Resveratrol promotes trophoblast invasion in pre‐eclampsia by inducing epithelial‐mesenchymal transition

Abstract Impairment spiral arteries remodelling was considered to be the underlying cause of pathogenesis of pre‐eclampsia (PE). Resveratrol (RE) was reported that it could modulate cellar phenotype to ameliorate diverse human diseases. However, the biological function of RE in PE remains poorly understood. In this report, we investigated the effect of RE on trophoblast phenotype both in vivo and in vitro. We conducted MTT and transwell assays to explore cell proliferation and invasion events in HTR‐8/SVneo. In mice model, the clinical characteristics of PE were established through the injection of NG‐nitro‐l‐arginine methyl ester (L‐NAME). Furthermore, related experiments were performed to detect cellar phenotype‐associated signalling pathway, including epithelial‐mesenchymal transition (EMT) and Wnt/β‐catenin. Cell assays indicated that RE could increase trophoblasts migration and invasion. In addition, hypertension and proteinuria were markedly ameliorated by RE compared with the controls in PE mice model. Moreover, treatment by RE in trophoblasts or in PE model, we found that RE activated EMT progress through the regulation of E‐cadherin, β‐catenin, N‐cadherin, vimentin expression, and further altered the WNT‐related gene expression, including WNT1, WNT3 and WNT5B. Our findings demonstrated that RE might stimulate the invasive capability of human trophoblasts by promoting EMT and mediating the Wnt/β‐catenin pathway in PE.


| INTRODUC TI ON
Pre-eclampsia (PE), a devastating multi-system syndrome, occurs in approximately 10% of pregnancies worldwide. 1 It is the major trigger leads to pregnancy-associated mortality and serious damage to the spleen, liver, kidney and many other organs. Previous evidence have established that diversified factors may involve in the occurrence and development of PE, such as impaired spiral artery remodelling, oxidative stress and oxygen dysregulation. [2][3][4] Among these factors, aberrant trophoblast invasion, which contributed to impaired spiral artery remodelling, is considered as the underlying cause of pathogenesis in PE. 5 The acquisition of invasive capabilities by trophoblasts is determined by the turnover of cell-cell junctions, degradation of the cell matrix and activation of pathways that control cytoskeletal dynamics. 6 Recently, epithelial-mesenchymal transition (EMT) has been known to play an essential role in the regulation of cell migration and invasion. It is characterized by the breakdown of cell-cell adhesion, loss of epithelial phenotypes and cell depolarization, which accelerate progression in multiple diseases, especially cancer. 7,8 EMT is important in the oncogenesis, metastasis and drug resistance of cancer progression. 9 During pregnancy, the acquisition of invasive phenotypes by extravillous trophoblast (EVT), which is involved in the pathogenesis of PE, has been proposed to be associated with EMT process. 10 However, the underlying regulatory mechanisms of EMT in trophoblasts in the pathogenesis of PE are still poorly understood.
Resveratrol (trans-3,4,5-trihydroxy-stilbene; RE), a traditional Chinese medicine, is known as a naturally occurring compound with potent biological properties. [11][12][13] It is found in plants, such as grapes, giant knotweed, peanuts and mulberries. 14 RE exerts protective effects against various types of cancers 15,16 partly by modulating cancer cell invasion, 17 and also can reduce platelet aggregation in haematological system diseases. 18,19 Moreover, longterm basic research and clinical research have confirmed that RE exerts protective effects against cardiovascular and cerebrovascular diseases (such as hypertension, ventricular hypertrophy, atherosclerosis, myocardial infarction and arrhythmias), inflammatory lesions and diabetes. 20,21 Also, many studies have reported that RE significantly ameliorates an oxidative-stress reaction in pregnant model with hypertension and proteinuria. 22 However, the underlying molecular mechanisms of RE remain to be elucidated.
Hence, PE mice model was first established through the intraperitoneal injection of NG-nitro-l-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase. 23  All efforts were made to minimize suffering in experimental animals.

