Phorbol 12-myristate 13-acetate (PMA; cat. no. P8139) and lipopolysaccharide (LPS; cat. no. L2630) were purchased from Sigma-Aldrich (Merck KGaA). The Luminex cytokine panel kit (cat. no. LXSAHM) was purchased from R&D Systems, Inc. Rabbit PRMT5 antibody (cat. no. 18436-1-AP) was purchased from ProteinTech Group, Inc. IL-6 (cat. no. BMS213-2) and IFN-γ-induced protein 10 (IP-10) ELISA kits (cat. no. KAC2361) were purchased from Thermo Fisher Scientific, Inc. SB431542 (cat. no. S1067), which is used as an inhibitor of Smad 2/3(16), was purchased from Selleck Chemicals. NF-κB inhibitor SN50 (cat. no. SML1471) and PRMT5 inhibitor EPZ015666 were obtained from MedChemExpress.

A total of 25 female patients with ovarian cysts or infertility were enrolled from the Department of Obstetrics and Gynecology of The Second Xiangya Hospital of Central South University (Changsha, China) between December 2018 and October 2019. These patients were diagnosed with endometriosis by laparoscopic and histopathological examination. According to the revised American Fertility Society endometriosis classification, the study population included 2 stage I-II cases, 7 stage III cases and 16 stage IV cases (17). Of these, 20 cases were in the proliferative phase and 5 cases were in the secretory phase. The control group consisted of 12 patients who were diagnosed with cervical neoplasia or uterus septum and underwent laparoscopy, during which no endometrial lesions were observed in the pelvic cavity. All patients (aged 20-45 years) had regular periods and did not receive hormone therapy within 3 months prior to surgery. There were no significant differences in age, pregnancy, gravidity or proliferative vs. secretory phase between the two groups (Table I).

During surgery, endometrial biopsies were collected via excision under sterile conditions from the control and research groups, and they were promptly transported to the laboratory on ice in PBS. A portion of each tissue sample was fixed in 10% formalin solution for 12 h at room temperature. and then processed for histological examination to exclude pathological abnormalities. Hematoxylin-eosin staining was performed for histological examination, and the staining was completed according to the a previously described protocol (18).

The present study was approved by the Ethics Committee of The Second Xiangya Hospital of Central South University (Changsha, China). Written informed consent was obtained from all participants.

During the laparoscopy, the peritoneal cavity was rinsed with 50 ml normal saline solution, and cell pellets were collected via centrifugation at 376 x g for 5 min at 4˚C. Monocytes were isolated following a standard protocol of Ficoll density gradient centrifugation (19-21). Briefly, Ficoll-loaded samples were centrifuged at 455 x g or 25 min at 20˚C. The buffy coat layer containing cells was then harvested and washed three times with PBS followed by centrifugation at 35 x g for 10 min at 20˚C to obtain monocytes. The monocytes were then stored at -80˚C until multiple samples were collected before performing subsequent reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses.

Samples of 5 ml peripheral blood were collected from each patient into a coagulation tube. The serum was separated by centrifugation at 845 x g for 5 min at 4˚C and then stored at -80˚C until further use.

Endometrial homogenates were prepared as described previously (22). Briefly, endometrial tissues were excised during laparoscopy. A total of ~200 mg tissue was minced and homogenized in 5 ml RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.), followed by centrifugation at 9,391 x g for 10 min at 4˚C. The supernatant was collected, and protein concentration was determined using the BCA method. The samples were then stored at -80˚C until further use.

THP-1 cells were generously provided by Dr Joseph Huang (University of South Florida). The cells were grown in RPMI-1640 supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin and 1% streptomycin at 37˚C in a humidified atmosphere with 5% CO₂. THP-1 cells were differentiated into macrophage-like cells through treatment with 25 ng/ml PMA for 3 days at 37˚C in a humidified incubator with 5% CO₂. THP-1-derived macrophage-like cells were pretreated with 100 ng/ml LPS for 24 h at 37˚C in a humidified incubator with 5% CO₂ and then cultured in RPMI-1640 containing 10% serum from controls and patients with endometriosis for 6 days or cultured in homogenized solution of endometrium from controls and eutopic endometrium from patients with endometriosis (100 µl homogenized solution/ml medium) for 3 days. The inhibitors SN50 (50 µg/ml; treated for 1 h), SB431542 (10 µM; treated for 24 h) and EPZ015666 (10 µM; treated for 24 h) were added to the medium before treatment with the homogenized solution.

