Objective: The objective of this study was to explore the inhibitory effect of total flavonoids of Polygala fallax Hemsl (PFHF) on human ectopic endometrial stromal cells (HEcESCs) and its mechanism. Design: The apoptosis, cell cycle, migration, and invasion ability of HEcESCs (Fresh human ovarian endometriosis tissue was used for primary culture) after PFHF treatment were detected, and the mechanism of action was explored. Materials: The Polygala fallax Hemsl (PFH), RPMI 1640 culture medium, Dulbecco’s modified Eagle’s medium (DMEM)/F-12, fetal bovine serum, penicillin/streptomycin, cell counting kit-8 (CCK-8) kit, trypsin, phenylmethylsulfonyl fluoride, radioimmunoprecipitation assay tissue/cell lysate, bicinchoninic acid protein concentration detection kits, protein loading buffer, the apoptosis and cell cycle extraction kits, the matrix glue, TRIzol Universal Reagent, the reverse transcription kit, AB HS Green qPCR Mix, the ECL chromogenic solution, enzyme labeling instrument, flow cytometry, automatic real-time fluorescence quantitative PCR instrument, Goat anti-rabbit, rabbit anti-β-actin, vimentin, phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), B-cell lymphoma-2 (Bcl-2), Bcl-extra long (Bcl-xl), Bcl-2 associated death promoter (Bad) antibody, Alexa Fluor 594-labeled secondary antibody, the inverted microscope, the constant temperature carbon dioxide cell incubator. Setting: Five parts included introduction, materials and methods, results, discussion, and conclusion. Methods: The potential targets and pathways of PFHF in the treatment of endometriosis were predicted by network pharmacology. The effect of PFHF on the proliferation, apoptosis and cell cycle, migration, and invasion of HEcESCs was detected by CCK-8 method, flow cytometry, and Transwell chamber experiment. Label-free quantitative proteomics based on mass spectrometry was used to analyze the protein mass spectrum of differential expression of HEcESCs before and after PFHF, and the biological information was analyzed. The effects of PFHF on the mRNA and protein expression of pathway-related genes predicted in HEcESCs were detected by reverse transcription-quantitative polymerase chain reaction and Western blotting. Results: The network pharmacology predicts that PFHF treats endometriosis through PI3K/AKT signaling pathway. Compared with control group (DMEM/F-12 medium alone), the high dose PFHF can significantly reduce the viability, migration, and invasion of HEcESCs, increase the apoptosis rate of HEcESCs, and make the HEcESCs accumulated in G0/G1 phase in a time- and dose-dependent manner (p < 0.05). The analysis of label-free quantitative proteomics indicated that PFHF flavonoids may induce apoptosis of EESCs through PI3K/AKT signaling pathway. The results of RT-qPCR and Western blotting showed that the expressions of PI3K, AKT, Bcl-2, and Bcl-xl were significantly downregulated, while the bad expression was upregulated in HEcESCs treated with PFHF (p < 0.05). Limitations: This research investigated the effects of PFHF on the stromal endometriotic cells only. So it is unknown how PFHF can affect the entire endometriotic lesion. And the research is carried out in vitro, which gives no impression about the bioavailability of the flavonoids. Conclusion: PFHF reduces the expression of PI3K, AKT, Bcl-2, and Bcl-xl through the PI3K/AKT/Bcl-2 signaling pathway to inhibit HEcESCs proliferation, migration, and invasion and promote their apoptosis.

© 2023 The Author(s). Published by S. Karger AG, Basel

Introduction

Endometriosis is a chronic benign disease affecting approximately 10–15% of women of reproductive age [1]. The disease can cause dysmenorrhea, chronic pelvic pain, and infertility, and even a series of mental symptoms, such as depression, anxiety [2], which can negatively affect daily life, sexual function, and interpersonal relationships [3]. Moreover, this benign disease is similar to malignant tumor in some biologic properties, such as progressive and invasive growth, estrogen-dependent growth, and the tendency of recurrence and metastasis [4]. Studies have shown that endometriosis is an independent risk factor for ovarian cancer, and some malignancies, such as endometrioid carcinoma and clear cell carcinoma of the ovary, may be derived from endometriosis [4]. Epidemiological data indicate that endometriosis does have malignant potential [5]. Since Von Rokitansky described endometriosis for the first time in 1860, the pathogenesis of the disease has not yet been fully clarified. The theory of menstrual reflux proposed by Sampson [6] has been widely accepted, but not all women with menstrual reflux develop endometriosis, suggesting that menstrual reflux may be a cause [7]. Moreover, endometriosis may occur without menstrual tubal reflux. Studies have shown that endometrial stromal cells of patients with endometriosis have stronger adhesion, invasion, and proliferation than those of normal women [8]. Attachment of the endometrium to the host peritoneum (associated with invasion and migration of endometrial stromal cells) is critical to the progression of endometriosis [9, 10]. Currently, the main treatment for reproductive endometriosis patients is ovarian cystectomy, but the recurrence rate is high, and it is easy to damage the ovarian cortex and reduce the ovarian reserve function [11]. Drug therapy has always been one of the important treatment options for endometriosis, which is used to improve the symptoms of patients or prevent recurrence [12], mainly including symptomatic treatment with nonsteroidal anti-inflammatory drugs and hormone suppression therapy that causes a low estrogen environment in the body. However, long-term use of hormone suppressive therapy can cause side effects, such as vasomotor symptoms, mood changes, sleep disorders, urogenital atrophy, and decreased bone mineral density [13, 14]. None of the current treatments can clearly cure endometriosis [12], and it is nearly inevitable that endometriosis will relapse after treatment has been terminated. So far, there are no specific drugs to treat endometriosis by inhibiting the growth of endometrial stromal cells in patients with endometriosis. Therefore, new potential drugs should be able to inhibit the proliferation of human ectopic endometrial stromal cells (HEcESCs), which has become one of the priorities in the research and development of anti-Endometriosis drugs.