| Cell culture and treatment
In this study, HTR-8/SVneo Cells were provided by Dr Charles

| RNA extraction and qPCR assays
Total RNA was extracted from approximately 0.1 g of placenta tissues or from trophoblasts using TRIzol reagent (Invitrogen Life Technologies).
A reverse transcription kit (Takara) and Power SYBR Green (Takara) were used for cDNA synthesis and amplification respectively. The expression of genes regulating migration, invasion, angiogenesis and EMT-related factors were detected by SYBR Green qRT-PCR. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. The primers used were as follows: E-cadherin An ABI 7500 system was used to carry out the qPCR and data collection. was used as a control. We used the Quantity One software (Bio-Rad) to quantify the intensity of the protein bands.

| Immunofluorescence
The placental tissues were removed from the rats and immediately fixed in 4% paraformaldehyde according to a standard protocol.
HTR-8/SVneo cells were also fixed through the same procedure.

| Cell invasion assays
Cell invasion assays were performed as previously reported by Xu et al. 24 The RE-treated HTR-8/SVneo cells were suspended in RPMI 1640 medium containing 1% FBS and subsequently seeded at a density of 5×10 5  Finally, the number of migrated cells was calculated under a digital microscope in five random fields for each sample.

| Tube formation assay
Tube formation assay was performed as previously described by Xu et al. 25 To determine the potential of blood vessel formation in spiral artery remodelling in vitro, this assay was introduced to simulate in

| Statistical analysis
All data are expressed as the mean ± SD. All statistical analyses were performed with the SPSS statistical software package (SPSS Inc, Chicago, IL, USA). Paired and unpaired Student's t tests were used to compare the results of two groups and ANOVA was used to compare the data of more than two groups. P < 0.05 was considered statistically significant.

| PE clinical phenotypes were ameliorated by RE in mice model
To explore the role of RE in PE, we first used L-NAME to simulate a PE mice model. After treatment with RE, the related clinical indicators, including systolic blood pressure, proteinuria, foetus number, foetus birth weight, placental weight and external malformations, were measured and observed in all groups (n = 16 per group) ( Table 1).
There are no significant differences in foetus number, foetus birth weight, placental weight and external malformations between the groups. The L-NAME + RE groups exhibited significantly lower BP and urine protein level compared with the L-NAME group ( Figure 1A and B), indicating the successful establishment of a PE model using L-NAME and the ameliorative effects of RE on PE clinical phenotypes.

| RE ameliorates the epithelial characteristics of the placenta in PE model
So far, aberrant trophoblast invasion in placenta was recognized that was associated with EMT process and could cause the impaired spiral arteries remodelling, and further promote the progression of PE.
Then, we wondered whether RE regulated EMT to ameliorate effects of RE on PE model. So, we performed the immunofluorescence assays and qPCR assays to detect the expression of the EMT-related Our resulting data demonstrated that in the L-NAME + RE group, E-cadherin expression was lower, whereas β-cadherin, N-cadherin, vimentin, snail and MMP-2/MMP-9 levels were higher than those in the L-NAME group ( Figure 2F and G), further proving the role of RE in the activation of EMT process. Simultaneously, the levels of the angiogenesis-related factors VEGF, sFlt-1, AngI and AngII in PE rats were significantly improved by RE ( Figure 2H). These results indicated the ability of RE in promoting the invasive capacity of trophoblasts, in partly through EMT activation, and thus RE increased the angiogenesis potential in the placenta of PE model.

