Pyrvinium pamoate induces in-vitro suppression of IL-6 and IL-8 produced by human endometriotic stromal cells

Abstract

Endometriosis, a chronic inflammatory disease, is identified by the presence of endometrial tissue outside the uterus. The prevalence of this disease among reproductive-age women is almost 10–15%. High levels of IL-6 and IL-8 have been found in the peritoneal fluid (PF) of women with endometriosis and are involved in its pathogenesis. Isolated stromal cells from 12 ectopic and eutopic endometrial biopsies of women with ovarian endometrioma and also 12 endometrial biopsies of nonendometriotic controls were treated with 1.1 µM pyrvinium pamoate, a Wnt/β-catenin signaling pathway inhibitor, for 72 hrs. Before treatment, mRNA gene expression and secretion of IL-6 and IL-8 were significantly higher in ectopic (EESCs) than eutopic (EuESCs) and control (CESCs) endometrial stromal cells. After treatment, mRNA gene expression and also secretion of IL-6 and IL-8 were significantly reduced. Our Findings showed that pyrvinium pamoate suppresses the mRNA gene expression and secretion of IL-6 and IL-8 in human endometriotic stromal cells. Additional investigations on this compound are required before clinical application.

Keywords

Endometriosis pyrvinium pamoate stromal cells interleukin-6 interleukin-8 Wnt signaling pathway

Introduction

Endometriosis is a complex gynecological disorder defined as unusual growth of endometrial tissue outside the uterine cavity on pelvic organs such as the ovaries or fallopian tubes and affects about 6–10% of general population of women of reproductive age. In spite of various studies, exact etiology and pathogenesis of endometriosis remain uncertain.^1,2 Sampson’s retrograde menstruation theory is the most widely accepted among several hypotheses. According to this theory menstrual blood composed of shedding endometrial cells and fragments pass toward peritoneal cavity through the fallopian tubes.³ Wnt/β-catenin signaling pathway regulates numerous biological process such as stem cell renewal, embryonic development, cell survival and differentiation, apoptosis and pathogenesis of some disease. The role of aberrant activation of Wnt/β-catenin signaling pathway in establishment and progression of endometriosis has been demonstrated in some researches.^4
–6 Therefore this signaling pathway can be a good target for examination of novel drugs to inhibit endometriosis development.

Increased level of some cytokines and chemokines such as IL-1β, IL-17A, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, and vascular endothelial growth factor (VEGF) which is produced by recruited immune cells such as activated macrophages, lymphocytes and also endometriotic lesions have observed in peritoneal fluid of women with endometriosis.^7
–9 These products have important roles in proliferation, invasion, adhesion, vascularization and progression of endometriotic implants. IL-6 is a multifunctional cytokine that is prevalently secreted by activated macrophages in the peritoneal fluid of women with endometriosis and also ectopic endometrial cells.^7,10 This proinflammatory cytokine is involved in angiogenesis of endometrial cells¹¹ and toxicity of high levels of IL-6 for mouse embryo is reported.^7,12,13 IL-8 as a chemokine produced by various cell types such as monocytes, fibroblasts, mesothelial, endometrial and endometriotic cells has been demonstrated to induce activation and migration of neutrophils toward the site of infection and inflammation.^14
–16 This inflammatory mediator contributes to the pathogenesis of endometriosis. Effective role of this chemokine in endometriotic cell attachment, proliferation and implantation has been mentioned in some studies.^16
–18

NF-κB Signaling Pathway is involved in inflammatory responses and the expression of IL-6 and IL-8 are induced by NF-κB.¹⁹ These cytokines also are target genes for Wnt/β-catenin signaling pathway^20

–23 and with respect to the interaction between WNT/β-catenin and NF-κB signaling pathway^24,25 pyrvinium pamoate can affect gene expression and secretion of these cytokines.

