Phenylbutyric acid inhibits hypoxia-induced trophoblast apoptosis and autophagy in preeclampsia via the PERK/ATF-4/CHOP pathway.

Preeclampsia (PE) is a common pregnancy complication with a high mortality rate. Abnormally activated endoplasmic reticulum stress (ERS) is believed to be responsible for the destruction of key placental cells-trophoblasts. Phenylbutyric acid (4-PBA), an ERS inhibitor, is involved in regulating the development of ERS-related diseases. At present, how 4-PBA affects trophoblasts and its mechanisms is still unclear. In this study, PE cell models were established by stimulating HTR-8/SVneo cells with hypoxia. To verify the underlying mechanisms of 4-PBA on PE, CCT020312, an activator of PERK, was also used. The results showed that 4-PBA restored hypoxia-induced trophoblast viability, inhibited HIF-1α protein expression, inflammation, and PERK/ATF-4/CHOP pathway. Hoechst 33342 staining and flow cytometry results confirmed that 4-PBA decreased hypoxia-induced apoptosis in trophoblasts. The results of the JC-1 analysis and apoptosis initiation enzyme activity assay also demonstrated that 4-PBA inhibited apoptosis related to the mitochondrial pathway. Furthermore, by detecting autophagy in trophoblasts, an increased number of autophagic vesicles, damaged mitochondria, enhanced dansylcadaverine fluorescence, enhanced levels of autophagy proteins Beclin-1, LC3II, and decreased p62 were seen in hypoxia-stimulated cells. These changes were reversed by 4-PBA. Furthermore, it was observed that CCT020312 reversed the effects of 4-PBA on the viability, apoptosis, and autophagosome number of hypoxia-induced trophoblasts. In summary, 4-PBA reduces autophagy and apoptosis via the PERK/ATF-4/CHOP pathway and mitochondrial pathway, thereby restoring the viability of hypoxic trophoblasts. These findings provide a solid evidence base for the use of 4-PBA in PE treatment and guide a new direction for improving the outcomes of patients with PE.

Ghrelin alleviates placental dysfunction by down-regulating NF-κB phosphorylation in LPS-induced rat model of preeclampsia.

In our previous study, we uncovered that ghrelin promotes angiogenesis in human umbilical vein endothelial cells (HUVECs) in vitro by activating the Jagged1/Notch2/VEGF pathway in preeclampsia (PE). However, the regulatory effects of ghrelin on placental dysfunction in PE are unclear. Therefore, we applied Normal pregnant Sprague-Dawley (SD) rats, treated with lipopolysaccharide (LPS), to establish a PE-like rat model. The hematoxylin-eosin (HE) staining method and immunohistochemistry (IHC) technology were used to detect morphological features of the placenta. IHC and Western blot were applied to examine Bax and Bcl-2 expression levels. The concentrations of serum soluble fms-like tyrosine kinase-1 (sFlt1) and placental growth factor (PIGF) were assessed by enzyme-linked immunosorbent assay (ELISA) kit. In addition, the apoptosis rates of JEG-3 and HTR-8/SVneo trophoblast cells were determined by Annexin V-FITC/PI apoptosis detection kit. Cell migratory capacities were assessed by scratch-wound assay, and RNA-sequencing assay was used to determine the mechanism of ghrelin in regulating trophoblast apoptosis. It has been found that ghrelin significantly reduced blood pressure, urinary protein, and urine creatinine in rats with PE, at the meanwhile, ameliorated placental and fetal injuries. Second, ghrelin clearly inhibited placental Bax expression and circulating sFlt-1 as well as elevated placental Bcl-2 expression and circulating PIGF, restored apoptosis and invasion deficiency of trophoblast cells caused by LPS in vitro. Finally, transcriptomics indicated that nuclear factor kappa B (NF-κB) was the potential downstream pathway of ghrelin. Our findings illustrated that ghrelin supplementation significantly improved LPS-induced PE-like symptoms and adverse pregnancy outcomes in rats by alleviating placental apoptosis and promoting trophoblast migration.

Cell type-dependent response to benzo(a)pyrene exposure of human placental cell lines under normoxic, hypoxic, and pro-inflammatory conditions.

