Changes in MCP‐1, HGF, and IGF‐1 expression in endometrial stromal cells, PBMCs, and PFMCs of endometriotic women following 1,25(OH)2D3 treatment

Abstract 1,25(OH)2D3 has anti‐inflammatory and growth inhibitory effects. Our study explored the effect of 1,25(OH)2D3 treatment on the expression of monocyte chemotactic protein‐1 (MCP‐1), hepatocyte growth factor (HGF), and insulin‐like growth factor‐1 (IGF‐1) by peripheral blood mononuclear cells (PBMCs), peritoneal fluid mononuclear cells (PFMCs), endometrial stromal cells (ESCs), and its effect on the proliferation of PBMCs and PFMCs of patients with endometriosis compared with controls. PBMCs, PFMCs, and ESCs were obtained from 10 endometriosis patients and 10 non‐endometriotic individuals. After treating cells with 0.1 μM of 1,25(OH)2D3 for 6, 24, and 48 h, the gene and protein expression of mentioned factors were evaluated by real‐time PCR and ELISA methods, respectively. 1,25(OH)2D3 treatment significantly reduced the protein expression of MCP‐1, HGF, and IGF‐1 in PBMCs and PFMCs of endometriotic patients at 48 h (p < 0.05–<0.01). Also, this treatment significantly reduced MCP‐1, HGF, and IGF‐1 gene and/or protein expression in EESCs and EuESCs at 24 and 48 h (p < 0.05–<0.01). 1,25(OH)2D3 treatment also reduced the proliferation of PBMCs and PFMCs of endometriotic patients compared with controls (p < 0.01). 1,25(OH)2D3 can be considered as a potentially effective agent in the prevention and treatment of endometriosis along with other therapies.

We also recently showed increased MCP-1, HGF, and IGF-1 serum and PF levels in endometriotic patients compared with controls. 11 MCP-1 affects endometriosis development by promoting proliferation and activating and recruiting mononuclear cells to secrete growth factors and cytokines. 12,13 HGF as a pleiotropic growth factor, can be involved in endometriosis development via its mitogenic, angiogenic, motogenic (migration), and morphogenic activities, 14 and IGF-1, as another growth factor, exerts its effect on endometriosis development by stimulating the growth and preventing apoptosis of endometrial cells. 15,16 Despite decades of research, treatment of endometriosis is currently limited to hormonal therapy or surgery which are non-curative and frequently lead to endometriosis recurrence after cessation of treatment. 17 Thus, recently, there has been an increasing emphasis on finding naturally occurring compounds for managing endometriosis.
One of these compounds which has considerable anti-inflammatory, anti-proliferative, and even immunomodulatory effects, is vitamin D. 18 Studies indicate that vitamin D influences women's reproductive health. Ectopic endometrium in endometriotic women, as well as endometriotic stromal cells, were shown to express 1alpha-hydroxylase (1α-OHase), which activates 25-hydroxyvitamin D3 (25(OH)D3). 19 A recent systematic review and meta-analysis showed lower 25(OH)D3 serum levels in endometriosis women compared with controls. Also, a negative correlation between vitamin D levels and the severity of endometriosis was observed in that study. 20 We also showed lower serum and PF levels of 25(OH)D3 in the patients with endometriosis compared with controls. 21 Regarding 1, (1,25(OH)2D3) serum concentration results were controversial. 22 The genomic pathway responsible for vitamin D activity is regulated by vitamin D receptor (VDR), which is expressed in many tissues and numerous tumours. 23 Based on Agic et al.'s findings, VDR expression in the endometrium of endometriotic women lay between the level seen in ovarian cancer and control groups. 24 Vitamin D can regulate the entire process of tumorigenesis through mechanisms such as proliferation, differentiation, apoptosis, migration, invasion, inflammation, and oxidative stress, 25 and based on findings of a recent systematic review, adequate vitamin D levels were associated with a lower risk of ovarian cancer and reduced cancer mortality in the general population. 26 Besides, recent studies pointed to the protective effects of vitamin D on endometriotic lesions 27,28 and also endometriotic stromal cells. 29,30 Considering the importance of MCP-1, HGF, and IGF-1 in the development of endometriosis and the inhibitory effect of vitamin

| Sample collection
Peripheral blood samples were collected in ethylenediaminetetraacetic acid (EDTA) Falcon tubes under sterile conditions from both groups before the administration of general anaesthesia and PF samples were collected before any operative manipulation to minimize blood contamination.
Ectopic lesions and eutopic endometrial tissues were collected from the same participant (endometriotic patient) using laparoscopy and biopsy curette, respectively. Eutopic endometrium also was collected from non-endometriotic women. In the case of vir-

| Enzyme-linked immunoassay (ELISA) procedure
The concentrations of MCP-1, HGF, and IGF-1 proteins were measured in PBMCs, PFMCs, and ESCs supernatant by a standard ELISA kit (Duoset; R&D Systems) based on the manufacturer's protocol.
The absorbance was measured at 570 nm by a microplate reader TA B L E 1 The MCP-1, HGF, IGF-1, and GAPDH primers sequences.

