Knocking down macrophages Caspase-6 through HMGB1 coordinates macrophage trophoblast crosstalk to suppress ferroptosis and alleviate preeclampsia.

OBJECTIVE: Caspase-6 is an important regulatory factor in innate immunity, inflammasome activation, and host defense, but its role in preeclampsia (PE) is unknown. This study aims to investigate the mechanism of Caspase-6 in the interaction between PE rats and macrophage-trophoblast cells, in order to provide a new theoretical basis for the treatment of PE. METHODS: Co-cultures of THP-1 cells and HTR8/SVneo cells were employed to investigate the HMGB1 signaling in macrophages (transfection with si-Caspase-6) and HTR8/SVneo cells. The PE rat model was constructed by using the reduced uterine perfusion pressure (RUPP) surgery to explore the therapeutic effects of bone marrow-derived macrophages (BMDM) transfected with si-Caspase-6 in PE rats. ELISA, Western blot, immunofluorescence, etc., were employed to characterize the expression of ferroptosis-related markers. RESULTS: Caspase-6 expression was significantly increased in CD14+ macrophages in the placental tissue of PE rats. Overexpression of Caspase-6 in THP-1 cells induced ferroptosis of HTR8/SVneo cells, but this process was blocked by anti-HMGB1 neutralizing antibody. Knockdown of Caspase-6 in macrophages could alleviate ferroptosis of HTR8/SVneo cells and restore its basic characteristics. Knockdown of Caspase-6 in BMDM downregulated ferroptosis in placental tissue of PE rats through HMGB1, thereby improving the disease phenotype in rats. CONCLUSION: Knocking down Caspase-6 in BMDM regulated the crosstalk between macrophages and HTR8/SVneo cells through HMGB1, inhibiting HTR8/SVneo cell ferroptosis, thereby improving adverse pregnancy outcomes of PE.

Insulin-like growth factor 1 ameliorates pre-eclampsia by inhibiting zinc finger E-box binding homeobox 1 by up-regulation of microRNA-183.

As a common hypertensive complication of pregnancy, preeclampsia (PE) remains one of the leading causes of maternal and fetal with high morbidity and mortality worldwide. Much research has identified the vital functions of insulin-like growth factor 1 (IGF-1) in PE treatment. However, the combined roles and molecular mechanism of IGF-1 and microRNAs (miRNAs) underlying PE remain unclear. Therefore, we first measured the expression of IGF-1, zinc finger E-box binding homeobox 1 (ZEB1) and microRNA-183 (miR-183) expression in the placenta tissues of patients with PE by Western blot analysis and RT-qPCR. Interactions among IGF-1, ZEB1 and miR-183 were assessed by Western blot analysis, ChIP-PCR and dual-luciferase reporter gene assay. The effect of IGF-1 on the biological characteristics of trophoblast cells was investigated by CCK-8, colony formation assay and in vitro angiogenesis experiments after cells were transfected with si-IGF-1. Finally, a mouse eclampsia model induced by knockdown of IGF-1 was established to confirm the in vitro effect of IGF-1 on PE. We found that IGF-1, ZEB1 and miR-183 were highly expressed in the placental tissues of patients with PE. The knockdown of IGF-1 resulted in reduced proliferation and invasion of trophoblast cells and was accompanied by inhibited angiogenesis. ZEB1 was positively regulated by IGF-1 via ERK/MAPK pathway, which in turn inhibited miR-153 expression by binding to the miR-183 promoter. The in vitro experiments further confirmed that IGF-1 knockdown could induce PE. To sum up, IGF-1 knockdown elevated expression of miR-183 by downregulating ZEB1, thereby promoting deterioration of PE.

miR-2115-3p inhibits ferroptosis by downregulating the expression of glutamic-oxaloacetic transaminase in preeclampsia.