Polygala fallax Hemsl (PFHF) is a unique plant in China, which integrates edible, medicinal, and ornamental. Its roots, leaves, and flowers are edible, with unique flavor and fragrance, with the effect of tonifying qi and replenishing blood, invigorating spleen and dampness, promoting blood circulation for regulating menstruation. It is used for post-disease body deficiency, waist and knee soreness, traumatic injury, icterohepatitis, nephrotic edema, uterine prolapse, irregular menstruation, and other diseases [15–17]. According to literature reports, the plant contains flavonoids, saponins, polysaccharides, amino acids, organic acids, and other active components [18–21] and has the effects of anti-oxidation [20, 22], antiviral [21], lipid regulation [23–26], immune enhancement [27, 28], anti-stress [29], blood-activating, and anti-inflammatory [30]. Studies have shown that the total flavonoids of Polygala fallax Hemsl (PFHF) are toxic to endometrial cancer cells and have a significant dose-effect relationship [31]. However, so far, no study has reported on the effect of PFH on endometriosis.

The aim of this study was to examine the mechanism of PFHF in inhibiting HEcESCs proliferation. In addition, we also explored the effect of PFHF on the migration and invasion of HEcESCs induced and evaluated its molecular mechanism. Our results showed that PFHF has an inhibitory effect on HEcESCs.

Materials and MethodsMain Reagents and Instruments

The Polygala fallax Hemsl (PFH) was collected in Wuming District, Nanning City, Guangxi Province and was authenticated by the author (Ph.D. Xiangpei Zhao). Specimens of these materials were deposited in the herbarium, Guangxi International Zhuang Medicine Hospital, China. RPMI 1640 culture medium, Dulbecco’s modified Eagle’s medium (DMEM)/F-12, fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco (USA). Cell counting kit-8 (CCK-8) kit was purchased from Nanjing KeyGEN Biotechnology Development Co., Ltd. Trypsin. Phenylmethylsulfonyl fluoride, radioimmunoprecipitation assay tissue/cell lysate, bicinchoninic acid (BCA) protein concentration detection kits, and protein loading buffer were purchased from Beijing Solarbio Co., Ltd. The apoptosis and cell cycle extraction kits were obtained from Shanghai Beyotime Biotechnology Co., Ltd. The matrix glue was purchased from Corning Company; TRIzol Universal Reagent was procured from Tiangen Biotechnology (Beijing) Co., Ltd; the reverse transcription kit and AB HS Green qPCR Mix were purchased from Hangzhou SIMGEN biological reagent development Co., Ltd; the ECL chromogenic solution was bought from Millipore (USA). Enzyme labeling instrument was purchased from Switzerland Deacon Trading Company. Flow cytometry was purchased from Merck Chemical Company, and automatic real-time fluorescence quantitative PCR instrument was purchased from Roche. Goat anti-rabbit, rabbit anti-β-actin, vimentin, phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), B-cell lymphoma-2 (Bcl-2), Bcl-extra long (Bcl-xl), and Bcl-2 associated death promoter (Bad) antibody were obtained from Wuhan Servicebio Technology Co., Ltd. Alexa Fluor 594-labeled secondary antibody was obtained from Zhongshan Golden Bridge (Beijing). The inverted microscope was purchased from Leica (Germany); the constant temperature carbon dioxide cell incubator was purchased from Sanyo (Japan).

Network Pharmacology

Using “Polygala fallax Hemsl” as the keyword, chemical composition of PFH was searched in chEMBL [32] (https://www.ebi.ac.uk/chembl/). The flavonoids of PFH were screened according to the structural formula of each component. The flavonoids were introduced into the Swiss target prediction database [33] (http://www.swisstargetprediction.ch/) to predict the targets. Using “endometriosis” as keyword, endometriosis targets were searched in OMIM [34] (https://omim.org/), DrugBank [35] (https://go.drugbank.com), and DisGeNET [36] (https://www.disgenet.org/) databases. The information extracted from the three databases was merged, and duplicate items were deleted. The predicted target protein was transformed into gene name by UniProt [37] (https://www.uniprot.org/), and the species was limited to “Homo sapiens.” The component and disease targets were imported into Venny 2.1.0 online mapping tool (https://bioinfogp.cnb.csic.es/tools/venny/) to obtain the intersection targets. The protein-protein interaction (PPI) network was constructed by analyzing the interaction targets through the STRING database [38] (https://www.string-db.org/). The network diagram was imported into Cytoscape 3.8.2 software [39], and the network analyzer was used to assess the topology of the network diagram. The core targets were selected according to the card value of two-times degree median, betweenness median, and closeness median. The obtained core targets were imported into the Metascape database [40] (https://metascape.org/), the species were limited to select “Homo sapiens,” and the Kyoto Encyclopedia of Genes and Genomes [41] (KEGG) pathway enrichment analysis was carried out. The top 10 p value pathways were visualized using an advanced bubble chart.