Benzo(a)pyrene (BaP) can be detected in the human placenta. However, little is known about the effects of BaP exposure on different placental cells under various conditions. In this study, we aimed to investigate the effects of BaP on mitochondrial function, pyrin domain-containing protein 3 (NLRP3) inflammasome, and apoptosis in three human trophoblast cell lines under normoxia, hypoxia, and inflammatory conditions. JEG-3, BeWo, and HTR-8/SVneo cell lines were exposed to BaP under normoxia, hypoxia, or inflammatory conditions for 24 h. After treatment, we evaluated cell viability, apoptosis, aryl hydrocarbon receptor (AhR) protein and cytochrome P450 (CYP) gene expression, mitochondrial function, including mitochondrial DNA copy number (mtDNAcn), mitochondrial membrane potential (ΔΨm), intracellular adenosine triphosphate (iATP), and extracellular ATP (eATP), nitric oxide (NO), NLPR3 inflammasome proteins, and interleukin (IL)-1ß. We found that BaP upregulated the expression of AhR or CYP genes to varying degrees in all three cell lines. Exposure to BaP alone increased ΔΨm in all cell lines but decreased NO in BeWo and HTR-8/SVneo, iATP in HTR-8/SVneo, and cell viability in JEG-3, without affecting apoptosis. Under hypoxic conditions, BaP did not increase the expression of AhR and CYP genes in JEG-3 cells but increased CYP gene expression in two others. Pro-inflammatory conditions did not affect the response of the 3 cell lines to BaP with respect to the expression of CYP genes and changes in the mitochondrial function and NLRP3 inflammasome proteins. In addition, in HTR-8/SVneo cells, BaP increased IL-1ß secretion in the presence of hypoxia and poly(I:C). In conclusion, our results showed that BaP affected mitochondrial function in trophoblast cell lines by increasing ΔΨm. This increased ΔΨm may have rescued the trophoblast cells from activation of the NLRP3 inflammasome and apoptosis after BaP treatment. We also observed that different human trophoblast cell lines had cell type-dependent responses to BaP exposure under normoxia, hypoxia, or pro-inflammatory conditions.

Arginine and its metabolites stimulate proliferation, differentiation, and physiological function of porcine trophoblast cells through ß-catenin and mTOR pathways.

Arginine, which is metabolized into ornithine, proline, and nitric oxide, plays an important role in embryonic development. The present study was conducted to investigate the molecular mechanism of arginine in proliferation, differentiation, and physiological function of porcine trophoblast cells (pTr2) through metabolic pathways. The results showed that arginine significantly increased cell viability (P < 0.05). The addition of arginine had a quadratic tendency to increase the content of progesterone (P = 0.06) and protein synthesis rate (P = 0.03), in which the maximum protein synthesis rate was observed at 0.4 mM arginine. Arginine quadratically increased (P < 0.05) the intracellular contents of spermine, spermidine and putrescine, as well as linearly increased (P < 0.05) the intracellular content of NO in a dose-dependent manner. Arginine showed a quadratic tendency to increase the content of putrescine (P = 0.07) and a linear tendency to increase NO content (P = 0.09) in cell supernatant. Moreover, increasing arginine activated (P < 0.05) the mRNA expressions for ARG, ODC, iNOS and PCNA. Furthermore, inhibitors of arginine metabolism (L-NMMA and DFMO) both inhibited cell proliferation, while addition of its metabolites (NO and putrescine) promoted the cell proliferation and cell cycle, the mRNA expressions of PCNA, EGF and IGF-1, and increased (P < 0.05) cellular protein synthesis rate, as well as estradiol and hCG secretion (P < 0.05). In conclusion, our results suggested that arginine could promote cell proliferation and physiological function by regulating the metabolic pathway. Further studies showed that arginine and its metabolites modulate cell function mainly through ß-catenin and mTOR pathways.

Maternal exposure to beta-Cypermethrin disrupts placental development by dysfunction of trophoblast cells from oxidative stress.

