The effects of resveratrol on the expression of VEGF, TGF-β, and MMP-9 in endometrial stromal cells of women with endometriosis

Resveratrol is a phytochemical with anti-angiogenic, anti-inflammatory, and antioxidant properties. The present study has evaluated the effect of resveratrol on the expression of vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and matrix metalloproteinase-9 (MMP-9) as factors related to endometriosis progression. Thirteen eutopic (EuESCs) and 8 ectopic (EESCs) endometrial stromal cells from women with endometriosis and 11 control endometrial stromal cells (CESCs) were treated with resveratrol (100 µM) for 6, 24 and 48 h. The gene and protein expression levels of VEGF, TGF-β, and MMP-9 were measured using real-time PCR and ELISA methods, respectively. Results showed that the basal gene and protein expression of VEGF and MMP-9 were higher in EESCs compared to EuESCs and CESCs (P < 0.01 to  < 0.001 and P < 0.05 to  < 0.01 respectively). Also, resveratrol treatment decreased the gene and protein expression of VEGF and MMP-9 in EuESCs, EESCs and CESCs (P < 0.05 to  < 0.01 and P < 0.05 to  < 0.01 respectively) and gene and protein expression of TGF-β in EESCs and EuESCs (P < 0.05 to  < 0.01). The effect of resveratrol in reduction of VEGF gene expression was statistically more noticeable in EESCs compared to EuESCs and CESCs (P < 0.05). According to the findings, resveratrol may ameliorate endometriosis progression through reducing the expression of VEGF, TGF-β, and MMP-9 in endometrial stromal cells (ESCs).


Scientific Reports
| (2021) 11:6054 | https://doi.org/10.1038/s41598-021-85512-y www.nature.com/scientificreports/ in the sterile condition, ectopic and eutopic endometrial tissues from endometriotic women and normal endometrial tissues from the control women were minced into smaller pieces and digested in the presence of 2 mg/ ml Collagenase A (Roche, Pleasanton, CA, USA) and 300 mg/ml DNase (Roche, Pleasanton, CA, USA). Then the obtained cells were cultured in T25 culture flasks (SPL Life Sciences, Korea), ans stored in an atmosphere of 5% CO2 at 37 °C in DMEM-F12 (Sigma-Aldrich, St. Louis, MO, USA) containing 1% Penicillin-Streptomycin antibiotics (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and adherent stromal cells were allowed to multiply. The cultured cells were passaged three times and when they reached to about 80% confluency they were used for the treatment. Some tissue samples especially ectopic tissues were excluded due to the cultural contamination, inproper pathology results, or not obtained the desired cells. At the end, from 40 endometriotic and 15 nonendometriotic control tissues, 8 ectopic, 13 eutopic, and 11 control tissues were treated. The purification of the ESCs was approved by immunofluorescent staining and flow cytometry. These cells were characterized as a panel of vimentin + , nestin + , cytokeratin − , CD10 + , CD44 + , CD73 + , CD105 + , CD34 − , and CD45 − cells. as we described earlier 23 . Based  Statistical analysis. Statistical analyses were carried out using GraphPad Prism software 6.01 (GraphPad Software. Inc). Based on the results of the Kolmogorov-Smirnov test, all data was analyzed using the non-parametric tests, including the Wilcoxon sign-ranked test, Mann-Whitney, and Kruskal-Wallis tests. For the gene expression analysis, the fold change and relative expression were compared by calculating the 2 −∆Δct and 2 −∆ct , respectively. P-value < 0.05 was considered as the statistically significant level.

Results
The Basal expression of VEGF gene and protein in ESCs. Based on the results of real-time PCR, the VEGF gene was expressed significantly more in EESCs compared to EuESCs and CESCs (Both P < 0.01) in the basic state (Fig. 1a). Moreover, according to the results of ELISA, the VEGF protein had significantly higher expression in EESCs compared to EuESCs and CESCs (P < 0.01 and P < 0.001 respectively) (Fig. 1b). and ELISA the basal gene and protein expression of TGF-β had no statistically significant differences among EESCs, EuESCs and CESCs (Fig. 1c,d). Resveratrol decreased the expression of VEGF gene in all ESCs. The real-time PCR method demonstrated that treatment with resveratrol (100 µM) reduced the expression of VEGF gene significantly in EESCs at 24 (P < 0.05) and 48 h (P < 0.01) and in EuESCs and CESCs only at 48 h (Both P < 0.05) ( Table 2). Also, the effect of 100 µM resveratrol treatment was more noticeable in EESCs in comparison with EuESCs and CESCs at 48 h (Both P < 0.05) (Supplementary file).