Preparation of the Total Flavonoids of Polygala fallax Hemsl

The dried root bark of Polygala fallax Hemsl (PFH) was taken and pulverized into a coarse powder. Then 1,000 g coarse powder was dissolved in 10 times the amount of solvent (10 L 90% ethanol) for extraction. The extract was obtained through ultrasonically extract at 55°C for 40 min three times. Then the extract was decompressed and concentrated to obtain crude ointment. The crude ointment was evaporated to dryness, eluted by D101 macroporous resin, eluted by water first, then eluted by 70% ethanol. The 70% ethanol eluate is recovered and evaporated to dryness to obtain PFHF dry extract. Put into a brown glass bottle for later use. PFHF powder was dissolved in DMEM/F-12 medium during administration.

Primary Culture of HEcESCs

This study has been approved by the Ethics Committee of the Guangxi International Zhuang Medicine Hospital (Permission no: [2021]-022) and conducted according to the WMA and the CIOMS guidelines. This study was conducted with the written informed consent of the patients. Fresh ectopic endometrial tissue (the wall of ovarian endometrioma) was obtained from 10 volunteers who underwent cystectomy in Guangxi International Zhuang Medicine Hospital (The volunteers were between 22 and 40 years old, had regular menstruation, had not taken hormones 6 months before surgery, and had no other diseases). Some tissues collected from each patient were diagnosed as “ovarian endometriosis cyst” by pathology department after surgery, and some were sent to laboratory for experiment. These tissues were temporarily stored in RPMI 1640 medium and transferred to the laboratory in time. The cells are cultured by adherence method, and the treatment steps are as follows: after rinsing the tissue with phosphate-buffered saline (PBS) solution, soak the tissue with a small amount of penicillin/streptomycin for 3–5 min, and then wash it with PBS two to three times. Transfer the tissue to DMEM/F-12 medium containing 10% FBS for soaking and cut the tissue into small pieces of about 1 mm3 with ophthalmic scissors. Attach the cut tissue block to the bottom surface of the culture flask moistened with 1–2 mL of DMEM/F-12 medium containing 10% FBS and place it vertically. After 2–3 h, wait until the tissue is completely attached to the bottom of the culture flask, and then carefully lay it flat and do not move it. After 2–3 days, after the liquid in the culture flask turns yellow, carefully add 2–3 mL of DMEM/F-12 medium to the culture flask and put it back into the incubator. After the cells crawled out of the edge of the tissue block, trypsinized cell suspension was used to inoculate the culture flask and placed in a 37°C, 5% carbon dioxide cell culture incubator. The culture medium was changed every 3 days. The cultured HEcESCs were morphologically observed under an inverted phase-contrast microscope. Immunofluorescence staining was performed with an anti-vimentin antibody and Alexa Fluor 594-labeled secondary antibody, immunofluorescence showed >95% vimentin-positive (Figure 1). All the experiments were conducted between the third and fifth passage of the cultures.

Fig. 1.

Morphology and immunofluorescence of HEcESCs (magnification, × 100). Blue fluorescence is nuclear, red fluorescence is vimentin positive.

Cell Proliferation Assay

HEcESCs in the logarithmic growth phase were inoculated in 96-well plates at the density of 1 × 105 cells per well and starved for 24 h. The HEcESCs were divided into the following groups: normal control group, PFHF dose group (0.4, 0.5, 0.6, 0.7, and 0.8 mg/mL). All cells were treated for 12 h, 18 h, and 24 h, respectively. After the treatment, 10 μL CCK-8 solution was added to each well; the solution was then shaken and incubated in an incubator for an additional 2 h. The optical density of each group was measured at 450 nm wavelength by using an enzyme labeling instrument: inhibition rate = (control well - assay well)/control well * 100%.

Flow Cytometry

HEcESCs were treated with different concentrations of the PFHF for 12 h. Trypsin digested cells were collected by centrifugation and precipitation with PBS. An equivalent of 5 × 104 suspended cells was mixed with 195 μL AnnexinV-FITC binding solution, 5 μL AnnexinV-FITC, and 10 μL propidium iodide staining solution and incubated at room temperature (20–25°C) for 10–20 min to detect the degree of cell apoptosis by flow cytometry. Another 5 × 104 cells resuspended in PBS were obtained by centrifugation and precipitation. Then 1 mL ice-bath consisting of precooled 70% ethanol was gently blown and mixed, and the cells were fixed at 4°C for 12 h. Subsequently, propidium iodide staining was carried out at 37°C for 30 min, and the cell cycle was detected by flow cytometry.