As a broad-spectrum and efficient insecticide, beta-Cypermethrin (ß-CYP) poses a health risk to pregnancy. It matters the mechanisms of maternal exposure to ß-CYP for impacting reproductive health. The placenta, a transient organ pivotal for maternal-fetal communication during pregnancy, plays a crucial role in embryonic development. The effect of ß-CYP exposure on the placenta and its underlying molecular mechanisms remain obscure. The objective of this study was to investigate the effect of ß-CYP exposure on placental development and the function of trophoblast, as well as the underlying mechanisms through CD-1 mouse model (1, 10, 20 mg/kg.bw) and in vitro HTR-8/SVneo cell model (12.5, 25, 50, 100 µM). We found slower weight gain and reduced uterine wet weight in pregnant mice with maternal exposure to ß-CYP during pregnancy, as well as adverse pregnancy outcomes such as uterine bleeding and embryo resorption. The abnormal placental development in response to ß-CYP was noticed, including imbalanced placental structure and disrupted labyrinthine vascular development. Trophoblasts, pivotal in placental development and vascular remodeling, displayed abnormal differentiation under ß-CYP exposure. This aberration was characterized by thickened trophoblast layers in the labyrinthine zone, accompanied by mitochondrial and endoplasmic reticulum swelling within trophoblasts. Further researches on human chorionic trophoblast cell lines revealed that ß-CYP exposure induced apoptosis in HTR-8/SVneo cells. This induction resulted in a notable decrease in migration and invasion abilities, coupled with oxidative stress and the inhibition of the Notch signaling pathway. N-acetylcysteine (an antioxidant) partially restored the impaired Notch signaling pathway in HTR-8/SVneo cells, and mitigated cellular functional damage attributed to ß-CYP exposure. Collectively, exposure to ß-CYP induced oxidative stress and then led to inhibition of the Notch signaling pathway and dysfunction of trophoblast cells, ultimately resulted in abnormal placenta and pregnancy. These findings indicate Reactive Oxygen Species as potential intervention targets to mitigate ß-CYP toxicity. The comprehensive elucidation contributes to our understanding of ß-CYP biosafety and offers an experimental basis for preventing and managing its reproductive toxicity.

Triphenyl phosphate interferes with the synthesis of steroid hormones through the PPARγ/CD36 pathway in human trophoblast JEG-3 cells.

Triphenyl phosphate (TPhP), a chemical commonly found in human placenta and breast milk, has been shown to disturb the endocrine system. Our previous study confirmed that TPhP could accumulate in the placenta and interference with placental lipid metabolism and steroid hormone synthesis, as well as induce endoplasmic reticulum (ER) stress through PPARγ in human placental trophoblast JEG-3 cells. However, the molecular mechanism underlying this disruption remains unknown. Our study aimed to identify the role of the PPARγ/CD36 pathway in TPhP-induced steroid hormone disruption. We found that TPhP increased lipid accumulation, total cholesterol, low- and high-density protein cholesterol, progesterone, estradiol, glucocorticoid, and aldosterone levels, and genes related to steroid hormones synthesis, including 3ßHSD1, 17ßHSD1, CYP11A, CYP19, and CYP21. These effects were largely blocked by co-exposure with either a PPARγ antagonist GW9662 or knockdown of CD36 using siRNA (siCD36). Furthermore, an ER stress inhibitor 4-PBA attenuated the effect of TPhP on progesterone and glucocorticoid levels, and siCD36 reduced ER stress-related protein levels induced by TPhP, including BiP, PERK, and CHOP. These findings suggest that ER stress may also play a role in the disruption of steroid hormone synthesis by TPhP. As our study has shed light on the PPARγ/CD36 pathway's involvement in the disturbance of steroid hormone biosynthesis by TPhP in the JEG-3 cells, further investigations of the potential impacts on the placental function and following birth outcome are warranted.

In vitro blastocyst implantation and trophoblast migration are disrupted by the UV filter benzophenone-3 (BP3).

Benzophenone-3 (BP3) is a common ingredient in personal care products (PCPs) due to its well-established effectiveness in absorbing UV radiation. Sunscreen products are among the most widely used PCPs-containing BP3 applied to the skin, resulting in significant human exposure to BP3 primarily through a dermal application. In the present work, we have tested the action of three environmentally relevant concentrations of BP3 (2, 20 and 200 µg/L) on an in vitro model of implantation of murine blastocysts and on migration ability of the human trophoblast cell line Swan 71. We showed that BP3 caused a significant reduction of blastocyst expansion and a delayed hatching in a non-monotonic way. Besides, embryos displayed a delayed attachment in the three BP3 groups, resulting in a smaller implantation area on the 6th day of culture: BP3(2) (0.32 ± 0.07 mm2); BP3(20) (0.30 ± 0.08 mm2) and BP3(200) (0.25 ± 0.06 mm2) in comparison to the control (0.42 ± 0.07 mm2). We also found a reduced migration capacity of the human first-trimester trophoblast cell line Swan 71 in a scratch assay when exposed to BP3: the lowest dose displayed a higher uncovered area (UA) at 6h when compared to the control, whereas a higher UA of the wound was observed for the three BP3 concentrations at 18 and 24 h of exposure. The changes in UA provoked by BP3 restored to normal values in the presence of flutamide, an androgen receptor (AR) inhibitor. These results indicate that a direct impairment on early embryo implantation and a defective migration of extravillous trophoblast cells through the androgen receptor pathway can be postulated as mechanisms of BP3-action on early gestation with potential impact on fetal growth.