Pyrvinium pamoate an FDA-approved compound with anthelmintic properties is an inhibitor of Wnt/β-catenin signaling pathway through activation of casein kinase 1 α (CK1α).²⁶ Casein kinase 1α (CK1α), a protein with Ser/Thr protein kinase activity, is a component of destruction complex of Wnt/β-catenin signaling pathway which initiate phosphorylastion of β-catenin in serine 45.²⁷

Based on the inhibitory effects of pyrvinium pamoate on Wnt/β-catenin signaling pathway and aberrant activation of this pathway in endometriosis, its effects were investigated on gene expression and production of IL-6, IL-8 by ectopic (EESCs) eutopic endometrial stromal cells (EuESCs) from endometriotic patients and also endometrial stromal cells of women without the disease endometriosis (CESCs).

Therefore, this signaling pathway can be a good target for examination of novel drugs to inhibit endometriosis development.

Materials and methods

Subjects and sample collection

Endometrial specimens were taken from ectopic and eutopic sites of 12 patients with ovarian endometrioma at the time of laparoscopy, and endometrial biopsies were also collected from 12 healthy control women with benign gynecological conditions without any sign of endometriosis, following assessment by laparoscopic surgery. All tissue samples were kept in ice-cold DMEM/F12 containing 1% penicillin-streptomycin (Gibco, Grand Island, New York, USA) and immediately transferred to the laboratory.

The Subjects were included in the study if any of the following criteria apply. They:

Had reproductive age 29–43 years old

Had regular menstrual cycles of 25–30 days

Had not received hormonal medication for at least 3 months before surgery

Had Ovarian Endometriosis (endometrioma)

The Subjects were excluded in the study if any of the following criteria apply. They:

Had autoimmunity

Had previous malignancy

Functional layer of uterine fundus was chosen for taking endometrial biopsies by a biopsy catheter (Pipelle® Endometrial Suction Curette, Cooper Surgical, USA).

In patients with endometriosis, stages III were confirmed following histopathological evaluation according to the revised American Fertility Society system.

The present study was approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.MSP.REC.1395. 589) and conducted in accordance with the Helsinki Declaration. All participants gave written informed consent.

Isolation and purification of ESCs

ESCs were isolated and cultured based on our previous protocol.²⁸ Briefly, endometrial biopsies from women with ovarian endometrioma and control individuals were minced into small fragments (1–2 mm) and treated with 0.1% collagenase I (Gibco, New York, New York, USA) for enzymatic digestion at 37°C for 1 h. Removal of epithelial cells was achieved by filtration through a 70 µm strainer and then a 40-mm cell strainer. Stromal cells were grown in T-25 flasks at 37°C in 5% CO₂ for 12 h. Purification of cells in all cases were analyzed by immunofluorescence staining for vimentin as positive marker and also cytokeratin as negative marker.

Pyrvinium pamoate cytotoxicity assay

After reaching 80% confluence, cells were trypsinized and seeded (8 × 10⁴/well) in six-well plate in 2 mL of DMEMF12 medium containing 10% FBS and antibiotics. Following attachment, all type of the cells were treated with different concentrations (10, 1, 0.1, and 0.01 mM) of pyrvinium pamoate (Sigma, New York, New York, USA) dissolved in 0.25%DMSO for 24, 48 or 72 h. The vehicle group received nontoxic concentration of DMSO (0.25%) and control group received no treatment. All analyses were carried out in triplicate, each being repeated at least three times. MTT assay was used for calculation of half-maximal inhibitory concentration (IC50) values using a nonlinear regression curve were calculated by the GraphPad Prism® 6.0 (GraphPad Software, San Diego, California, USA) software and 1.1 µM concentration of pyrvinium pamoate for 72 h was chosen.

Measurement of IL-6 and IL-8 in culture medium by ELISA

The concentrations of IL-6 and IL-8 in the conditioned media from cultured ESCs were measured in triplicate with commercial kit DuoSet® ELISA Development Systems (R&D Systems, Minneapolis, MN, USA) according to manufacturers’ protocol. The minimum detectable levels of IL-6, IL-8 (sensitivity of ELISA) were 9.4 and 31.2 respectively. The protein content was quantified in the cell lysates by the Bradford protein assay following the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, USA). The results were calculated using standard curves fitted with sigmoidal four-parameter logistic (4PL) regression for each cytokine.

