Effects of Nrf-2 expression in trophoblast cells and vascular endothelial cells in preeclampsia

Abstract

The present study aimed to explore the role of kelch-like ECH-associated protein-1 (Keap1)/Nuclear factor erythroid 2-related factor 2 (Nrf-2) signaling pathway in regulating heme oxygenase-1 (HO-1) expression in adverse outcomes of preeclampsia (PE). Adult Wistar rats, HTR-8/SVneo and hESC cells were used for models in vitro and in vivo, respectively. Inhibition of Nrf-2 could slightly reduce the elevation of systolic blood pressure (SBP) and urinary protein in PE rats. The percentages of dead fetuses during pregnancy and within seven days of birth were decreased by Nrf-2 inhibitor. There was no significant effect on the pathology and HO-1 expression of Nrf-2 in placental tissue. Deficiency of Nrf-2 increased significantly the levels of chemokine 2 (CCL2), interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), angiotensin II receptor type 1 (AT1R) and reactive oxygen species (ROS) in the embryonic tissues. Knockdown of Nrf-2 suppressed cell proliferation, improved cell apoptosis and invasion with an increase of ROS and HO-1, but the effect on cells apoptosis was greater. Activation of Nrf-2 pathway could reduce oxidative stress in PE rats and trophoblast cells induced by Ang II, and enhance the adverse outcome of PE via increasing HO-1. Nrf-2 silence reshaped blood vessels and achieved the effect of treating PE. Our results might provide theoretical guidance for the application of Nrf-2 in the treatment of PE.

Keywords: Preeclampsia; apoptosis; heme oxygenase-1; nuclear factor erythroid 2-related factor 2; oxidative stress.

Figure 1

The effects of Nrf-2 on SBP, proteinuria and fetal weight. The level of SBP (A) and 24 h proteinuria (B) in rats of different groups on the day 6, 12 and 21 of pregnancy. The fetal weight (C) of rats in different groups. The rate of dead fetuses of rats during pregnancy (D) and within 7 days after birth (E) in different groups. n=8, *P<0.05, **P<0.01 and ***P<0.001 vs. Control; #P<0.05 and ###P<0.001 vs. PE. SBP, systolic blood pressure; CDDO-Im, Nrf-2 agonist; ML385, Nrf-2 inhibitor.

Figure 2

The effect of Nrf-2 on the changes in placental pathology and HO-1 expression. A. Representative photographs of HE-stained placental sections (200 ×). B. Immunohistochemical detection of HO-1 (brow) in the placental tissue in different groups (200 ×). C. The expression of HO-1 in the placental tissue in different groups were examined by western blot. n=8. Scale bar =100 μm. CDDO-Im, Nrf-2 agonist; ML385, Nrf-2 inhibitor.

Figure 3

The effect of Nrf-2 on the level of CCL2, IL-1β, TNF-α, AT1R and ROS in placental tissue. A. The expression of CCL2, IL-1β, TNF-α, and AT1R of placental tissue in different groups were examined by western blot. B. The level of ROS in placental tissue was detected using ELISA kits. n=8, *P<0.05, **P<0.01 and ***P<0.001 vs. Control; #P<0.05 and ###p<0.001 vs. PE. ROS, Reactive oxygen species; CDDO-Im, Nrf-2 agonist; ML385, Nrf-2 inhibitor.

Figure 4

Validation of transfection efficiency of Nrf-2 overexpressed or low-expressed plasmids in human trophoblast cells (HTR-8/SVneo) and human normal endometrial stromal cells (hESC). A, B. The analysis results of RT-qPCR for Nrf-2 in HTR-8/SVneo and hESC cells after transfected with siRNA-Nrf-2-1 and siRNA-Nrf-2-2 plasmids. C, D. Western blot images and quantitative analyses of Nrf-2 in HTR-8/SVneo and hESC cells. E, F. The analysis results of RT-qPCR for Nrf-2 in HTR-8/SVneo and hESC cells after transfected with siRNA-Keap-1 and siRNA-Keap-2 plasmids. G, H. Western blot images and quantitative analyses of Nrf-2 in HTR-8/SVneo and hESC cells. *P<0.05, **P<0.01 and ***P<0.001 vs. Normal; #P<0.05, ##P<0.01 and ###P<0.001 vs. siRNA-NC.

Figure 5

The effect of Nrf-2 on cell proliferation of HTR-8/SVneo and hESC cells. In HTR-8/SVneo cells (A) and hESC cells (B) transfected with siRNA-Nrf-2-1 and siRNA-Keap1-1, a three-day MTT assay was carried out to detect the proliferation rates. (C, D) Western blot images and quantitative analyses of PCNA and Ki67 expressions in HTR-8/SVneo and hESC cells. *P<0.05, **P<0.01 and ***P<0.001 vs. Normal; #P<0.05, ##P<0.01 and ###P<0.001 vs. Angll.

Figure 6

The effect of Nrf-2 on cell apoptosis and ROS level in HTR-8/SVneo and hESC cells. After staining with Annexin V-FITC and PI, apoptotic cells in HTR-8/SVneo cells (A) and hESC cells (B) transfected with siRNA-Nrf-2-1 and siRNA-Keap1-1 were analyzed using a flow cytometer. Histograms of data statistics of A and B are showed in (C) and (E). The level of ROS in HTR-8/SVneo cells (D) and hESC cells (F) transfected with siRNA-Nrf-2-1 and siRNA-Keap1-1 was detected using ELISA kits. *P<0.05, **P<0.01 and ***P<0.001 vs. Normal; #P<0.05, ##P<0.01 and ###P<0.001 vs. Angll. ROS, Reactive oxygen species.

Figure 7

The effect of Nrf-2 on cell invasion and the related proteins in HTR-8/SVneo and hESC cells. Representative images of Transwell membranes (magnification, × 200; stain 0.05% crystal violet) and quantification of migrated cells of HTR-8/SVneo (A) and hESC cells (B) in each group. Scale bar =100 μm. Western blot analysis was performed to detect the expression levels of MMP2 and MMP9, and statistical analysis of relative expression in HTR-8/SVneo (C) and hESC (D) cells in different groups. *P<0.05, **P<0.01 and ***P<0.001 vs. Normal; #P<0.05, ##P<0.01 and ###P<0.001 vs. Angll.

Figure 8

The effect of Nrf-2 on the expression of related molecules in HTR-8/SVneo and hESC cells. Western blot analysis was performed to detect the expression levels of Nrf-2, HO-1, VEGFR, TNF-α, Bax, Bcl-2 and Cleaved caspase-3 proteins and statistical analysis of relative these proteins expression in HTR-8/SVneo (A) and hESC (B) cells in different groups. *P<0.05, **P<0.01 and ***P<0.001 vs. Normal; #P<0.05, ##P<0.01 and ###P<0.001 vs. Angll.