An Examination of the Effect of Aspirin and Salicylic Acid on Soluble Fms-like Tyrosine Kinase-1 Release from Human Placental Trophoblasts.

Low-dose aspirin (LDA) is efficacious in preventing preeclampsia, but its mechanism of action is unclear. Conflicting evidence suggests that it may inhibit placental trophoblast release of soluble fms-like tyrosine kinase-1 (sFlt1), a key mediator of preeclampsia. We examined whether, and at what concentrations, aspirin and its principal metabolite, salicylic acid, modulate sFlt1 release and/or expression in trophoblasts. Human trophoblast lines BeWo and HTR-8/SVneo were cultured; BeWo cells were also treated with 1% oxygen vs. normoxia to mimic hypoxia in preeclamptic placentas. Cells were treated with aspirin or salicylic acid vs. vehicle for 24 h at concentrations relevant to LDA and at higher concentrations. Protein concentrations (ELISA) and mRNA expression (RT-PCR) of sFlt1 were determined. Under normoxia, LDA-relevant concentrations of aspirin (10-50 µmol/L) or salicylic acid (20-100 µmol/L) had no significant effect on sFlt1 protein release or mRNA expression in BeWo cells. However, inhibition was observed at higher concentrations (1 mmol/L for aspirin and ≥200 µmol/L for salicylic acid). Hypoxia enhanced sFlt1 protein release and mRNA expression in BeWo cells, but these responses were not significantly affected by either aspirin or salicylic acid at LDA concentrations. Similarly, neither drug altered sFlt1 protein secretion or mRNA expression in normoxic HTR-8/SVneo cells at LDA concentrations. We suggest that direct modulation of trophoblast release or expression of sFlt1 is unlikely to be a mechanism underlying the clinical efficacy of LDA in preeclampsia.

The Effect of the cAMP Signaling Pathway on HTR8/SV-Neo Cell Line Proliferation, Invasion, and Migration After Treatment with Forskolin.

Pre-eclampsia (PE) is thought to be related to placental dysfunction, particularly poor extravillous trophoblast (EVT) invasion and migration abilities. However, the pathogenic mechanism is not fully understood. This article describes the impact of the cyclic adenosine monophosphate(cAMP) signaling pathway on EVT behavior, focusing on EVT proliferation, invasion, and migration. Here, we used the HTR8/SV-neo cell line to study human EVT function in vitro. HTR8/SV-neo cells were treated with different concentrations of forskolin (cAMP pathway-specific agonist) to alter intracellular cAMP levels, and dimethyl sulfoxide (DMSO) was used as the control. First, a cAMP assay was performed to measure the cAMP concentration in HTR8/SV-neo cells treated with different forskolin concentrations, and cell proliferation was assessed by constructing cell growth curves and assessing colony formation. Cell invasion and migration were observed by Transwell experiments, and intracellular epithelial-mesenchymal transition (EMT) marker expression was evaluated by quantitative real-time polymerase chain reaction (qPCR) and Western blotting (WB). According to our research, the intracellular cAMP levels in HTR8/SV-neo cells were increased in a dose-dependent manner, and HTR8/SV-neo cell proliferation, invasion and migration were significantly enhanced. The expression of EMT and angiogenesis markers was upregulated. Additionally, with the increase in intracellular cAMP levels, the phosphorylation of intracellular mitogen-activated protein kinase (MAPK) signaling pathway components was significantly increased. These results suggested that the cAMP signaling pathway promoted the phosphorylation of MAPK signaling components, thus enhancing EVT functions, including proliferation, invasion, and migration, and to a certain extent, providing a novel direction for the treatment of PE patients.

IL­1ß­induced pentraxin 3 inhibits the proliferation, invasion and cell cycle of trophoblasts in preeclampsia and is suppressed by IL­1ß antagonists.