Resveratrol decreased the expression of TGF-β gene in EuESCs and EESCs.
The TGF-β gene expression had significant reduction by treatment with resveratrol (100 µM) in EuESCs and EESCs at 48 h (P < 0.05 and P = 0.01 respectively). The TGF-β gene expression had no significant changes in EuESCs and EESCs at 6 and 24 h, and in CESCs at all three time intervals (Table 2). There was no significant difference in the effect of resveratrol treatment between EESCs and EuESCs at 48 h (Supplementary file).
Resveratrol decreased the expression of MMP-9 gene in all ESCs. The MMP-9 gene expression was significantly reduced by resveratrol (100 µM) in EuESCs at 24 (P < 0.01) and 48 h (P < 0.05) and in EESCs and CESCs at 48 h (Both P < 0.05). The gene expression of MMP-9 did not show significant changes in EuESCs at 6 h, and in EESCs and CESCs at 6 and 24 h ( Table 2). In addition, resveratrol had a greater effect on EESCs compared with EuESCs and CESCs at 48 h, but this was not statistically significant (Supplementary file).

Resveratrol decreased the expression of VEGF protein in EuESCs and
EESCs. The use of ELISA method revealed that the protein expression of VEGF was significantly reduced in EuESCs and EESCs at 48 h by www.nature.com/scientificreports/ 100 µM resveratrol (P < 0.05 and P < 0.01 respectively). Resveratrol treatment had no significant effect at 6 and 24 h in these cells. The VEGF protein expression did not change significantly in CESCs at any of the treatment times (Fig. 2). Although, the effect of resveratrol treatment on reducing VEGF protein expression in EESCs was greater than that of EuESCs, this difference was not statistically significant. (Supplementary file).

Resveratrol decreased the expression of TGF-β protein in EuESCs and
EESCs. The effect of treatment with 100 µM resveratrol on the expression of TGF-β protein was the same as its gene expression. Resveratrol could reduce the expression of this factor in EuESCs and EESCs at 48 (Both P < 0.05). The expression of TGF-β protein showed no significant changes at 6 and 24 h in EuESCs and EESCs, and at any treatment times in CESCs (Fig. 3). The effect of resveratrol treatment between EESCs and EuESCs had no significant difference at 48 h (Supplementary file).
Resveratrol decreased the expression of MMP-9 in all ESCs. Resveratrol (100 µM) decreased significantly the MMP-9 protein in EuESCs, at 24 and 48 h and in EESCs and CESCs at 48 h (All P < 0.05). The MMP-9 protein production had no significant changes at 6 in EuESCs at 6 and 24 h in EESCs and CESCs (Fig. 4).
In addition, the effect of treatment with 100 µM resveratrol at 48 h on the reduction of MMP-9 protein production was not statistically significant among three groups (Supplementary file).