THP-1 cells and the isolated peritoneal monocytes were washed with cold PBS and lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology; cat. no. P0013B) on ice. Cell lysates were collected by centrifugation at 13,523 x g for 10 min at 4˚C, after which the supernatant was obtained and the protein concentration was determined using a BCA assay kit (Beyotime Institute of Biotechnology; cat. no. P0010S). Samples were then heated at 95˚C for 5 min in loading buffer. Protein samples (10 µg) were separated via 10% SDS-PAGE, transferred to a 0.2-µm nitrocellulose membrane, blocked with 5% nonfat milk in TBS containing 0.1% Tween-20 (TBST) for 2 h at room temperature, and incubated overnight with primary antibodies against PRMT5 (1:1,000) and β-actin (ProteinTech Group, Inc.; cat. no. 20536-1-AP; 1:10,000; internal control) at 4˚C. The membranes were then washed with TBST three times, incubated with HRP-Goat Anti-Rabbit IgG (H+L) (ProteinTech Group, Inc.; cat.no. SA00001-2; 1:5,000) for 1 h at room temperature, and then washed again with TBST three times. The chemiluminescence signal, visualized using SuperSignal West Pico PLUS (Thermo Fisher Scientific, Inc.; cat. no. 34580) was detected using the ChemiScope 5300 chemiluminescence system (Clinx Science Instruments Co. Ltd.) and quantified using Quantity One software (version 4.6.6; Bio-Rad Laboratories, Inc.).

Total RNA was extracted from THP-1 cells and the isolated peritoneal monocytes using TRIzol^® reagent (Thermo Fisher Scientific, Inc.; cat. no. 15596026). cDNA was synthesized using the PrimeScript RT reagent kit with gDNA Eraser (Perfect Real Time; Takara, cat. no. RR047A) from 1 µg total RNA in accordance with the manufacturer's protocol. Relative gene expression levels were determined using the SYBR green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific; cat. no. 4309155) using the 2^-ΔΔCq method (23) with normalization to β-actin expression. The thermocycling conditions were as follows: Initial denaturation at 95^°C for 30 sec, followed by 40 cycles of 95^°C for 5 sec, 60^°C for 20 sec, 95^°C for 1 sec, 65^°C for 15 sec and 95^°C for 1 sec for the dissociation curve. The primer sequences were as follows: PRMT5 forward, 5'-GGTGAACGCTTCCCTG-3' and reverse, 5'-TGAGACTACGGTCACTTGG-3'; β-actin forward, 5'-GAGCGCGGCTACAGCTT-3' and reverse, 5'-TCCTTAATGTCACGCACGCACGATTT-3'.

After being treated as aforementioned, THP-1 cells with eutopic endometrial tissue extract from patients with endometriosis and the PRMT5 inhibitor EPZ015666, IL-6 and IP-10 levels in these cells were measured using ELISA kits (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

TNF-α, IL-6, IL-10 and C-C motif chemokine 20 (CCL20) levels in endometrial extracts from patients with endometriosis and control individuals were determined using the Luminex cytokine kit (R&D Systems, Inc.) according to the manufacturer's instructions. Briefly, frozen endometrial extracts were homogenized in RIPA lysis buffer [Beyotime Institute of Biotechnology; cat. no. P0013B; 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate] with protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). Total protein levels were measured using the BCA method. TNF-α, IL-6, IL-10 and CCL20 levels were measured using a Luminex 200 analyzer (Luminex Corporation). The ratio of cytokine concentration to total protein concentration (pg/ml/mg protein) was used to determine differences between the two groups.

All quantitative data are presented as the mean ± SD. Statistical analysis was performed using SPSS software (version 17.0; SPSS, Inc.) and visualized using GraphPad Prism software (version 8.0; GraphPad Software, Inc.). In vitro cell experiments were repeated three times per sample, and the average values were used. Quantitative data were firstly examined for normal distribution using the Kolmogorov-Smirnov test, and data with equal variances were compared using one-way ANOVA followed by the Least Significant Difference test for comparisons among three groups, while unpaired t-test was used for comparisons between two groups. Alternatively, Mann-Whitney U tests were used for comparisons. Categorical variables were compared using the χ² test. The Fisher's exact test was used to analyze the menstrual cycle variable in Table I. P<0.05 was considered to indicate a statistically significant difference.