Pentraxin 3 (PTX3), a member of the c­reactive protein family, is a long pentraxin protein and a pro­inflammatory marker. However, the role of PTX3 in preeclampsia (PE) remains to be elucidated. Thus, the present study aimed to investigate the biological role and mechanisms underlying PTX3 in PE. In the present study, PTX3 was overexpressed in trophoblasts and the subsequent changes in cell proliferation, cycle distribution and invasion were observed using Cell Counting Kit­8, flow cytometry and Transwell assays, respectively. Moreover, the expression levels of MMP2 and MMP9, proteins associated with the development of PE, were detected using reverse transcription­quantitative PCR and western blot analysis. Following treatment with interleukin (IL)­1ß, the expression levels of PTX3 were measured. Furthermore, subsequent changes in cell proliferation, cycle distribution and invasion were investigated following overexpression of PTX3 and treatment with IL­1 receptor antagonist (IL­1Ra). Overexpression of PTX3 inhibited the proliferation, cycle and invasion of HTR­8/SV neo and JEG3 cells. Moreover, treatment with IL­1ß increased the expression of PTX3 in HTR­8/SV neo and JEG3 cells, which was suppressed following treatment with the IL­1ß antagonist. Following PTX3 overexpression and treatment with IL­1Ra, the inhibitory effects of PTX3 overexpression alone on the invasion of HTR­8/SV neo and JEG3 cells were attenuated. In conclusion, these results indicated that IL­1ß could induce PTX3 upregulation, which led to the inhibition of the proliferation, invasion and cell cycle of trophoblasts, thereby promoting the progression of PE.

Nickel nanoparticles affect the migration and invasion of HTR-8/SVneo cells by downregulating MMP2 through the PI3K/AKT pathway.

Proper migration and invasion of extravillous trophoblast cells into the endometrium in early gestation is essential for successful embryo implantation. The development of nanotechnology has led to the emergence of nickel nanoparticles (Ni NPs), for which attendant health concerns are widespread. Ni NPs are known to affect reproduction and be embryotoxic, but whether they affect the migration and invasion functions of trophoblast cells is unclear. We investigated the effects of Ni NPs on the migration and invasion of HTR-8/SVneo in extravillous trophoblast cells and explored the possible role of the PI3K/AKT/MMP2 signaling pathway in this regard. Results showed that the migration and invasion of cells was significantly inhibited by the exposure of Ni NPs. The protein and mRNA levels of PI3K/AKT/MMP2 signaling pathway were significantly reduced with the increase in Ni NPs concentration. The presence of the PI3K activator 740Y-P partially attenuated the inhibition of cell migration and invasion by Ni NPs, confirming the involvement of this pathway. Thus, Ni NPs inhibit migration and invasion of human trophoblast HTR-8/SVneo cells by downregulating the PI3K/AKT/MMP2 signaling pathway. This study is important for the development of safety evaluation criteria for Ni NPs.

Effects of tumor necrosis factor on undifferentiated and syncytialized placental choriocarcinoma BeWo cells.

Tumor necrosis factor (TNF) regulates trophoblast turnover during the formation of the placental syncytium and can be a potentially relevant target for adverse effects of xenobiotics. We mimicked syncytialization in vitro by stimulating BeWo cells with 50 µM forskolin. Undifferentiated and syncytialized BeWo cells were exposed to TNF (10 pg/mL-10 ng/mL) for 48 h after which cell viability, progesterone release and gene expression of a selected set of markers representative for placental function were assessed. In undifferentiated BeWo cells, high TNF levels (1-10 ng/mL) increased gene expression of TNF, NF-κB, and TNFRSF1B to maximally 99 ± 17, 2.2 ± 0.2, and 3.0 ± 0.4 of control values, respectively (p < 0.001). These effects were also found in syncytialized BeWo cells but less pronounced. Additionally, TNF may induce syncytialization in BeWo cells as it upregulated ERVW-1 expression by 1.55 ± 0.14-fold (p < 0.05). On the contrary, TNF levels of 10 and 100 pg/mL did not affect gene expression in both undifferentiated and syncytialized BeWo cells, but did enhance cell viability in syncytialised BeWo cells (p < 0.001). In conclusion, we found that high TNF levels (1-10 ng/mL) increased gene expression of TNF, NF-κB, and TNFRSF1B especially in undifferentiated BeWo cells, while physiological TNF concentrations positively affected cell viability and while there was no effect on any of the investigated functional markers.