RNA extraction and real-time reverse transcriptase-polymerase chain reaction

The RiboEx (GeneAll, Seoul, Korea) Reagent was used to extract and purify the RNA from endometrial samples, and complementary DNA (cDNA) was generated from 500 ng of total RNA using Synthesis kit (Qiagen, Hilden, Germany). RT-PCR was carried out in triplicate with corresponding primers and SYBR® Green Master Mix (Ampliqon, Odense, Denmark) and using Applied Biosystems StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, California, USA). Gene expression values were normalized against GAPDH of the same sample as control housekeeping gene. The primer sequences and the size of amplicons are provided in Table 1. Reactions were initial denaturation at 95°C for 10 min; followed by 40 cycles of 95°C for 15 s and 60°C for 40 s. The relative gene expression of IL-6 and IL-8 genes were analyzed using the DDCT method and REST-RG software (version 3). For each gene, negative control included a qPCR reaction without cDNA.

Table 1.

List of primer sequences used in real-time PCR.

Primer name	Primer sequences
H-GAPDH-F	5′-TCTGACTTCAACAGCGACACC-3′
H-GAPDH-R	5′-GTTGCTGTAGCCAAATTCGTT-3′
H-IL6-F	5′-GTGTGAAAGCAGCAAAGAGGC-3′
H-IL6-R	5′-CAGGCAAGTCTCCTCATTGAATC-3′
H-IL8-F	5′-ACCACCGGAAGGAACCATCT-3′
H-IL8-R	5′-AAAACTGCACCTTCACACAGAG-3′

Phosphorylated β-catenin (serine 45) assay

Quantification of Phospho β-Catenin was performed using ELISA kit InstantOne™ (eBioscience, San Diego, CA, USA) according to manufacturer’s protocol. Briefly, cells at a density of 3 × 10⁴ were seeded in 96-well microplates. Following attachment, cells were lysed with 100 µL of freshly prepared cell lysis buffer Mix (1×), then 50 µL of this cell lysate, cell lysis mix (1×) as negative control and positive control (kit component) were added to wells of 96-well microplates in triplicate. Next, 50 µL of prepared Antibody Cocktail (kit component) to all testing wells, microplate was covered with adhesive seal and incubate for 1 hour at room temperature on a microplate shaker (∼300 rpm). After three washes with 200µL 1× wash buffer per well, detection reagent (100 µL) was added to each well and 20 minutes incubated with shaking at 300 rpm. In appropriate time 100 µL of stop solution (kit component) was added to each well and finally the absorbance of the samples in the plate was read by using a plate reader at a 450-nm wavelength a colorimetric plate reader set at 450 nm.

Statistical analyses

All results are presented as mean ± standard deviation (SD) and were analyzed by SPSS v.19 software (Chicago, IL, USA). All experiments were repeated at least three times. To compare cytokine levels and messenger RNA (mRNA) gene expression between experimental groups, one-way analysis of variance (ANOVA) was used and comparisons between different dosages of pyrvinium pamoate were performed using two-way ANOVA. The level of significance was indicated at P < 0.05.

Results

ESCs characterization

Immunofluorescent staining of isolated ESCs exhibits antibody reactivity against vimentin (Figure 1(a)) and no reactivity against cytokeratin (Figure 1(b)).

Figure 1.

Immunofluorescent staining in isolated CESCs, EuESCs and EESCs. (a) Vimentinpositive cells. (b) Cytokeratin negative cells. CESCs: endometrial stromal cells from endometrium of nonendometriotic individuals; EuESCs: endometrial stromal cells from endometrium of endometriotic patients; EESCs: endometrial stromal cells from ovarian endometrioma.

IC50 calculation

ESCs incubated with different concentrations of pyrvinium pamoate (10, 1, 0.1, and 0.01 µM) in 24-, 48-, and 72-h time periods. The IC50 values for pyrvinium pamoate at 24, 48 and 72 hrs were 3.5 µM (CI: 1.589 to 8.008), 2.6 µM (CI: 1.193 to 5.937) and 1.1 µM (CI: 0.4542 to 2.998), respectively. There was no statistically significant difference between these IC50 values (P = 0.1646; Figure 2). Concentration of 1.1 µM and incubation time of 72 hrs were selected as treatment dosage for further examination.

Figure 2.

