Resveratrol treatment reduces expression of MCP‐1, IL‐6, IL‐8 and RANTES in endometriotic stromal cells

Endometriosis is an inflammatory disease affecting reproductive‐aged women. Immunologic disturbance, as well as inflammation, have crucial roles in the pathogenesis of endometriosis. In this study, we evaluated the effects of resveratrol treatment on expression of monocyte chemotactic protein‐1 (MCP‐1), interleukin‐6 (IL‐6), IL‐8, and regulated upon activation, normal T cell expressed and secreted (RANTES) in endometrial stromal cells from patients with endometriosis compared with non‐endometriotic controls. Thirteen eutopic (EuESCs) and nine ectopic (EESCs) endometrial stromal cells from endometriotic patients as well as eleven endometrial stromal cells from non‐endometriotic controls (CESCs) were treated with resveratrol (100 μmol/L) or ethanol, and gene and/or protein expression of MCP‐1, IL‐6, IL‐8 and RANTES was examined at 6, 24 and 48 hours following treatment in the cells from all origins. Resveratrol treatment significantly reduced gene and protein expression of MCP‐1, IL‐6, and IL‐8 in EuESCs and EESCs compared with CESCs (P < .05‐.001, P < .05‐.001 and P < .05‐<.01, respectively), and this reduction was more noticeable in EESCs than EuESCs (P < .05‐<.001). Besides, resveratrol treatment significantly reduced RANTES protein expression in EESCs in all time intervals (P < .05). Resveratrol treatment significantly reduced the expression of MCP‐1, IL‐6, IL‐8 and RANTES in EESCs.


| INTRODUC TI ON
Endometriosis is an enigmatic gynaecological disease characterized by ectopic implantation of endometrial like tissues in extra-uterine sites. 1 It afflicts approximately 10% of reproductive-aged women, 2%-11% of asymptomatic women, and almost half of the women experiencing chronic pelvic pain and associated with dysmenorrhoea, dyspareunia, pelvic pain and infertility. 1,2 The pathogenesis of endometriosis is not precisely understood. 3 Among numerous theories proposed to elucidate the pathophysiology of endometriosis, peritoneal implantation of viable endometrial cells during retrograde menstruation is the generally accepted one. 4 However, retrograde menstruation occurs in almost all reproductive-aged women, but only 10%-20% of them develop endometriosis, so other factors must be involved in implantation and survival of the displaced endometrial cells. 5 Based on recent findings, immune dysregulation in the peritoneal microenvironment and chronic inflammation have crucial roles in endometriosis development 6 as increased levels of pro-inflammatory cytokines and chemokines such as interleukin-6 (IL-6), IL-8, monocyte chemotactic protein-1 (MCP-1), and regulated upon activation, normal T cell expressed and secreted (RANTES) have been detected in peritoneal fluid (PF) of endometriotic patients compared to non-endometriotic participants, suggesting that these secretory products may be involved in endometriosis initiation and progression through the promotion of growth, adhesion, invasion and proliferation of endometrial cells. [7][8][9] Current treatment modalities for endometriosis are surgical and hormonal treatments, but the high incidence of disease recurrence and side effects of these therapies limit their usage in a long period. 10 Thus, in recent years, there has been an increasing emphasis on finding naturally occurring compounds for the management of endometriosis.
Resveratrol (trans-3,5,4′-trihydroxystilbene), a nutraceutical found in significant amounts in red grapes, berries, peanuts and red wine, is one of these substances. 11 Protective effects of resveratrol on various diseases have been widely investigated in preclinical and clinical studies and attributed to anti-oxidative, anti-inflammatory, anti-tumorigenic, anti-atherogenic and anti-ageing properties of resveratrol. 12 The first animal study of the effect of resveratrol on endometriosis was reported by Bruner-Tran et al in 2011. 13 In that study, resveratrol treatment decreased the number and volume of endometriotic lesions in a nude mouse model of endometriosis. In subsequent studies, resveratrol treatment in animal models of endometriosis decreased the number and size of endometriotic implants and showed anti-inflammatory, anti-angiogenic, anti-proliferative, anti-oxidative, and pro-apoptotic activities 12 and in just one in vitro study resveratrol treatment showed anti-inflammatory effect through suppression of IL-8 expression in endometriotic stromal cells. 14 So in this study, for the first time, we sought to investigate and compare the effect of resveratrol treatment on MCP-1, IL-6, and IL-8 gene expression and protein production and RANTES protein expression in ectopic and eutopic endometrial stromal cells of endometriotic women (EESCs and EuESCs, respectively) and non-endometriotic control endometrial stromal cells (CESCs).