Bisphenol S Impairs Invasion and Proliferation of Extravillous Trophoblasts Cells by Interfering with Epidermal Growth Factor Receptor Signaling.

The placenta supports fetal growth and is vulnerable to exogenous chemical exposures. We have previously demonstrated that exposure to the emerging chemical bisphenol S (BPS) can alter placental endocrine function. Mechanistically, we have demonstrated that BPS interferes with epidermal growth factor receptor (EGFR) signaling, reducing placenta cell fusion. Extravillous trophoblasts (EVTs), a placenta cell type that aids with vascular remodeling, require EGF to invade into the maternal endometrium. We hypothesized that BPS would impair EGF-mediated invasion and proliferation in EVTs. Using human EVTs (HTR-8/SVneo cells), we tested whether BPS could inhibit the EGF response by blocking EGFR activation. We also evaluated functional endpoints of EGFR signaling, including EGF endocytosis, cell invasion and proliferation, and endovascular differentiation. We demonstrated that BPS blocked EGF-induced phosphorylation of EGFR by acting as a competitive antagonist to EGFR. Transwell assay and a three-dimensional microfluidic chip invasion assay revealed that BPS exposure can block EGF-mediated cell invasion. BPS also blocked EGF-mediated proliferation and endovascular differentiation. In conclusion, BPS can prevent EGF-mediated EVT proliferation and invasion through EGFR antagonism. Given the role of EGFR in trophoblast proliferation and differentiation during placental development, our findings suggest that maternal exposure to BPS may contribute to placental dysfunction via EGFR-mediated mechanisms.

Prothioconazole induces cell cycle arrest by up-regulation of EIF4EBP1 in extravillous trophoblast cells.

Prothioconazole (PTC) is a new broad-spectrum triazole antibacterial agent that is being widely used in agriculture. PTC has been linked to a number of reproductive outcomes including embryo implantation disorder; however, the exact mechanism underlying this relationship has yet to be determined. Proper trophoblast proliferation and migration is a prerequisite for successful embryo implantation. To elucidate the underlying molecular perturbations, we detect the effect of PTC on extravillous trophoblast cells proliferation and migration, and investigate its potential mechanisms. Exposure to different concentrations of PTC (0-500 µM) significantly inhibited the cell viability and migration ability (5 µM PTC exposure), and also caused the cell cycle arrest at the lowest dose (1 µM PTC exposure). Transcriptome analysis revealed that PTC exposure disturbed multiple biological processes including cell cycle and apoptosis, consistent with cell phenotype. Specifically, eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1, 4E-BP1) was identified as up-regulated in PTC exposure group and knockdown of EIF4EBP1, and attenuated the G1 phase arrest induced by PTC exposure. In summary, our data demonstrated that 4E-BP1 participated in PTC-induced cell cycle arrest in extravillous trophoblast cells by regulating cyclin D1. These findings shed light on the potential adverse effect of PTC exposure on the embryo implantation.

TCDD-induced IL-24 secretion in human chorionic stromal cells inhibits placental trophoblast cell migration and invasion.

Environmental pollutant dioxins are potentially harmful to pregnant women and can lead to severe adverse outcomes in pregnancy, such as spontaneous abortion and stillbirth. However, little is currently known about the underlying toxicological mechanism. Our previous study reported that the IL-24 gene is a dioxin response gene during 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) treatment. Here, we further tested the effect of TCDD on IL-24 expression in human chorionic stromal cells. We also investigated the effect of IL-24 on the behaviors of human placental trophoblast cells and predicted the potential mechanism underlying these behaviors using functional network analysis. We found that TCDD stimulates IL-24 expression in human chorionic stromal cells in an AhR (aromatic hydrocarbon receptor)-related manner. We also found that IL-24 inhibits the migration and invasion of human placental trophoblast cells, the possible mechanism of which involves thirteen key proteins and mitochondrial function. Our findings suggest that IL-24 is a potential factor induced by TCDD to regulate trophoblast cell invasion, which potentially involves in TCDD-induced abortion.

Forkhead-box C1 attenuates high glucose-induced trophoblast cell injury during gestational diabetes mellitus via activating adenosine monophosphate-activated protein kinase through regulating fibroblast growth factor 19.