Transwell Chamber ExperimentMigration

HEcESCs in the logarithmic growth phase were seeded in the upper chamber at a density of 5 × 104 cells/mL, 200 μL/well, and DMEM/F-12 medium containing 10% FBS was added in the lower chamber. PFHF was added to each upper chamber at varying concentrations and incubated at 37°C under 5% CO2. After 24 h, the filter membrane was removed and fixed with methanol for 20 min before Giemsa staining. Under the light microscope, the number of transmembrane cells in the upper, lower, left, right, and middle visual fields was counted randomly at ×200, and the mean value was calculated.

Invasion

HEcESCs in the logarithmic growth phase were seeded in the upper chamber (matrix glue was laid in advance to simulate extracellular matrix in vivo) at a density of 5 × 104 cells/mL, 200 μL/well, and DMEM/F-12 medium containing 10% FBS was added in the lower chamber. PFHF was added to each upper chamber at varying concentrations and incubated at 37°C under 5% CO2. After 48 h, the filter membrane was removed and wiped off the matrix glue, fixed with methanol for 20 min before Giemsa staining. Under the light microscope, the number of transmembrane cells in the upper, lower, left, right, and middle visual fields was counted randomly at ×200, and the mean value was calculated.

Label-Free Quantitative ProteomicsProtein Preparation

HEcESCs were treated with different concentrations of PFHF (0, 0.5 mg/mL) for 24 h, and the cell pellets were collected by centrifugation. Cells were digested using the filter-aided sample preparation procedure, as described previously [42]. The cells were precipitated by SDT lysate (2% SDS, 0.1 m Dithiothreitol, 0.1 m Tris/HCL, pH 7.6). The lysate was swirled in a low-temperature homogenizer for 60 s, ultrasonic extraction for 3 min, heating at 100°C for 10 min for reduction reaction to open disulfide bond, and then centrifuged at 12,000×g for 10 min. The supernatant was used for proteomics sample preparation. The BCA method measures the protein content and balances the difference in protein content between samples. Take 200 μg protein and replace it with a 10 kd ultrafiltration tube with 8M UA solution (prepared temporarily), then add 50 mm iodoacetamide 100 μL (IAA, final concentration not less than 20 mm) and incubate at room temperature in dark for 30 min for alkylation reaction to block the sulfhydryl group. Then transfer it to a 10 kd ultrafiltration tube of 50 mm NH4HCO3 enzymatic digestion solution and add about 4 μg trypsin, incubated at 37°C and oscillated overnight for enzyme digestion. Trifuoroacetic acid was added to the ultrafiltrate to terminate enzyme digestion, and SEP Pak C18 was used for desalination. After the desalted peptide solution is drained by a centrifugal concentrator, it is frozen and stored at −20°C for later use. Reconstitute with 0.1FA% before testing.

Mass Spectrometry

The protein samples were analyzed using the Q Exactive™ liquid chromatography-mass spectrometry (LC/MS) system (Thermo Fisher Scientific, USA). The samples were inhaled through an automatic sampler and bound to A C18 capture column (3 μm, 75 μm × 20 mm, 100 Å). Elution was carried out on an analytical column (50 μm × 150 mm, 2 μm particle size, 100 Å pore size, Thermo) for separation. Using two mobile phases (mobile phase A: 99% H2O, 0.1% formic acid and mobile phase B: 80% acetonitrile (ACN), 0.1% formic acid) established a 100-min analytical gradient (0 min in 3% B, 0–5 min of 3–5% B; 5–70 min of 5–23% B, 70–90 min of 23–55% B, 90–92 min of 55–90% B, 90% B for 8 min). The flow rate of the liquid phase was set at 300 nL/min. In the data dependent analysis mode analysis of MS, each scan cycle consisted of one MS full scan (m/z range: 350–1800, ion accumulation time: 200 ms) followed by 40 MS/MS scans (M/Z range: 100–1,500, ion accumulation time: 50 ms). MS/MS acquisition conditions are set as the parent ion signal is greater than 3e6 and the charge number is +2∼+5. The exclusion time of ion repeated collection was set to 35 s.

Data Analysis

The data were retrieved through Protein Discover (version 2.2), and the database retrieval algorithm was Percolator. The database used for the search is the human proteome reference database in UniProt (UniProt_human_20190515.fasta). The retrieval parameters are as follows: Scan Event: Msaa Analyzer (FTMS), MS Order (MS2), Activation Type (HCD), scan type (full); Sequest HT: Enzyme (Trypsin full), Dynamic Modification (Oxdation, Acetyl, Carbamidomethyl). The retrieval results were screened based on the Maximum Delta Cn and Maximum Rank of PSM with card value ≥0.05, and the retrieved items and contaminated proteins in the reverse database were deleted. The volcano plot combines a measure of statistical significance from a statistical test with the magnitude of the change, enabling quick visual identification of those data-points. KEGG pathway enrichment analysis was performed for the differential expression of proteins.