INTRODUCTION: Recently, ferroptosis has been reported to be closely related to preeclampsia (PE). However, the mechanisms associated with ferroptosis in PE have been poorly studied. METHODS: A PE rat model was established via reduced uterine perfusion pressure (RUPP) surgery. A Ferroptosis inhibitor was used to treat the model rats. Blood pressure, urine protein, sFlt-1, GSH, GSH-PX, MDA, total Fe, Fe (II), and Fe (Ⅲ) levels were detected. Placenta morphological changes and fetal survival rate were observed and counted, respectively. miRNA sequencing and bioinformatical analysis were conducted to establish the ferroptosis-related interaction network of miRNAs-mRNAs in PE. After hypoxia treatment, cells were silenced glutamic-oxaloacetic transaminase1 (GOT1) or overexpressed miR-2115-3p/GOT1. Cellular proliferation, invasion, and reactive oxygen species (ROS) were determined. The ferroptosis-related factors levels (ACSL4, TfR1, GPX4, SCL7A11, GSH, GSH-PX, Fe (II), and MDA) were quantified. RESULTS: In vivo, ferrostatin-1 attenuated ferroptosis in the PE models, significantly increasing fetal survivability. The miR-2115-3p/GOT1 pathway was screened for possible association with abnormal ferroptosis in PE. miR-2115-3p was discovered to interact with the mRNA of GOT1. Inhibition of GOT1 and overexpression of miR-2115-3p reversed the decrease in cell proliferation capacity, GSH, GSH-PX, and GPX4 levels, and the increase in ROS, ACSL4, TfR1, MDA, total Fe, and Fe (II) levels induced by hypoxia. However, simultaneous overexpression of miR-2115-3p and GOT1 reversed the above results of overexpression of miR-2115-3p. DISCUSSION: miR-2115-3p might interact with the GOT1 mRNA to downregulate its expression, further inhibiting the hypoxia-promoted ferroptosis in a PE model.

Elevated MicroRNA 183 Impairs Trophoblast Migration and Invasiveness by Downregulating FOXP1 Expression and Elevating GNG7 Expression during Preeclampsia.

Preeclampsia (PE) is a hypertensive disorder of uncertain etiology that is the leading cause of maternal and fetal morbidity or mortality. The dysregulation of microRNAs (miRNAs) has been highlighted as a potential factor involved in the development of PE. Therefore, our study investigated a novel miRNA, miRNA 183 (miR-183), and its underlying association with PE. Expression of miR-183, forkhead box P1 (FOXP1), and G protein subunit gamma 7 (GNG7) in placental tissues of patients with PE was determined. Gain- and loss-of-function experiments were conducted to explore modulatory effects of miR-183, FOXP1, and GNG7 on the viability, invasion, and angiogenesis of trophoblast cells in PE. Finally, we undertook in vivo studies to explore effects of FOXP1 in the PE model. The results revealed suppressed expression of FOXP1 and significant elevations in miR-183 and GNG7 expression in placental tissues of PE patients. FOXP1 was observed to promote proliferation, invasion, and angiogenesis in human chorionic trophoblastic cells. miR-183 resulted in depletion of FOXP1 expression, while FOXP1 was capable of restraining GNG7 expression and promoting the mTOR pathway. The findings confirmed the effects of FOXP1 on PE. In conclusion, miR-183 exhibits an inhibitory role in PE through suppression of FOXP1 and upregulation of GNG7.

GNG7 silencing promotes the proliferation and differentiation of placental cytotrophoblasts in preeclampsia rats through activation of the mTOR signaling pathway.

Preeclampsia (PE) is a pathological condition that manifests during pregnancy as the occurrence of an abnormal urine protein level and increased blood pressure due to inadequate cytotrophoblast invasion. To elucidate the mechanism underlying PE, the present study primarily focused on the regulatory effects and mechanism of the G protein γ 7 (GNG7) on placental cytotrophoblasts in a rat PE model. Initially, the PE model was established with 45 specific pathogen­free Sprague­Dawley rats (30 females and 15 males). The expression patterns of GNG7, 4E­binding protein 1 (4E­BP1), phosphoprotein 70 ribosomal protein S6 kinase (p70S6K) and mammalian target of rapamycin (mTOR) were examined in the PE rats. Placental cytotrophoblasts isolated from normal and PE rats were treated with a small interfering RNA against GNG7, mTOR signaling pathway activator (HIV­1 Tat) or inhibitor (rapamycin). Following treatment, cell proliferation, differentiation and apoptosis were evaluated, and mTOR signaling pathway­related factors (4E­BP1, p70S6K and mTOR), cell proliferation­related factors (vascular endothelial growth factor and transforming growth factor­ß1), differentiation­related factors [activator protein­2 (AP­2)α and AP­2γ], and apoptosis­related factors [B­cell lymphoma 2 (Bcl­2) and Bcl­2­associated X protein] were determined. Finally, soluble fms­like tyrosine kinase 1 (sFlt­1) and soluble endoglin (sEng) levels were measured via enzyme­linked immunosorbent assay. Initially, the mTOR signaling pathway was inactivated in the placental tissues and cytotrophoblasts in the PE rats. Silencing GNG7 reduced the levels of sFlt­1 and sEng and activated the mTOR signaling pathway. Silencing of GNG7 or activation of the mTOR signaling pathway enhanced cell proliferation and differentiation, but inhibited the apoptosis of placental cytotrophoblasts in the PE rats. Taken together, the results showed that GNG7 silencing repressed apoptosis and enhanced the proliferation and differentiation of placental cytotrophoblasts in PE rats through activation of the mTOR signaling pathway.