Gestational diabetes mellitus (GDM) is a complication developed during pregnancy and recover after childbirth. The purpose of this study was to investigate the protective role of FOXC1 during GDM and the underlying mechanism. FOXC1 was downregulated in GDM placental tissues and HG-treated HTR-8/SVneo cells. Overexpression of FOXC1 prevented HG-induced inhibition of cell proliferation, migration and invasion. FOXC1 suppressed HG-induced cell apoptosis in HTR-8/SVneo cells. The apoptosis-related proteins: cleaved caspase-3, cleaved caspase-9 and BAX, were also downregulated by FOXC1 overexpression. FOXC1 increased glucose uptake and improved insulin sensitivity. The expression of FOXC1 was positively correlated with FGF19 expression. FOXC1 regulated the expression of FGF19 and phosphorylation of AMPK. Inhibition of FGF19 attenuated the biological functions of FOXC1 through inactivation of AMPK. In conclusion, this study demonstrates that FOXC1 attenuates HG-induced trophoblast cell injury through upregulating FGF19 to activate the AMPK signaling pathway during GDM, suggesting that FOXC1 is a potential therapeutic target for drug discovery in the future.

Cyclosporin A alleviates trophoblast apoptosis and senescence by promoting autophagy in preeclampsia.

INTRODUCTION: Abnormal extravillous trophoblast (EVT) function is closely related to preeclampsia (PE) and may be caused by inadequate autophagy, apoptosis, and senescence. Cyclosporin A (CsA) is an effective immunosuppressant that has been reported to stimulate autophagy and exert benign biological effects on EVTs. Therefore, we hypothesized that CsA may display therapeutic efficacy against PE by activating autophagy. METHODS: We established the nitro-l-arginine methyl ester (l-NAME)-induced preeclamptic mice model and a hypoxia-reoxygenation (H/R) model in vitro. The effects of CsA on autophagy were evaluated by western blotting (WB). The effects of CsA on apoptosis were analyzed by Hematoxylin-eosin (H&E) staining, cell apoptosis assay and WB. Senescence-associated ß-galactosidase (SA-ß-gal) staining, RT-qPCR and WB were used to examine the senescence level. RT-qPCR were used to detect the senescence-associated secretory phenotype (SASP) level. DCFH-DA fluorescent probe, dihydroethidium (DHE) staining and mitochondrial membrane potential (ΔΨm) were used to detect senescence-associated mitochondrial dysfunction (SAMD). RESULTS: CsA alleviated PE-like symptoms and reduced placental necrosis and senescence in mice injected with l-NAME. CsA ameliorated placental SASP and SAMD level induced by l-NAME. CsA also upregulated the expression of autophagic proteins in mouse placentas disrupted using l-NAME. In vitro, we found that CsA reversed H/R-induced apoptosis and senescence, as well as decreasing SASP and SAMD levels and upregulating autophagic proteins levels. Notably, 3-methyladenine (3-MA), an early phase inhibitor of autophagosome formation, abolished the protective effects of CsA against H/R. DISCUSSION: CsA may display some therapeutic effects against PE by activating autophagy in vivo and in vitro.

Ryanodine receptor calcium release channels in trophoblasts and their role in cell migration.

Trophoblasts are specialized epithelial cells of the placenta that are involved in invasion, communication and the exchange of materials between the mother and fetus. Cytoplasmic Ca2+ ([Ca2+]c) plays critical roles in regulating such processes in other cell types, but relatively little is known about the mechanisms that control this second messenger in trophoblasts. In the current study, the presence of RyRs and their accessory proteins in placental tissues and in the BeWo choriocarcinoma, a model trophoblast cell-line, were examined using immunohistochemistry and Western immunoblotting. Contributions of RyRs to Ca2+ signalling and to random migration in BeWo cells were investigated using fura-2 fluorescent and brightfield videomicroscopy. The effect of RyR inhibition on reorganization of the F-actin cytoskeleton elicited by the hormone angiotensin II, was determined using phalloidin-labelling and confocal microscopy. RyR1 and RyR3 proteins were detected in trophoblasts of human first trimester and term placental villi, along with the accessory proteins triadin and calsequestrin. Similarly, RyR1, RyR3, triadin and calsequestrin were detected in BeWo cells. In this cell-line, activation of RyRs with micromolar ryanodine increased [Ca2+]c, whereas pharmacological inhibition of these channels reduced Ca2+ transients elicited by the peptide hormones angiotensin II, arginine vasopressin and endothelin 1. Angiotensin II increased the velocity, total distance and Euclidean distance of random migration by BeWo cells and these effects were significantly reduced by tetracaine and by inhibitory concentrations of ryanodine. RyRs contribute to reorganization of the F-actin cytoskeleton elicited by angiotensin II, since inhibition of these channels restores the parallelness of these structures to control levels. These findings demonstrate that trophoblasts contain a suite of proteins similar to those in other cell types possessing highly developed Ca2+ signal transduction systems, such as skeletal muscle. They also indicate that these channels regulate the migration of trophoblast cells, a process that plays a key role in development of the placenta.