Quantitative Real-Time Polymerase Chain Reaction

TRIzol was used to extract the total RNA of HEcESCs treated with different concentrations of PFHF for 24 h. D (λ) 260/D (λ) 280 was determined by enzyme labeling instrument. If D (λ) 260/D (λ) 280 was 1.8–2.0, it was stored for later use. An equivalent of 1 μg total RNA was used to synthesize cDNA according to the instructions of the reverse transcription kit. The residual DNA was removed at 42°C for 5 min after which reverse transcription was performed at 42°C for 15 min. The reverse transcriptase enzyme was inactivated at 95°C for 5 min. A total of 1.2 μL reverse transcription reaction product was used for the real-time fluorescence PCR reaction. According to the instructions of AB HS green qPCR mix kit, a two-step thermal cycle was used. The reaction conditions were as follows: predenaturation at 95°C × 3 min for 1 cycle, denaturation at 95°C × 10 s, and annealing at 60°C × 30 s, and repeated for 40 cycles. All samples were added to 96-well PCR plates, each sample was repeated for 3 wells, and all reactions were carried out in a Roche LightCycler Sequence Detection System. The primer sequence is shown in Table 1, and GAPDH was used as an internal reference. The relative gene expression amount of target RNA was calculated by 2−ΔΔ Ct method [43].

Table 1.

Primer/probe sequence for qRT-PCR

Target geneSequences (5’→3’)PIK3CA Forward primerGGT​TGT​CTG​TCA​ATC​GGT​GAC​TGT Reverse primerGAA​CTG​CAG​TGC​ACC​TTT​CAA​GCAKT1 Forward primerTTC​TGC​AGC​TAT​GCG​CAA​TGT​G Reverse primerTGG​CCA​GCA​TAC​CAT​AGT​GAG​GTTBad Forward primerCAGGGGCCTCGTTATCGG Reverse primerGGA​CTC​TGG​ATC​AGA​CCT​CABcl-2 Forward primerTCG​CCC​TGT​GGA​TGA​CTG​A Reverse primerCAG​AGA​CAG​CCA​GGA​GAA​ATC​ABcl-xl Forward primerAGT​TTG​AAC​TGC​GGT​ACC​GG Reverse primerGCA​TTG​TTC​CCA​TAG​AGT​TCGAPDH Forward primerCTG​GGC​TAC​ACT​GAG​CAC​C Reverse primerAAG​TGG​TCG​TTG​AGG​GCA​ATGWestern Blotting

HEcESCs were treated with different concentrations of PFHF for 24 h. The protein was extracted according to the instructions of the protein extraction kit. The protein concentration was measured using the BCA method and stored at −80°C for later use. Proteins were separated on 10% SDS-PAGE. Immunoblotting was used to transfer samples to the PVDF membrane, which was incubated in 5% skimmed milk powder solution overnight at 4°C. The membrane was then incubated with PI3K, AKT, Bcl-2, Bcl-xl, Bad, and β-actin antibodies at room temperature for 4 h, after which it was washed for 3 times in TBST and incubated again with the corresponding horseradish peroxidase-labeled secondary antibody. Samples were analyzed using a chemiluminescent protein detection method and image software [44].

Statistical Analysis

Each experiment was repeated at least three times. All data were analyzed using GraphPad Prism 8.0.2 and expressed as mean ± standard deviation. One-way analysis of variance and unpaired t test were used to compare each group. p ≤ 0.05 was considered statistically significant.

ResultsNetwork Pharmacology Predicts that PFHF Treats Endometriosis by Regulating the PI3K/AKT Signaling Pathway

A total of 79 chemical components of Polygala fallax Hemsl (PFH) were retrieved from chEMBL database, including flavonoids ingredients 40 kinds (Raw DataS1, available at www.karger.com/doi/10.1159/000530104). The obtained chemical structure formulas of flavonoids were imported into Swiss Target Prediction database. A total of 1,709 targets were predicted, and 276 targets were obtained after deleting duplicates. Using “endometriosis” as the keyword, 1206 disease targets were obtained by searching OMIM, DrugBank, and DiGeNET databases and deleting duplicate items. Among these, 116 intersection targets of drugs and diseases were identified, suggesting that these could be potential targets for the treatment of endometriosis (Fig. 2a).

Fig. 2.

The network pharmacology results (a–d) of PFHF and endometriosis, include the intersection targets of PFHF and ovarian endometriosis (a), the PPI map of intersection targets (b) and core targets (c), the top 10 pathways with p value which the PFHF treat endometriosis (d).

These 116 intersection targets of PFHF and endometriosis were entered into the STRING database for analysis, and the PPI map was constructed (Fig. 2b; Table 2, Raw DataS2, available at www.karger.com/doi/10.1159/000530104). Topological analysis was conducted on the network diagram of the intersection targets, and 16 core targets were selected according to the card value of 2-fold degree median, betweenness median, and closeness median (Table 3), and PPI core gene target map was constructed (Fig. 2c; Table 2).

Table 2.

The score of intersection targets and core targets in PPI map

Intersection targetsCore targetsNodes numbera11616Edges numberb1812119average node degreec31.214.9PPI enrichment p-value<1.0e−164.72e−12Table 3.

The degree, betweenness, and closeness of core targets

GeneDegreeaBetweennessClosenessAKT191886.957340.81560284TP5387810.546630.7986111VEGFA79403.483430.7565789ESR1771123.44760.751634STAT377328.177030.7419355EGFR76387.935150.73717946MAPK376413.742650.7419355IL673428.620360.7278481HIF1A72299.19780.7232704EGF70219.80990.70987654CCND170318.25780.70987654MMP965480.66080.6886228PTGS260292.858760.6764706ERBB260184.11350.6647399PPARG59301.54660.6686047MAPK157201.066280.6571429

KEGG pathway enrichment analysis was performed on the core target, and the top 10 pathways (Table 4) with p value were selected to construct an advanced plot chart (Fig. 2d). Based on the KEGG results, we found that the PI3K/AKT signaling pathway, HIF-1 signaling pathway, and FOXO signaling pathway are closely related to endometriosis. Previous studies have shown that PI3K/AKT signaling pathway is extensively involved in all stages of endometriosis [45]. Thus, it is speculated that PFHF may treat endometriosis by regulating the PI3K/AKT signaling pathway.