The half maximal inhibitory concentration (IC50) values for different concentrations of pyrvinium pamoate (10 μM, 1 μM, 0.1 μM and 0.01 μM) were determined on in vitro cultured human ESCs after incubation for 24-, 48-, and 72-h using GraphPad Prism® 6.0. Each point was in triplicate and represented as mean ± SD.

Inhibitory effect of pyrvinium pamoate on Wnt/β-catenin signaling pathway

The level of phospho-β-catenin (Ser45) in ESCs was assessed by ELISA kit InstantOne™ (eBioscience, San Diego, CA, USA) to determine activating impact of pyrvinium pamoate on Casein kinase 1 α and thereby increasing the level of phospho-β-catenin (Ser45). The level of phospho-β-catenin (Ser45) in both EESCs and EuESCs groups was significantly downregulated (P < .01 & P < .05 respectively, Figure 3(a)) in comparison with CESCs group. Following treatment of EESCs group with pyrvinium pamoate at a dosage of 1.1 µM for 72 hrs the level of phospho-β-catenin (Ser45) was significantly elevated compared to vehicle group (P < .05, Figure 3(b)).

Figure 3.

The level of phospho-β-catenin (Ser45) in cultured CESCs, EuESCs and EESCs after treatment with 1.1 μM PP (pyrvinium pamoate) treatment for 72 hrs. ELISA assay was performed before (a) and after drug treatment on isolated ectopic (b) stromal cells from endometriotic subjects OD (optical density). *P < .05 and **P < .01. CESCs: endometrial stromal cells from endometrium of nonendometriotic individuals; EuESCs: endometrial stromal cells from endometrium of endometriotic patients; EESCs: endometrial stromal cells from ovarian endometrioma.

Effect of pyrvinium pamoate on IL-6 and IL-8 production by ESCs

The levels of secreted IL-6 and IL-8 in cultured ESCs were evaluated using ELISA assay to determine the impact of pyrvinium pamoate on IL-6 and IL-8 secretion.

The levels of secreted IL-6 and IL-8 in EESCs group was significantly higher than EuESCs and CESCs groups (P < .001, Figures 4(a) and 5(a)). In treatment groups pyrvinium pamoate significantly decreased the IL-6 (P < .01, Figure 4(b)) produced by CESCs, similar significant decrease was also observed for IL-8 (P < .05, Figure 5(b)). Pyrvinium pamoate also significantly reduced the IL-6 and IL-8 produced by EuESCs (P < .01 & P < .05, Figures 4(c) and 5(c)) and EESCs (P < .001, Figures 4(d) and 5(d)) groups.

Figure 4.

The level of secreted IL-6 by cultured CESCs, EuESCs and EESCs after treatment with 1.1 μM PP (pyrvinium pamoate) treatment for 72 hrs. ELISA assay was performed before (a) and after drug treatment on isolated normal (b), eutopic (c) and ectopic (d) stromal cells from endometriotic and nonendometriotic subjects. All values are normalized against total protein content of cell lysates. **P < .01 and ***P < .001. CESCs: endometrial stromal cells from endometrium of nonendometriotic individuals; EuESCs: endometrial stromal cells from endometrium of endometriotic patients; EESCs: endometrial stromal cells from ovarian endometrioma.

Figure 5.

The level of secreted IL-8 by cultured CESCs, EuESCs and EESCs after treatment with 1.1 μM PP (pyrvinium pamoate) treatment for 72 hrs. ELISA assay was performed before (a) and after drug treatment on isolated normal (b), eutopic (c) and ectopic (d) stromal cells from endometriotic and nonendometriotic subjects. All values are normalized against total protein content of cell lysates. *P < .05 and ***P < .001. CESCs: endometrial stromal cells from endometrium of nonendometriotic individuals; EuESCs: endometrial stromal cells from endometrium of endometriotic patients; EESCs: endometrial stromal cells from ovarian endometrioma.

Effect of pyrvinium pamoate on mRNA expression of IL-6 and IL-8 in ESCs

The IL-6 and IL-8 mRNA levels in ESCs were evaluated by qRT-PCR to determine the impact of pyrvinium pamoate on IL-6 and IL-8 mRNA expression.