| Patient recruitment and tissue collection
This study included fifty-five patients admitted to gynaecology ward of Rassoul Akram hospital, who were allocated to two groups based on laparoscopy or hysterectomy findings: Group I (endometriosis group) consisted of forty women with stage III-IV peritoneal endometriosis, and group II (control group) consisted of fifteen women with benign gynaecological diseases and no evidence of endometriosis.
All women enrolled were at reproductive age (19-45 years old), with regular menstrual cycles, and were at the proliferative phase of the menstrual cycle. Those who had received hormonal treatment or antioxidant supplements within the last three months before sampling, or had the pelvic inflammatory disease, adenomyosis, malignancy, or were pregnant and breastfeeding were excluded. The diagnosis of endometriosis was initially evaluated by an expert clinician during laparoscopy and then confirmed by histopathological examination, and the severity of endometriosis was identified according to the revised American Society for Reproductive Medicine (rASRM). 15 Before enrolling in the study, informed consent was obtained from each woman using protocols approved by the Human Ethics Committees of the Iran University of Medical Sciences (Code: IR.IUMS.REC.1395.28108).
Ectopic and eutopic endometrial samples were obtained through laparoscopic sampling and biopsy curette, respectively. Endometrial tissues were collected in sterile Falcon tubes containing Dulbecco's modified Eagle's medium (DMEM)-F12 (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin antibiotics (Gibco, Grand Island, NY, USA) and immediately transported to the laboratory on ice, and a portion of tissue was sent to pathology for confirmation of endometriosis.

| Isolation, culture and purification of endometrial stromal cells (ESCs)
Isolation, culture and purification of ESCs described previously. 16 Briefly, fresh endometriotic tissue was minced to pieces of about

| Determining the appropriate concentration for treatment of ESCs with resveratrol
In a pilot study, various concentrations of resveratrol (Sigma-Aldrich) (12.5, 25, 50, 100, 200 and 400 µmol/L) were tested to find safety dose of resveratrol by MTT assay, 17 based on MTT assay findings, 16 Table 1. A total reaction system of 20 μL contained syber premix Extaq (Biofact, Daejeon, Korea), 10 μL; primer pairs, 1 μL; cDNA template, 1 μL; and DNase-free water, 8 μL. Glyceraldehyde-3phosphate dehydrogenase (GADPH) was used as a housekeeping gene to normalize the amount of total RNA present in each reaction. Thermocycler conditions included an initial holding at 95°C for 15 minutes, followed by 40 cycles of 95°C for 20 seconds, annealing and elongation for 40 seconds and the melting step was from 60° to 99°C. Melting curve analyses were performed after amplification cycles to ensure the purity and specificity of the products. The efficiency of qRT-PCR reaction was determined by the standard curve, which was derived from serial dilutions of cDNA and qRT-PCR product in triplicate. The PCR amplification efficiency of these candidate genes ranged from 95% to 97%, and the regression coefficient (R 2 value) of the standard curve ranged from 0.968 to 0.998, well within the acceptable range of qRT-PCR. 21

| Statistical analysis
The statistical analysis of data was carried out using the GraphPad

| The basal gene/protein expression levels of MCP-1, IL-6, IL-8 and RANTES in ESCs
Based on our findings, the basal gene and protein expression of MCP-1, IL-6 and IL-8 were significantly higher in EESCs compared with EuESCs and CESCs (P < .01-<.0001; P < .0001; and P < .05-<.001, respectively). RANTES protein expression was also higher in EESCs compared with CESCs, but the difference was not significant ( Figure 1).

