Regulation of Keap-1/Nrf2 Signaling Pathway Is Activated by Oxidative Stress in Patients with Premature Rupture of Membranes

Abstract

BACKGROUND The potential mechanisms underlying premature rupture of membrane (PROM) is still unknown. The aim of this study was to determine the role of Keap-1/Nrf2 signaling pathway activation by oxidative stress in patients with preterm premature rupture of membranes. MATERIAL AND METHODS Placental tissues from preterm premature rupture of membranes (PPROM) (n=20), full-term premature rupture of membranes (FPROM) (n=20), and normal-term births (n=20) were collected and amniotic tissues were separated from the placental tissues from pregnant women at Shandong Provincial Qianfoshan Hospital. RT-PCR and Western blot were used to detect the levels of factors in the Keap-1/Nrf2 signaling pathway. To investigate the roles of Nrf2, we downregulated Nrf2 expression using siRNA in primary human amniotic epithelial (HAE) cells. RESULTS Among the control group, FPROM group, and PPROM group, the reactive oxygen species (ROS) levels were significantly increased in the FPROM and PPROM groups. The differences indicated higher levels of oxidative stress in amniotic tissues with FPROM and PPROM after downregulation of si-Nrf2 in HAE cells. Antioxidants were lower in amniotic tissues with the FPROM group and PPROM group than in the control group. The antioxidant enzymes catalase (CAT), glutathione (GSH), glutathione peroxidase (GSHPx), and superoxide dismutases (SOD1 and SOD2) were examined in amniotic tissues. We found that the ROS levels were significantly increased after downregulation of si-Nrf2 compared with the control group. We found that the expression of Heme Oxygenase-1 (HO-1) and Glycogen Synthase Kinase-3ß (GSK-3ß), which is critical in the Keap-1/Nrf2 signaling pathway, increased significantly after downregulation of si-Nrf2 in HAE cells. CONCLUSIONS We found that increased ROS levels and decreased antioxidant enzymes in the PPROM and FPROM patients compared with the control group.

Figure 1

The ROS levels were detected and compared in the control group (A), FPROM group (B), and PPROM group (C) (P<0.01).

Figure 2

(A–F) Antioxidants were detected among the control group, FPROM, and PPROM group; (G) Keap-1 mRNA levels were significantly different in the control group, FPROM group, and PPROM group (P<0.05); (H) Nrf2 mRNA levels were significantly different in the control group, FPROM group, and PPROM group (P<0.05).

Figure 3

(A–C) Expressions of Keap-1 and Nrf2 were significantly different in the control group, FPROM group, and PPROM group as assessed by Western blot (P<0.05); The ROS levels were detected and compared in the control group (D) and si-Nrf2 group (E) (P<0.01).

Figure 4

Knockdown of the expression of si-Nrf2 significantly increased the expression of Keap-1 (A) and decreased the expression of Nrf2 (B), HO-1 (C), and GSK-3β (D) at both mRNA and protein levels.