Discussion
The results revealed that the basal expression of VEGF and MMP-9, but not TGF-β in EESCs were significantly higher compared to EuESCs and CESCs. To date, some studies assessed the concentration of these factors in PF or endometrial implants of the endometriotic patients 21,33-35 and according to our knowledge, the present study is the first to compare the expression of VEGF, TGF-β and MMP-9 in EESCs, EuESCs, and CESCs. The findings of the previous studies, consistent with the present study, have shown that VEGF expression in endometrial tissue and PF of patients with endometriosis is increased compared to controls, although, it does not differ significantly between the different stages of the disease 33,36 . In the only discordant study, the VEGF concentration in PF of patients with genital endometriosis and healthy control women was not significantly different 34 . VEGF receptors gene expression was also higher in ectopic endometrial lesions than in eutopic tissue 21,37 .
VEGF is one of the most important angiogenic factors in endometriosis. It can increase cell proliferation, cell migration, and vascular permeability 13,38 . The most important cells secreting this factor in endometriosis are eutopic and ectopic stromal cells, peritoneal macrophages, and neutrophils that increase the expression of this factor in response to elevated inflammatory conditions 13 . Increased levels of reactive oxygen species (ROS) due to oxidative stress in endometriosis can also increase VEGF expression and its angiogenesis in in-vivo and in-vitro 39 .
The few studies that have examined the expression of TGF-β in endometriosis have shown contradictory findings. For example, in a study of Sokolov et al. the concentration of TGF-β in PF did not differ significantly between the women with genital endometriosis and healthy controls 34 , but two other studies, showed that levels of TGF-β in serum and PF were higher in patients than in controls, and this level, especially in PF, increased with increasing severity of the disease 35,40 .  13 . Increased expression of this factor in endometriosis seems to occur in response to increased inflammatory conditions and oxidative stress in the peritoneal cavity 42 . As is evident in our study, there was no significant difference in TGF-β expression in the stromal cells of the study groups. Previous studies reported that peritoneal mesothelial cells are the most important source of this factor in peritoneum-related diseases such as peritoneal endometriosis, followed by peritoneal macrophages, ectopic endometrial tissue including ESCs 17 , it appears that the increased expression of this factor in the serum and PF of patients with endometriosis than in controls has been reported in some previous studies 35,40 , may be due to the increased production of this cytokine by peritoneal mesothelial cells and then other sources and in the meantime, the ESCs evaluated in the present study, have less role in the production of this factor. Previous studies have also shown that the concentration of TGF-β in the peritoneum of individuals with endometriosis changes during the menstrual cycle and its highest concentration is seen in the secretory phase and in the premenstrual phase 18,41 . However, in our study, we measured the expression of TGF-β in the proliferative phase.
In the case of MMP-9, the only study comparing the expression of this factor in ectopic endometrial lesions with eutopic endometrium is Machado's study on an induced model of endometriosis in rats and reported findings consistent with the present study 21 .
Studies have shown that chronic inflammation increases MMP-9 expression. Expression of MMP-9 by EESCs and EuESCs increases in endometriosis in response to inflammatory conditions in the peritoneal cavity, which is higher in ectopic than eutopic lesions and activation of the NF-κB and MAP-kinase signaling and other www.nature.com/scientificreports/ inflammatory pathways, as well as to increased oxidative stress 43,44 ; This assists the replacement, growth, and invasion of endometriotic implants 20,21 . Increased production and activity of MMP-9 increase the degradation and regeneration of extracellular matrix, angiogenesis, and VEGF secretion 19,45 .
The present study revealed that the gene and protein expression of VEGF, TGF-β, and MMP-9 in EESCs and EuESCs were reduced by resveratrol treatment. According to our knowledge, this is the first study to investigate the effect of resveratrol on VEGF expression in ESCs of patients with endometriosis. In the only animal study, resveratrol significantly reduced VEGF expression in endometriosis-induced rats 25 . Other previous in-vivo and in-vitro studies on the effect of resveratrol on VEGF expression in other diseases have also reported findings consistent with the present study 29,[46][47][48][49] .