Table 4.

The top 10 pathways with p value which the PFHF treat endometriosis

PathwayRelated genep valuePathways in cancerAKT1,CCND1,EGF,EGFR, ERBB2,ESR1,HIF1A,IL6,MMP9,PPARG, MAPK1,MAPK3,PTGS2,STAT3,TP53,VEGFA2.44014E−28Proteoglycans in cancerAKT1,CCND1,EGFR, ERBB2,ESR1,HIF1A,IL6,MMP9,MAPK1,MAPK3,STAT3,TP53,VEGFA5.42785E−26Pancreatic cancerAKT1,CCND1,EGF,EGFR, ERBB2,MAPK1,MAPK3,STAT3,TP53,VEGFA9.60391E−23Bladder cancerCCND1,EGF,EGFR, ERBB2,MMP9,MAPK1,MAPK3,TP53,VEGFA1.38244E−22HIF-1 signaling pathwayAKT1,EGF,EGFR, ERBB2,HIF1A,IL6,MAPK1,MAPK3,STAT3,VEGFA4.74458E−21Non-small cell lung cancerAKT1,CCND1,EGF,EGFR, ERBB2,MAPK1,MAPK3,STAT3,TP531.26131E−20EGFR tyrosine kinase inhibitor resistanceAKT1,EGF,EGFR, ERBB2,IL6,MAPK1,MAPK3,STAT3,VEGFA3.98418E−20Endocrine resistanceAKT1,CCND1,EGFR, ERBB2,ESR1,MMP9,MAPK1,MAPK3,TP532.50101E−19Prostate cancerAKT1,CCND1,EGF,EGFR, ERBB2,MMP9,MAPK1,MAPK3,TP534.41263E−19Endometrial cancerAKT1,CCND1,EGF,EGFR, ERBB2,MAPK1,MAPK3,TP534.77518E−18MelanomaAKT1,CCND1,EGF,EGFR,IL6,MAPK1,MAPK3,TP532.87687E−17Breast cancerAKT1,CCND1,EGF,EGFR, ERBB2,ESR1,MAPK1,MAPK3,TP533.21339E−17Focal adhesionAKT1,CCND1,EGF,EGFR, ERBB2,IL6,MAPK1,MAPK3,VEGFA4.29536E−16PI3K-Akt signaling pathwayAKT1,CCND1,EGF,EGFR, ERBB2,IL6,MAPK1,MAPK3,TP53,VEGFA6.52962E−16FOXO signaling pathwayAKT1,CCND1,EGF,EGFR,IL6,MAPK1,MAPK3,STAT31.79984E−15GliomaAKT1,CCND1,EGF,EGFR, MAPK1,MAPK3,TP536.46788E−15Central carbon metabolism in cancerAKT1,EGFR, ERBB2,HIF1A,MAPK1,MAPK3,TP536.46788E−15MicroRNAs in cancerCCND1,EGFR, ERBB2,MMP9,MAPK1,PTGS2,STAT3,TP53,VEGFA8.61674E−15Hepatitis CAKT1,CCND1,EGF,EGFR, MAPK1,MAPK3,STAT3,TP539.9693E−15Hepatitis BAKT1,CCND1,IL6,MMP9,MAPK1,MAPK3,STAT3,TP531.45173E−14PFHF Inhibits the Proliferation, Migration, and Invasion of HEcESCs, Blocks Their Cell Cycle, and Induces Their Apoptosis

Although endometriosis is a benign disease, it has malignant biological characteristics such as invasive growth, recurrence, and metastasis [4]. In this study, CCK-8 detection was carried out to determine the inhibitory effect of PFHF on the proliferation of HEcESCs (Figure 3). Compared with the control group, the cell growth of each dose group was significantly inhibited after 12 h, and the inhibition rate was in direct proportion to the drug concentration, with a statistically significant difference (p < 0.05). The inhibitory effect of PFHF on the growth of HEcESCs was significantly stronger than that of 12 h after 18 h of treatment; however, the growth inhibition effect of HEcESCs was the most obvious after 24 h of PFHF. It can be seen that the PFHF inhibited the proliferation of HEcESCs in a time-dose dependent manner. When the PFHF concentration was 0.6 mg/mL, the 24-h inhibition rate was more than 50%. Therefore, drug concentration in the range of 0.4–0.5 with inhibition rate less than 30% was selected to explore the effects of PFHF on cell cycle, apoptosis, invasion, and migration ability and its mechanism.

Fig. 3.

Effect of PFHF on HEcESCs proliferation. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with control group (DMEM/F-12 medium alone).