| Effect of RE on the migration and invasion in vitro
In conjunction with the results of in vivo study, we choose trophoblast cells to further invalidate the signalling pathway in vitro. Here, to unravel whether RE regulates the EMT in trophoblast cells, we first selected RE and doxorubicin (DOX, a kind of EMT inhibitor 26 ) to treat HTR-8/SVneo respectively. After HTR-8/SVneo cells were treated with RE, the capacity of cell migration and invasion, and proliferation were detected. As shown in Figure 3A and B, we conducted transwell assays and revealed that cells in the group treated with RE could significantly promote trophoblast migration and invasion compared with those in control. Moreover, we further confirmed that protein levels of MMP-2 and MMP-9 were significantly higher in the RE-treated group than those in the control by qRT-PCR and Western blotting assays ( Figure 3D and E). In addition, MTT assays were conducted to examine the effect of RE on cellular growth activity. As shown in Figure 3C, there was no significant difference All data are presented as the mean ± SD. a Analysed by one-way analysis of variance.

TA B L E 1 Clinical Parameters of pregnant mice among three groups
F I G U R E 1 RE ameliorated many PE phenotypes in PE mice model. The L-NAME group exhibited significantly higher blood pressure and urinary protein level than the control and L-NAME + RE groups. **P < 0.01 F I G U R E 2 RE ameliorates the epithelial characteristics of the placenta in PE model. L-NAME activated EMT in the mice placenta. A-E, Immunofluorescence analysis of E-cadherin, β-cadherin, N-cadherin, vimentin and snail expression, respectively, in the placenta of mice treated with L-NAME or L-NAME + RE. F, qRT-PCR analysis of E-cadherin, β-cadherin, N-cadherin, vimentin and snail expression in the placenta. G, qRT-PCR analysis of MMP-2 and MMP-9. H, qRT-PCR analysis of the angiogenesis-related factors VEGF, sFlt-1, AngI and AngII. All experiments were performed in triplicate with three technical replicates. *P < 0.05, **P < 0.01 in cell growth activity between the RE-treated and control groups (P > 0.05).

| RE promoted invasion and tube formation by EMT in vitro
Our previous work has reported that the EMT was involved in trophoblast cell invasion and migration. 27 qRT-PCR assays were performed to detect the expression of EMT biomarkers. As shown in Figure 4A, the DOX+RE group showed elevated levels of the mesenchymal biomarkers N-cadherin, β-cadherin, vimentin and snail, as well as a decreased level of the epithelial biomarker E-cadherin. Moreover, these observations were further confirmed by Western blotting ( Figure 4B, P < 0.05). In addition, the number of branch point was significantly higher in trophoblasts treated with RE than that of the control, indicating a significant increase network formation ability ( Figure 4D and E). The expression of the angiogenesis-related factors VEGF, sFlt1, AngI and AngII was also higher in the DOX+RE group than those in the DOX group (Figure 4C, Effect of RE on the migration and invasion of trophoblast cells. A and B, The transwell assay was conducted to investigate the changes in migratory and invasive abilities of HTR-8/SVneo cells. The migration and invasion capacities of the cells transfected with DOX were significantly lower than that of the normal and DOX + RE groups (data are presented as mean ± SD; **P < 0.01). C, There were no significant differences among the three groups, as observed using the MTT assay. D and E, qRT-PCR and Western blotting analysis of MMP-2 and MMP-9 expression levels. **P < 0.01 P < 0.05). Taken together, these findings suggested that RE could ameliorate cell invasion and tube formation by promoting the EMT process.

| Effects of RE on the WNT/β-catenin pathway
Previous studies have reported that β-catenin is the key transcriptional activator in canonical Wnt signalling in the nucleus, and L-NAME represses Wnt/β-catenin-mediated transcriptional activity by promoting the cytoplasmic localization of β-catenin. 28  mRNA decreased by 31%, 60% and 25%, respectively, following L-NAME injection, and they increased by 2.2-, 4.6-and 1.9-fold, respectively, following RE treatment ( Figure 5A) in PE mice model. Extravillous cytotrophoblast (EVT) in the uterine wall is limited to the superficial parts of the uterine vascular invasion or incomplete invasion. 18 Epithelial-mesenchymal transition process is generally associated with the pathogenesis and progression of various humans disorders, included PE. 19

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.