The results of non-treated groups demonstrated that mRNA expression of IL-6 and IL-8 in EESCs group was significantly increased compared to EuESCs group (P < .05, Figures 6(a) and 7(a)). The mRNA expression of IL-6 and IL-8 was significantly enhanced in EESCs group compared with CESCs group (P < .01 & P < .001, Figures 6(a) and 7(a)).

Figure 6.

mRNA expression of IL-6 in cultured CESCs, EuESCs and EESCs after treatment with 1.1 μM PP (pyrvinium pamoate) treatment for 72 hrs. Relative mRNA expression was measured before (a) and after drug treatment on isolated normal (b), eutopic (c) and ectopic (d) stromal cells from endometriotic and nonendometriotic subjects. *P < .05 and **P < .01. CESCs: endometrial stromal cells from endometrium of nonendometriotic individuals; EuESCs: endometrial stromal cells from endometrium of endometriotic patients; EESCs: endometrial stromal cells from ovarian endometrioma.

Figure 7.

mRNA expression of IL-8 in cultured CESCs, EuESCs and EESCs after treatment with 1.1 μM PP (pyrvinium pamoate) treatment for 72 hrs. Relative mRNA expression was measured before (a) and after drug treatment on isolated normal (b), eutopic (c) and ectopic (d) stromal cells from endometriotic and nonendometriotic subjects. *P < .05, **P < .01, and ***P < .001. CESCs: endometrial stromal cells from endometrium of nonendometriotic individuals; EuESCs: endometrial stromal cells from endometrium of endometriotic patients; EESCs: endometrial stromal cells from ovarian endometrioma.

Pyrvinium pamoate had no significant effect on mRNA expression of IL-6 and IL-8 in CESCs group (Figures 6(b) and 7(b)), but this drug significantly reduced mRNA expression of IL-6 in EuESCs (P < .01, Figure 6(c)) and EESCs (P < .01, Figure 6(d)) groups. Pyrvinium pamoate also lowered mRNA expression of IL-8 in EuESCs (P < .05, Figure 7(c)) and EESCs (P < .01, Figure 7(d)) groups.

Discussion

Endometriosis is a chronic gynecologic problem in which proinflammatory cytokines such as IL-6 and IL-8 are elevated in the peritoneal fluid.^8,9,29,30 and have pivotal role in its pathogenesis.^29,31,32 Immune cells in the peritoneal fluid and also eutopic and ectopic endometrial cells are main sources of IL-6 and IL-8.^16,33 Tomio Iwabe and his colleagues studied the role of IL-8 in the progression of endometriosis and found that this chemokine has proliferative and angiogenic effects on endometriotic stromal cells. This research also discovered the autocrine and paracrine action of IL-8, because of gene expression of IL-8 receptor type A in both endometrial and endometriotic cells.⁹ Another study showed that development and proliferation of in vitro cultured endometrial stromal cells could be induced by IL-8.³⁴ IL-6 in peritoneal fluid of patients with endometriomas can cause chronic pelvic pain and dysmenorrhea.³⁰ This cytokine can act as a trigger to initiate the inflammatory response through B and T-cell activation.³⁵

Wnt/β-catenin signaling pathway has been aberrantly activated in cancers and also endometriosis.^4,36 Given the crucial role of aberrant activation of this important signaling pathway in the pathogenesis of endometriosis,^4,5,37,38 targeting this cellular signaling pathway as an efficacious approach for treating this complex condition has been conducted in some studies.^4
–6,28 Due to adverse effects of current medications on reproductive function, novel compounds for treatment of endometriosis without impairing fertility of women with this problem have attracted much attention by researchers for further evaluation.^39,40

In the present study, for the first time the effect of pyrvinium pamoate on the mRNA expression of IL-6 and IL-8 and the secretion of these cytokines in cultured human endometrial cells isolated from both patients with and without endometriosis was investigated. We confirmed inhibitory effect of this compound on Wnt/β-catenin signaling through the measurement of phospho-β-catenin (Ser45) level in treated cells. Immunofluorescent staining demonstrated that the EESCs, EuESCs, and CESCs were positive for vimentin as a marker for cells with mesenchymal origin and no expression of cytokeratin (as an epithelial marker) was observed.