The effect of PFHF on apoptosis and cell cycle of HEcESCs was detected by flow cytometry. As shown in the results of Figures 4 and 5a, compared with the control group, there was no significant change in the proportion of necrotic cells, which was maintained within 0.5% (p > 0.05). However, the percentage of apoptotic cells in the late stage was significantly increased and maintained at about 1% (p < 0.05). The percentage of normal HEcESCs decreased significantly and gradually decreased with the increase of PFHF concentration (p < 0.05). At the same time, the proportion of apoptotic cells in the early stage was significantly higher than that in the control group, and gradually increased with the increase of PFHF concentration (p < 0.05). This indicates that PFHF induces apoptosis of HEcESCs in a dose-dependent manner, mainly in the form of early apoptosis. As shown in the results of Figures 6 and 5b, compared with the control group, the proportion of HEcESCs in G0/G1 phase showed a significant upward trend (p < 0.05), and increased with the increase of PFHF concentration; the percentage of G2/M cells decreased with the increase of PFHF concentration (p < 0.05). The proportion of S phase cells had no significant change in the control group and the drug group (p > 0.05). It can be seen that PFHF can effectively block the progress of HEcESCs cell cycle in a dose-dependent manner, and make them accumulate in G0/G1 phase to prevent cell mitosis.

Fig. 4.

Effect of PFHF on the apoptosis of HEcESCs. In each apoptosis diagram, the upper left quadrant is necrotic cells, the lower left quadrant is normal cells, the upper right quadrant is late apoptotic cells, and the lower right quadrant is early apoptotic cells.

Fig. 5.

Apoptotic rate (a) and cell cycle rate (b) of PFHF on HEcESCs. The number of migration and invasion of HEcESCs treated with PFHF (c) and the expression of mRNA by qRT-PCR (d). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with control group (DMEM/F-12 medium alone).

Fig. 6.

Effect of PFHF on the cell cycle of HEcESCs.

The effect of PFHF on migration and invasion of HEcESC was tested by Transwell chamber experiment (Fig. 5c; 7). Compared with the control group, the number of cell migration and invasion in HEcESC treated with PFHF decreased significantly and was positively correlated with the concentration of PFHF (p < 0.05). As we know, the reduced number of cells behind the membrane may be related to the ability of drugs to promote cell apoptosis and inhibit its migration. It can be seen from Figure 5c that the reduced number of cells behind the membrane by more than 50% after the treatment of 0.5 mg/ml PFHF for 24 h, which was significantly higher than the apoptosis rate at this concentration. This indicates that PFHF inhibits the migration and invasion of HEcESCs in a dose-dependent manner.

Fig. 7.

Effect of PFHF on the migration (a) and invasion (b) of HEcESCs.

Identification of Differential Expressed Proteins by Label-Free Quantitative Proteomics

To further analyze the potential molecular mechanism of PFHF inhibition on HEcESCs, label-free quantitative proteomics was used to screen differential proteins and influence pathways. After mass spectrometry detection, the proteins with sum PEP score ≥1.5 were screened out. After deleting the contaminating proteins, a total of 937 proteins were identified, including 920 proteins in the control group and 618 proteins in the PFHF administration group (Fig. 8a). After comparing and analyzing with the database, it is confirmed that the mass deviation of the searched peptides conforms to the normal distribution. The distribution of sample peptides between the two groups is shown in Figure 8b. Based on the number of peptide spectra and scores detected by mass spectrometry, screening PV ≥0.05, FC ≥1, a total of 363 DEPs were identified. Among them, 54 were upregulated proteins and 309 were downregulated proteins (Fig. 8c). Using the KEGG database to identify the functional pathway information of DEPs (Fig. 8d), it was found that these DEPs were enriched in PI3K/AKT signaling pathway, HIF-1 signaling pathway, and other pathways. This is consistent with the results of network pharmacology and provides an important theoretical basis for further confirming that the PFHF can regulate the PI3K/AKT signaling pathway to treat endometriosis.

Fig. 8.

Identification of differential expressed proteins (DEPs) by label-free quantitative proteomics, include intergroup protein cross plot (a), the peptide mass distribution among samples (b), the volcano plot (c), and the KEGG analysis (d) of the DEPs. In picture C, gray indicates insignificant proteins, red indicates upregulated proteins, and green indicates downregulated proteins.

PFHF Inhibits the Expression of Genes and Proteins Related to the PI3K/AKT/Bcl-2 Signaling Pathway

To further verify the mechanism of PFHF inhibiting HEcESCs proliferation and migration, qRT-PCR and Western blotting were used to detect the mRNA and protein expression of key genes of PI3K/AKT signaling pathway (PI3K, AKT, Bcl-2, Bad, Bcl-xl) (Fig. 5d; 9). The results showed that with the increase of PFHF concentration, the relative expression of PI3K and AKT decreased significantly (p < 0.05), while the downstream genes Bcl-2, Bad, and Bcl-xl showed no obvious trend (p > 0.05). The protein expression level of PI3K/AKT signaling pathway was detected by Western blot. The results showed that the expression of PI3K, AKT, Bcl-2, and Bcl-xl protein was significantly reduced, while the expression of Bad protein increased in a dose-dependent manner (p < 0.05). This shows that PFHF can induce the degradation of PI3K, AKT, Bcl-2, Bcl-xl proteins and promote the expression of Bad protein to inhibit HEcESCs proliferation and migration by regulating the PI3K/Akt/Bcl-2 signaling pathway. However, the mRNA expression of Bcl-2, Bad, and Bcl-xl has no obvious trend, which may be due to the fact that RNA splicing produces different splicing bodies (that is, mRNA is processed) to encode different protein isomers. The mRNA detected by the primers we have designed may not be the protein detected by the target antibody. This indicates that more in-depth molecular biology research is needed in the future.