| Carboxyfluorescein diacetate, succinimidyl ester (CFSE)
Cell proliferation was evaluated using CFSE assay. Briefly, PBMCs and PFMCs were stained with 5 μM CFSE in 1 ml phosphatebuffered saline (PBS; Biolegend) for 20 min in a CO 2 incubator at 37°C and 5% CO 2 (keep protected from the light). For quenching the staining and removal of the remaining free dye, cells were incubated with 5 ml complete growth medium for 10 min at 37°C and 5% CO 2 . The cells were washed three times and resuspended in the complete growth medium, then treated with 1,25(OH)2D3.
After 72 h, cells were stimulated with ionomycin (1 μg/ml) and PMA (50 ng/ml; Sigma-Aldrich). Five days later, PBMCs and PFMCs were harvested and cell proliferation was analysed on BD FACSCalibur.
The flow cytometry data were analysed using the software FlowJo (Tree Star version 10.1r5 Inc.).

| Statistical analysis
Statistical analyses were performed using the GraphPad Prism software 8 (GraphPad Software, Inc.). Kolmogorov-Smirnov test was applied to assess the normal distribution of data. All data were analysed using the non-parametric tests, including the Wilcoxon signed-rank test, Mann-Whitney, and Kruskal-Wallis tests. The mRNA expression analysis was performed using the 2 −ΔΔCt method.
A p-value of <0.05 was considered statistically significant.

| The effect of 1,25(OH)2D3 treatment on cell proliferation
Data obtained from the CFSE assay showed that 1,25(OH)2D3 can reduce the proliferation of PBMCs and PFMCs of endometriosis patients (p < 0.01, for both cell types; Figure 4).

| DISCUSS ION
In this study, 1,25(OH)2D3 treatment significantly reduced the HGF, also known as the scatter factor, is an important growth factor related to endometriosis. 14 The HGF receptor is the c-met proto-oncogene product (c-Met). 14 Peritoneal macrophages appear to be the major source of most endometriosis-related cytokines.
But regarding HGF, the peritoneum and endometriotic stromal cells seem to be primary production sources of this cytokine in endometriosis. 46 Inflammatory cytokines such as IL-6, LPS, and prostaglandins known as HGF inducers stimulate HGF production in the pelvic cavity of patients with endometriosis. 46 We recently showed increased HGF gene and protein expression in PFMCs of endometriotic patients compared with controls. 11 Overexpression of HGF in ectopic lesions compared with eutopic endometrium of endometriotic patients has been shown in one study. 47  IGF-1, which is synthesized by different cells, such as endometrial cells, prevents apoptosis and acts as a mitogen on ESCs cultured in vitro. 15,16 Studies on the association between IGF-1 and endometriosis were inconsistent, but according to a recent prospective study, IGF-1 and IGFBP-3 were associated with a higher risk of endometriosis among younger women. 54 Furthermore, it has been previously reported that endometriotic cysts significantly expressed lower levels of IGF-I, both at the mRNA and protein levels compared with eutopic endometrium. 55,56 Contrary to these findings, in a study by Zhou et al. IGF-I and IGF-1R were expressed both in paired eutopic endometrium and ovarian endometrioma tissues. 57 In addition, in that study, mRNA levels of IGF-I, but not IGF-1R, in EESCs were significantly higher than those in EuESCs. 57 In line with these results, we recently showed higher gene and/or protein expression of Previously, we showed that the rate of the proliferation of EESCs and EuESCs significantly decreased after treatment with 1,25(OH)2D3 in the fibronectin-coated plate. 29 In the present study,

ACK N OWLED G EM ENTS
We thank all the participants in the present study. This work was supported by a grant from Iran University of Medical Sciences with the grant number 1394.26098.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Ali-Akbar Delbandi https://orcid.org/0000-0003-0012-9333