The mechanisms of the effect of resveratrol on VEGF expression seems to be through activation of the sirtuin-1 molecule and inhibition of the NF-κB pathway 50 . Resveratrol can also inhibit VEGF through ACE-I-like activity. Thus, resveratrol inhibits positive feedback between angiotensin-II and VEGF. The in-vitro studies have shown that ACE-I-like factors can inhibit VEGF-induced endothelial cell migration and invasion and inhibit VEGF mRNA expression 49,51 . Resveratrol may also block the VEGF receptor response pathway by reducing MAP-kinase phosphorylation and inhibiting VEGF-induced angiogenesis by blocking tyrosine phosphorylation in the cadherin molecule 46 . Besides, resveratrol reduces VEGF expression and its invasion and angiogenesis by preventing the production and eliminating the ROS and reactive nitrogen species (RNS) 39,52 .
It seems that the difference in the effect of resveratrol on the reduction of VEGF gene expression in EESCs compared to EuESCs and CESCs is due to differences in inflammatory and micro-environmental conditions of these cells. Previous studies have shown that EESCs, EuESCs, and CESCs differ in cytokine expression, cell proliferation, invasion, metastasis, and response to nutritional interventions 53,54 .
The present study is the first to investigate the effect of resveratrol on TGF-β expression in ESCs of patients with endometriosis, and it is not possible to compare the results with similar studies. Therefore, the findings of this study were compared with those of animal studies on the effect of resveratrol on TGF-β levels in other diseases. Most of these studies consistent with the present study have shown that resveratrol can decrease TGF-β gene and protein expression 28,55,56 . In the only inconsistent study, a single-dose intraperitoneal injection of resveratrol had no significant effect on TGF-β levels in rats with acute liver injury, possibly due to the amount and timing of the intervention 57 .
Resveratrol has been reported to inhibit TGF-β transcription by blocking the NF-κB pathway 55 . Resveratrol can also reduce TGF-β expression by blocking the activator protein 1 (AP-1) molecule and removing ROS and reducing oxidative stress 58 . Resveratrol also down-regulates TGF-β expression and activity by down-regulating TGF-β signaling pathway molecules, including, Smad-2, 3,4 59 . TGF-β is a pro-fibrotic factor that can increase the production of type IV collagen and fibrin 28 . Resveratrol treatment can prevent TGF-β-induced fibrotic tissue growth in ectopic lesions 55 .
The present study is the first to assess the effect of resveratrol treatment on MMP-9 expression in ESCs. The only animal study that investigated the effect of resveratrol on MMP-9 expression in endometriosis also reported the same results 25 . Other in-vivo and in-vitro studies also shown that resveratrol decreases MMP-9 mRNA and protein expression and suppresses the activity of this enzyme 30,60,61 . In the only inconsistent study, Gweon and Kim reported that resveratrol at different concentrations increased the activity and expression of MMP-9 in human fibrosarcoma cells. The cause of this contradictory finding may be the different inflammatory condition 62 .
MMP-9 is one of the proteins whose expression is enhanced by activation of the NF-κB pathway. It appears that resveratrol decreases the expression of this factor by suppressing the expression and activity of the NF-κB pathway 60 . Resveratrol inhibits NF-κB transcriptional activity by blocking phosphorylation and degradation of the IκB inhibitor molecule, thereby inhibiting NF-κB translocation and DNA binding and preventing expression of inflammatory cytokines and growth factors and angiogenesis including MMP-9 63 . Resveratrol can also prevent MMP-9 expression by decreasing TGF-β expression, inhibiting MAP-kinase signaling pathway, reabsorption of ROS, and reducing oxidative stress 64,65 .
The present study had some advantages and limitations: As we mentioned earlier, it was the first study investigated the basal gene and protein expression and also the effect of resverstrol treatment on the gene and protein expression of VEGF, TGF-β and MMP-9 in ectopic (EESCs), and eutopic (EuESCs) endometrial stromal cells of women with endometriosis in comparison with non-endometriotic controls (CESCs). One of the limitations was that the present study was carried out only in the severe (III and IV) stages of the EM and at the proliferative phase. Also, it would have been better if we could assess the MMP-9 activity. It is also better to investigate the effect of resveratrol treatment on the expression of VEGF, TGF-β and MMP-9 in the peritoneal fluid mononuclear cells (PFMCs) and mesothelial cells as the important sources of these factors. Moreover, in order to better determine the effect of resveratrol on EM, further studies are needed on the effect of resveratrol treatment on cell proliferation, angiogenesis, invasion, adhesion, apoptosis, and other processes involved in the pathogenesis of EM.

Conclusion
The present study showed that the basal gene and protein expression of VEGF and MMP-9 were higher in EESCs compared to EuESCs and CESCs. The treatment of EESCs and EuESCs with resveratrol could reduce the gene and protein expression of VEGF, TGF-β, and MMP-9. Further in-vitro and in-vivo studies are needed to determine the possible beneficial effects of resveratrol on EM progression.

Data availability
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