Mechanism of endogenous digitalis-like factor­induced vascular endothelial cell damage in patients with severe preeclampsia.

Although endogenous digitalis­like factor (EDLF) is associated with the development of various physical disorders, the role in preeclampsia remains unclear. This study investigated the effects of EDLF on vascular endothelial cell damage in patients with preeclampsia and the potential mechanisms. From July 2014 to July 2015, 120 singleton pregnancy cases underwent a prenatal examination, inpatient delivery and had normal blood pressure were included in the study, either as patients with severe preeclampsia or the control patients. Serum EDLF levels were compared in these two groups, and an in vitro hypoxic trophocyte­induced vascular endothelial cell damage model was established to explore the changes in hypoxic trophocyte EDLF level and the subsequent effects on human umbilical vein endothelial cells (HUVECs). Nuclear factor­κB (NF­κB) p65 gene expression was silenced in hypoxic trophocytes, and EDLF levels and HUVEC damage were subsequently assessed. Serum EDLF levels were significantly higher in the severe preeclampsia cases than in the controls at the same gestational week (P<0.001). EDLF levels in hypoxic trophocytes increased with the increasing co­culture duration. Damage to the biofunctions of HUVECs co­cultured with hypoxic trophocytes also increased with co­culture duration. However, silencing of NF­κB p65 in the hypoxic trophocytes reduced the EDLF levels. Annexin A2 was highly expressed in HUVECs, and no biofunctions were significantly damaged (P<0.05) compared with the group without receiving NF­κB p65 silencing. Serum EDLF levels were significantly higher in patients with severe preeclampsia compared with the controls. The results of the current study indicate that NF­κB p65 has a role in regulating EDLF production in hypoxic trophocytes.

Tegafur Substitution for 5-Fu in Combination with Actinomycin D to Treat Gestational Trophoblastic Neoplasm.

Although 5-fluorouracil (5-Fu) combination chemotherapy provides a satisfactory therapeutic response in patients with gestational trophoblastic neoplasms (GTNs), it has severe side effects. The current study analyzed the therapeutic effects and side effects of tegafur plus actinomycin D (Act-D) vs. 5-Fu plus Act-D for the treatment of GTNs based on controlled historical records. A total of 427 GTN cases that received tegafur and Act-D combination chemotherapy at the Second Xiangya Hospital of XiangYa Medical School between August 2003 and July 2013 were analyzed based on historical data. A total of 393 GTN cases that received 5-Fu plus Act-D between August 1993 and July 2003 at the same hospital were also analyzed, which constituted the control group. The therapeutic effects, toxicity and side effects after chemotherapy were compared between the groups. The overall response rate was 90.63% in the tegafur+Act-D group (tegafur group) and 92.37% in the 5-Fu+Act-D group (5-Fu group); these rates were not significantly different (P > 0.05). However, the incidence rates of myelosuppression (white blood cell decline), gastrointestinal reactions (nausea, vomiting, dental ulcer, and diarrhea), skin lesions and phlebitis were lower in the tegafur group than in the 5-Fu group (P < 0.05). The results of this study may provide useful data for the clinical application of tegafur in GTN treatment.

Corticotropin-releasing hormone activates connexin 43 via activator protein-1 transcription factor in human myometrial smooth muscle cells.

Corticotropin-releasing hormone (CRH) and connexin 43 (Cx43) play crucial roles in uterine contraction and the onset of labor. The aim of the present study was to investigate the regulatory effects of CRH on Cx43 expression in human myometrial smooth muscle cells (SMCs) and, potentially, its activation of the c-Fos/activator protein (AP)-1 signaling pathway. Human myometrial SMCs collected from nonpregnant women were treated with different concentrations of CRH. Transient transfection of AP-1 decoy oligodeoxynucleotide (ODN) was used to block AP-1 sites of Cx43. The transcriptional activity of AP-1 was detected by luciferase assay. Cx43 protein expression was visualized by immunofluorescence staining. mRNA and protein expression of c-Fos and Cx43 were demonstrated by real-time quantitative RT-PCR and Western blot, respectively. CRH facilitated Cx43 expression and enhanced AP-1 promoter activity in human uterine SMCs. After CRH treatment, Cx43 expression in the cytoplasm increased significantly. CRH significantly increased mRNA and protein expression of c-Fos and Cx43 in a dose-dependent manner (P < 0.01). A transient transfection of AP-1 decoy ODN did not affect CRH regulation of c-Fos (P > 0.05) but almost completely abolished CRH-induced enhancement of Cx43 expression (P < 0.01). In human primary myometrial SMCs, CRH enhances Cx43 mRNA and protein expression through upregulation of c-Fos expression. Blockade of AP-1 sites to the Cx43 promoter can neutralize the CRH-induced upregulation of Cx43.