Fig. 9.

Expression of protein by Western blotting analysis. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with control group (DMEM/F-12 medium alone).

Discussion

The exact mechanism of endometriosis is unclear. It is noteworthy that the pathogenesis of endometriosis is now considered to be multifactorial. Genetic predisposition and epigenetic dysregulations are suggested to be the most important part of the pathogenesis and etiology of endometriosis [46]. The most widely accepted doctrine of menstrual blood reflux is that the endometrial tissue passes through the fallopian tube to reach the peritoneal cavity when menstruation falls off, adheres to the wall of the peritoneal cavity, and invades the extracellular matrix to form endometriosis lesions [6]. On this basis, Professor Lang Jinghe put forward the “eutopic endometrial determinism” [7]. The theory believes that the endometrial cells that flow back through the menstrual are planted on the pelvic and abdominal cavity membranes, organs, and tissues through adhesion, invasion, and blood vessel formation. With the influence of hormones, these ectopic implanted endometrial cells undergo changes such as bleeding, inflammatory response, and immune response, thereby forming endometriosis. The adhesion, invasion, and angiogenesis of eutopic endometrium in patients with endometriosis are stronger than those in patients without endometriosis. The retrograde menstruation of stem/progenitor cells from endometrial niches to the peritoneal cavity may underlie the development of endometriosis within the peritoneal cavity; bone marrow-derived stem cells may migrate through peripheral circulation, cause endometriosis in remote sites, and also infiltrate eutopic endometrium [47–49]. Accumulating evidence suggests that immune cells, adhesion molecules, extracellular matrix metalloproteinase, and pro-inflammatory cytokines activate/alter peritoneal microenvironment, creating the conditions for differentiation, adhesion, proliferation, and survival of ectopic endometrial cells [50]. Therefore, inhibiting the growth of ectopic endometrial stromal cells is of great significance in the treatment of endometriosis. The traditional medicines are often used by women with endometriosis as therapeutic supplements or alternatives to conventional medicines to improve treatment outcomes and reduce side effects [51]. But the clinical effectiveness and pharmacological mechanisms of most traditional herbal medicines remain to be further elucidated.

Polygala fallax Hemsl (PFH) is a perennial deciduous shrub belonging to Polygala tenuifolia of Polygala family, which mostly grows under sparse forests on hillsides or in valley forests. Its roots, leaves, and flowers are edible, with unique flavor and fragrance. As a traditional medicine, Polygala fallax Hemsl (PFH) is widely used in the treatment of icterohepatitis, nephritis edema, uterine prolapse, menstrual irregularities, and other diseases [15–17]. In Guilin, Guangxi, people often use their roots and meat to stew together to treat endometriosis by dietotherapy, but there is no pharmacological activity study related to this. At present, the research on the chemical constituents of PFH has been more detailed. Some scholars found that the genus is rich in flavonoids, and some related studies have confirmed that the flavonoids of this genus have anti-oxidation, antiviral, immune enhancement, anti-stress, blood-activating, and anti-inflammatory effects. Studies have shown that the total flavonoids of Polygala fallax Hemsl (PFHF) are toxic to endometrial cancer cells and have a significant dose-effect relationship [31]. However, so far, no study has reported on the effect of PFH on endometriosis.

In this study, the functions and possible mechanisms of PFHF against endometriosis were predicted using a network pharmacology strategy. As shown by the network pharmacology analysis, PFHF may treat endometriosis through PI3K/AKT signaling pathway, HIF-1 signaling pathway, and FOXO signaling pathway. To further verify the prediction, this study investigated the effect of PFHF on HEcESCs by CCK-8 method, flow cytometry, and Transwell method. The experimental results showed that PFHF inhibited cell growth in a dose- and time-dependent manner (Fig. 3), induced G0/G1 cell cycle arrest and apoptosis (Figure 4, Fig. 5a, b; 6), and inhibited cell migration and invasion activities (Fig. 5c; 7). Subsequently, proteomic results (Fig. 8) were used to predict whether PFHF might inhibit the proliferation and survival of HEcESCs through the PI3K/AKT signaling pathway.

Phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway is an important signaling pathway for cell survival. It plays an important role in cell proliferation, differentiation, and apoptosis [52–54]. When PI3K is activated, phosphatidylinositol-4,5-bisphosphate (PIP2) on the inner surface of the cell membrane is catalyzed to produce phosphatidylinositol-3, 4,5-triphosphate (PIP3). As a second messenger [55], PIP3 changes the conformation of AKT, exposes its phosphorylation sites, causes a cascade of signal transduction pathways, and then regulates cell proliferation, differentiation, and apoptosis [52, 56, 57]. Previous studies have shown that the PI3K/AKT signaling pathway is related to the development of diseases such as cancer [53,