PE is a common complication of pregnancy which is characterized by maternal endothelial cell dysfunction, hypertension and proteinuria in the 20th week (16). ROS production, oxidative stress and mitochondrial dysfunction have been various implicated as the underlying mechanism of PE and abnormal placental development(17), although the exact pathophysiology still unclear.
The most important function of the placenta is the exchange of nutrients and oxygen between a maternal and fetal tissues. Placental oxygen content lower than 2% triggers the initial human trophoblast invasion (18). Sustained hypoxia can promote inflammation, and inhibit angiogenesis and cell migration, suggesting a crucial pathological role in PE (2). In our study, we established an in vitro model of placental hypoxia by treating human trophoblasts with CoCl2 (19), which is known to stabilize hypoxia-inducible factor-1α (HIF-1α) in cells (20). Previous studies have shown that CoCl2 can mimic the hypoxic condition over a concentration range of 100–800 mM (13). Consistent with a previous report, EDA rescued the trophoblasts from CoCl2-induced toxicity, indicating that it can promote cell growth in the hypoxic placenta.
Short-term hypoxia in the placenta also increases the production of ROS, including peroxides, superoxides and hydroxyl radicals, which result in signficant protein, lipid and DNA damage(9, 21). Therefore, antioxidant supplements taken during pregnancy can mitigate oxidative stress in the placenta. However, most antioxidants have not been applied clinically due to their toxicity and variability(22). EDA (3-methyl-1-phenyl-2-pyrazoline-5-one) is a free radical scavenger(23) that alleviates oxidative stress-induced degeneration of both motor neurons and muscles in ALS patients (24) In addition, EDA inhibited ROS accumulation in oligodendrocyte precursor cells and alleviated the resulting damage (25). Consistent with these reports, we observed a marked increase in ROS following CoCl2 treatment, which was reversed by EDA. ROS generation is also closely associated with apoptosis induction. CoCl2 significantly upregulated the pro-apoptotic Bax and downregulated Bcl-2 that is known to decreases apoptosis by inhibiting the permeability transition complex (26). EDA reversed the CoCl2-induced apoptosis and restored the levels of the Bcl-2 family of proteins. Thus, EDA protected trophoblasts from CoCl2-induced cytotoxicity, which can be attributed its anti-oxidative and anti-apoptotic effects.
PI3K/AKT regulates multiple cellular processes such as cell division, autophagy, apoptosis, cell survival, cell growth and differentiation (27)(28). Overexpression of RBP4 increased HTR-8/SVneo cell proliferation and invasion via inhibition of the PI3K/AKT signaling pathway (29). Furthermore, hypoxia-induced oxidative stress (30) promotes the preeclamptic state through the PI3K/ AKT pathway (31). Consistent with this, we detected high levels of PI3K, p-AKT, AKT and HIF-1αin the placental tissues of PE patients relative to that of healthy subjects. EDA blocked CoCl2-induced activation of the PI3K/AKT pathway in the cultured trophoblasts, as indicated by changes in the levels of PI3K, p-AKT, AKT and HIF-1α. These results suggest that the PI3K/AKT pathway is the central target of EDA.
There are several limitations in our study that ought to be considered. Our sample size was relatively small, and larger cohorts are needed to validate our findings. In addition, the mechanism through which EDA inhibits the PI3K/AKT signaling pathway needs to be elucidated. The potential therapeutic effects of combining EDA with specific PI3K/AKT inhibitors also needs to be investigated. Furthermore, the tissue in this study lacked placenta histology and authenticated. Finally, the effect of EDA may be dependent on the cell type and/or influenced by the simulated anoxic environment. Further studies are needed to clarify these issues.
In conclusion, EDA protected trophoblasts against CoCl2-induced toxicity, apoptosis and oxidative stress by targeting the PI3K/AKT pathway, and is a promising therapeutic agent against PE.