To investigate the differential expression of cytokines in patients with endometriosis and control individuals, the Luminex assay was used to compare the levels of TNF-α, IL-6, IL-10 and CCL20 in endometrial tissue extracts from these patients. The results demonstrated that the endometrial extracts of patients with endometriosis contained significantly higher levels of TNF-α, IL-6 and IL-10 and a decreased level of CCL20 compared with the endometrial extracts of control individuals (Fig. 1), suggesting that there are differences in the expression of TNF-α, IL-6, IL-10 and CCL20 that may contribute to the inflammatory response under pathological conditions.

To investigate the possible role of macrophage-derived PRMT5 in endometriosis development, the expression pattern of PRMT5 in THP-1 cells treated with serum from controls and patients with endometriosis in the presence of LPS was examined. As indicated in Fig. 2, both the mRNA and protein levels of PRMT5 were significantly decreased in THP-1 cells treated with serum from patients with endometriosis, compared with the levels in cells treated with serum from controls. These results revealed that PRMT5 expression in macrophages could be downregulated in the context of endometriosis, suggesting a potential role for macrophage-derived PRMT5 in the pathogenesis of endometriosis.

To further confirm the possible involvement of macrophage-expressed PRMT5 in endometriosis, PRMT5 expression was examined in THP-1 cells treated with extracts of normal and eutopic endometrium in the presence of LPS. As presented in Fig. 3, the extracts of normal endometrium induced a substantial increase in PRMT5 expression in THP-1 cells. However, treatment with extracts of eutopic endometrium significantly inhibited PRMT5 mRNA and protein expression in macrophages compared with extracts of normal endometrium, suggesting that macrophage-derived PRMT5 may serve a role in the interaction between macrophages and the eutopic endometrial microenvironment, which likely contributes to the inflammatory response under pathological conditions.

To investigate the mechanisms underlying macrophage PRMT5 expression in endometriosis, the NF-κB inhibitor SN50 or the Smad2/3 inhibitor SB431542 was added to cells together with homogenized endometrial tissue to examine PRMT5 expression in THP-1 cells in the presence of LPS. The results revealed that SB431542 significantly inhibited the mRNA expression of PRMT5 in cells treated with extracts of normal endometrium. However, SB431542 treatment demonstrated no inhibition of PRMT5 protein expression in cells treated with extracts of normal endometrium. Additionally, no inhibition was demonstrated in cells treated with extracts of eutopic endometrium at both the mRNA and protein level. The results demonstrated that SN50, but not SB431542, markedly inhibited PRMT5 mRNA and protein expression in THP-1 cells treated with extracts of both normal and eutopic endometrium (Fig. 4). The results indicated that the endometrial microenvironment regulated macrophage PRMT5 expression through NF-κB signaling, but not Smad2/3 signaling.

To examine the function of PRMT5 in the interaction between macrophages and the endometrial microenvironment, THP-1 cells were treated with eutopic endometrial tissue extract from patients with endometriosis containing the PRMT5 inhibitor EPZ015666. As presented in Fig. 5, EPZ015666 significantly inhibited IL-6 secretion and promoted IP-10 secretion from THP-1 cells activated by eutopic endometrial tissue extract. These data indicated that PRMT5 is essential for endometrial tissue extract-induced macrophage activation, suggesting an important role for PRMT5 in mediating the interaction between macrophages and the endometrial microenvironment.

To further investigate the possible role of PRMT5 expression in macrophages in endometriosis, peritoneal monocytes were collected from controls and patients with endometriosis for measuring PRMT5 expression. Previous studies have indicated that among the isolated CD68⁺ monocytes in peritoneal fluid, 85-95% are macrophages (20,21). As demonstrated in Fig. 6, the protein levels of PRMT5 were significantly increased in the peritoneal monocytes from patients with endometriosis, compared with those in the peritoneal monocytes from controls. However, while the mRNA levels of PRMT5 appeared to also be increased, the difference was not statistically significant.