Wnt/β-catenin pathway regulate the secretion of IL-6 as an inflammatory cytokine.²³ According to the role of NF-κB and Wnt/β-catenin pathway in inflammation responses and also interaction between these two pathways,^{24,41

–45} suppression of these pathways could affect inflammatory responses and also secretion of IL-6 and IL-8 as pro-inflammatory cytokines. The expression of IL-6 and IL-8 were down-regulated by DKK-1 and LGK974 as Wnt/β-catenin pathway inhibitors.⁴⁶ Delbandi et al. reported that EESCs group secreted significantly higher levels of IL-6 and IL-8 compared to EuESCs, and CESCs groups,⁴⁷ which is in accordance with the findings of the our study.

In the current study pyrvinium pamoate decreased IL-6 production by EESCs, EuESCs, and CESCs groups at a dosage of 1.1 µM after 72 h treatment, but reduction of IL-8 production only observed in EESCs and EuESCs groups.

Li et al. showed that resveratrol, as a Wnt/β-catenin signaling pathway inhibitor with anti-proliferation and anti-inflammation effects⁴⁸ suppressed IL-6 secretion in osteoarthritis chondrocytes following induction with IL-1β.⁴⁹

Curcumin is an herbal compound which could suppress WNT/β-catenin and nuclear factor-κB (NF-κB) signaling pathways and also has anti-inflammatory properties.^50,51 Hidaka and his colleagues showed that after treatment of human pancreatic tumor cells with curcumin, IL-8 production was reduced.⁵² Curcumin also caused down-regulation of TNF-α-induced IL-6 gene expression in HaCaT keratinocytes.⁵³ Inhibitory effect of curcumin on IL-6 has been reported in several studies.^54

–57 Sutrisno et al. in their investigation demonstrated that genistein as an inhibitor of Wnt/β-catenin signaling pathway decreased high levels of IL-6 and IL-8 in mice model of endometriosis and in vitro cultured endometriotic cells.^58,59 Additionally, another study showed that artemisinin with anti-inflammatory properties could suppresses mRNA expression of IL-6 in chondrocytes extracted from patients with osteoarthritis through the interference with Wnt/β-catenin signaling pathway.⁶⁰ Delbandi et al. revealed that the 1,25(OH)2D3, a Wnt/β-catenin signaling pathway inhibitor,⁶¹ could suppress the IL-6 secretion by endometriotic stromal cells isolated from women with ovarian endometriosis, but this vitamin had no significant effect on IL-8 production.⁶² This finding is in line with our data which pyrvinium pamoate decreased the secretion of IL-6 and also IL-8 in EESCs group.

Apigenin, a natural flavonoid product with inhibitory effect on WNT/β-catenin signaling pathway, significantly repressed mRNA expression and also secretion of IL-6 in human esophagus cancer cell lines.⁶¹

Another research conducted by Sharma et al. demonstrated that plasma concentration and mRNA levels of IL-6 was diminished in sepsis-induced mice after treatment with iCRT3 via targeting Wnt/β-catenin signaling pathway.⁶³

CCN1 as an extracellular matrix (ECM) molecule enhanced IL-8 production by Beas2B cells (Human bronchial epithelial cell line) via Wnt/β-catenin signaling and cessation of this mechanism was observed after treatment with a Wnt pathway inhibitor, XAV.⁶⁴

In summary, our findings in the present research provided evidence indicating that pyrvinium pamoate, an inhibitor for WNT/β-catenin signaling pathway, attenuated mRNA expression and secretion of IL-6 and IL-8 in all treatment groups through suppressive effect on WNT/β-catenin signaling pathway. Furthermore, activation of this signaling pathway was observed in ectopic and eutopic endometrial stromal cells which was confirmed by quantification of phospho-β-catenin (Ser45). Our observations provide new information about the effect of a WNT/β-catenin signaling pathway inhibitor on behavior of endometriosis, but further investigations are required to clarify toxicity and effectiveness of this product for treatment before clinical application.

Footnotes

Authors’ note

This article has been extracted from the thesis written by Mr Amin Karamian at the School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran (Registration No:12).

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The present study was financially supported by a grant from the Biology and Anatomical Sciences Department of the School of Medicine, Shahid Beheshti University of Medical Sciences (Project No: 11104).

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