| The effect of resveratrol treatment on MCP-1 gene and protein expression in ESCs
As shown in Figure Figure 2D, the reducing effect of resveratrol treatment on MCP-1 protein expression was more remarkable in EESCs than EuESCs at 6 and 48 hours (P < .01).

| The effect of resveratrol treatment on IL-6 gene and protein expression in ESCs
As shown in Figure 3A, resveratrol treatment reduced IL-6 gene expression in EESCs at 6, 24 and 48 hours (P < .05-<.01) and in EuESCs at 24 and 48 hours (P < .05) and had no effect on CESCs.
The effect of resveratrol treatment on IL-6 gene expression reduction was more significant in EESCs compared with EuESCs in all time intervals (P < .05-<.01; Figure 3C). However, the differential effect of resveratrol treatment on IL-6 protein expression reduction was not statistically significant between EuESCs and EESCs at 6, 24 and 48 hours ( Figure 3D).

| The effect of resveratrol treatment on IL-8 gene and protein expression in ESCs
Resveratrol treatment reduced IL-8 gene expression in EESCs at 6, 24 and 48 hours (P < .05-<.01) and in EuESCs at 6 and 24 hours (P < .05-<.01) and had no effect on CESCs ( Figure 4A). Besides,
The effect of resveratrol treatment on IL-8 gene expression reduction was more significant in EESCs compared with EuESCs at all time intervals (P < .05-<.001, Figure 4C), although the differential effect of resveratrol treatment on IL-8 protein expression reduction was only significant at 48 hours in EESCs compared with EuESCs (P < .05, Figure 4D).

| The effect of resveratrol treatment on RANTES protein expression in ESCs
Resveratrol treatment reduced RANTES protein expression in EESCs at 6, 24 and 48 hours (P < .05) and had no effect on EuESCs and CESCs ( Figure 5).

| D ISCUSS I ON
We demonstrated in this study that EESCs express higher levels of MCP-1, IL-6 and IL-8 under basal conditions than EuESCs and CESCs.

RANTES protein expression was also higher in EESCs than EuESCs
and CESCs, but the difference was not significant. To the best of our knowledge, only one study compared the in vitro production on an experimental rat model of endometriosis. 46 Resveratrol treatment in that study significantly reduced plasma and PF levels of IL-6 compared to control. 46 In our study for the first time, resveratrol treatment reduced gene and protein expression of IL-6 in EuESCs and EESCs and had no effect on CESCs.
IL-8 is a chemokine with potent neutrophil and T cell chemotactic activities. 47 Monocytes, macrophages, 48 eutopic and ectopic endometrial stromal cells are principal sources of IL-8. 23 Inflammatory cytokines like IL-1, tumour necrosis factor-α (TNF-α) and LPS can also affect the release of this chemokine. 49,50 Many studies have pointed to increased PF levels of IL-8 and its correlation with disease stage. 51 Besides consistent with our findings, other studies have shown increased IL-8 production by EESCs than EuESCs or CESCs. 23  production. 61 Based on the literature, eutopic endometrium of endometriotic patients express more COX-2 than disease-free women, and COX-2 protein is highly expressed in ectopic than eutopic endometrium in endometriotic women. 62

| CON CLUS ION
Our results showed that EESCs differed significantly from EuESCs

ACK N OWLED G EM ENTS
We would like to appreciate all patients who participated in this research. This work was supported by Iran University of Medical Sciences (grant number 28108).

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data supporting the findings of this study are available from the corresponding author on request.