Inhibition of the cleaved half of tRNAGly enhances palmitic acid-induced apoptosis in human trophoblasts.

Palmitic acid (PA) induces apoptosis in the human trophoblast cell line HTR8/SVneo. However, the molecular mechanism underlying this effect remains unclear. Although small noncoding RNAs are involved in trophoblast growth and invasion during early pregnancy, the functional roles of tRNA-derived species are currently unknown. Therefore, the purpose of this study was to examine the involvement of tRNA-derived species in PA-induced apoptosis in human trophoblasts. In this study, we investigate the expression and function of tRNA-derived stress-induced RNAs (tiRNAs) in HTR8/SVneo. We determined the expression of tiRNAs in HTR8/SVneo cells in response to PA. Then, we transfected inhibitor of target tiRNA in HTR8/SVneo with or without PA to examine the tRNA-derived species-regulated intracellular signal transduction by detecting calcium homeostasis, mitochondrial membrane potential, and signaling proteins. We found that the expression of tRNAGly-derived tiRNAs decreased in PA-treated human trophoblasts. Moreover, inhibition of tiRNAGlyCCC/GCC enhanced the PA-induced apoptosis along with the induction of DNA fragmentation and mitochondrial depolarization. Inhibition of tiRNAGlyCCC/GCC enhanced the expression of endoplasmic reticulum stress-related proteins and increased Ca2+ levels in the cytoplasm and mitochondria. Moreover, the levels of cytochrome c released from the mitochondria were synergistically affected by tiRNAGlyCCC/GCC inhibitor and PA. Furthermore, artificial regulation of ANG inhibited the expression of tiRNAGlyCCC/GCC and similar effects were observed upon the inhibition of tiRNAGlyCCC/GCC in human trophoblasts. These results suggest that tiRNAGlyCCC/GCC might be the molecule via which PA induces its effects in human trophoblasts.

Elevated Serum SFRP5 Levels During Preeclampsia and Its Potential Association with Trophoblast Dysfunction via Wnt/ß-Catenin Suppression.

Preeclampsia (PE) is a life-threatening pregnancy complication associated with diminished trophoblast migration and invasion. Wnt signalling is one of the most important regulators of placentation. Secreted frizzled-related protein 5 (SFRP5) is an anti-inflammatory adipokine that may inhibit Wnt signalling. In this study, we aimed to investigate the relationship between SFRP5 and PE and its effect on trophoblast function, as well as the underlying signalling pathways. SFRP5 levels in the serum and placental tissues were detected using enzyme-linked immunosorbent assay and immunohistochemistry, respectively. To evaluate the effect of SFRP5 on Wnt signalling, the human trophoblast cell line HTR8/SVneo was treated with recombinant human SFRP5 and Dickkopf-related protein 1 (Dkk-1, canonical Wnt inhibitor) proteins and lithium chloride (LiCl, canonical Wnt agonist). The migration and invasion ability of HTR8/SVneo cells was evaluated using wound-healing and Matrigel Transwell assays. The activities of multiple matrix metalloproteinases (MMP)-2/9 were detected using gelatin zymography. Expression of glycogen synthase kinase-3 beta (GSK3ß) and ß-catenin proteins was investigated using western blotting. The serum SFRP5 levels were elevated in patients with PE, but SFRP5 expression was not detected in the placental tissues. Furthermore, SFRP5 inhibited the migration and invasion of HTR8/SVneo cells in vitro, increased GSK3ß, and decreased ß-catenin expression and MMP-2/9 activity in HTR8/SVneo cells. In conclusion, this study suggests that SFRP5 inhibits trophoblast migration and invasion potentially via the inhibition of Wnt/ß-catenin signalling, which might be involved in the development of PE. However, the primary cause of the increased SFRP5 levels needs to be investigated.