The authors declare no conflict of interest.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.