Trophoblast function is the key to normal placental development and successful pregnancy. After embryo implantation, cytotrophoblasts proliferate and differentiate into syncytial trophoblasts or extravillous trophoblasts (EVTs). Then, EVTs invade the mother's decidua and the uterine spiral artery to reshape the blood vessels to provide enough blood and nutrients for the fetus. Studies have shown that trophoblast proliferation, invasion, and migration and restricted angiogenesis are the key reasons for the occurrence of preeclampsia. We found that IL-6 levels were increased in the placental tissue of the preeclampsia group; we also observed that on the one hand, IL-6 can inhibit the proliferation and invasion of gestational trophoblast cells, and on the other hand, it can promote angiogenesis. which indicates that the mechanisms underlying the role of IL-6 in the pathogenesis of preeclampsia cannot be explained via a single mode. Therefore, we further screened the possible downstream regulators of IL-6 involved in the pathogenesis of preeclampsia through TMT-based proteomics analysis and provided new ideas for perfecting the pathophysiological study of preeclampsia.
IL-6 is a typical cytokine involved in inflammation and immune response. At the same time, IL-6 has a close relationship with pregnancy and preeclampsia. However, there is still controversy about whether the level of IL-6 in preeclampsia is elevated. Some studies have shown that there is no significant difference between the expression of IL-6 in placental tissues during preeclampsia and normal pregnancy, and other studies have found that IL-6 level increases  or decreases in preeclampsia. However, these studies only revealed the relationship between the IL-6 expression level and preeclampsia and did not thoroughly explore the mechanism whereby IL-6 participates in the pathogenesis of preeclampsia. We found that the IL-6 level was significantly increased in the placental tissue of the preeclampsia group, which is consistent with the results of the study by Ruby Aggarwal et al.. At the same time, our study also found that IL-6 can inhibit the proliferation and invasion of trophoblasts, which mediates the occurrence of disease. Trophoblast proliferation and invasion can lead to insufficient spiral arterial remodeling and insufficient blood perfusion, which are important causes of preeclampsia. Most research reports have shown that IL-6 promotes the proliferation and invasion of tumor cells , but it has an inhibitory effect on the invasion of trophoblast cells, which indicates that the mechanism whereby IL-6 inhibits the proliferation and invasion of trophoblast cells is quite different from that in case of tumor cells. The TMT-based proteomics analysis showed that the proteins encoded by FLNB and DKK1 were significantly downregulated in the IL-6 stimulation group in our study. FLNs are networked cytoskeletal proteins connected to the cell membrane surface and play an important role in the integration of cell mechanics and signal transduction. Jufeng Wei et al. found that the mRNA and protein expression levels of FLNB decreased significantly in the placenta tissues during preeclampsia. The downregulation of FLN-b has been reported to inhibit the ERK/MMP-2 and MMP-9 pathways, leading to a reduction in the invasion ability of trophoblasts in the placenta during preeclampsia .DKK1 (Dickkopf-1) is a secreted glycoprotein closely related to Wnt signaling. Mariz Kasoha, et al. reported that the expression level of DKK1 decreased significantly in the placenta tissue of patients with preeclampsia, and its decreased level was closely related to the severity of the disease。
At the same time, using KEGG pathway enrichment analysis, we found that the involvement of IL-6 in the pathogenesis of preeclampsia may be related to the Wnt signaling pathway. In the process of placenta formation, there are a variety of signaling pathways involved in regulating the proliferation, differentiation, invasion, and apoptosis of trophoblasts, among which the Wnt signaling pathway is an important one. In addition, the Wnt signaling pathway is also involved in placental implantation. Its excessive activation can lead to trophoblast cell tumors . In recent years, many studies have shown that the Wnt signaling pathway is involved in the invasion of trophoblast cells during pregnancy . Xiaofang Wang et al. detected Wnt1 and β-catenin, the key molecules of the Wnt signaling pathway in placental tissues, and found lower expression of these proteins in preeclampsia patients, further indicating that the Wnt/β-catenin signaling pathway may regulate the invasion and proliferation of trophoblasts, which play an important role in the pathogenesis of preeclampsia.
We co-cultured HTR-8/SVneo cells with HUVECs and found that IL-6 can promote angiogenesis in our study. Preeclampsia is related to superficial placental implantation and oxidative stress induced by ischemia-reperfusion, which leads to increased levels of pro-inflammatory cytokines, excessive inflammatory response, and unbalanced angiogenesis. These changes result in endothelial cell dysfunction and inflammation, which ultimately led to poor maternal and fetal outcomes . IL-6 is a pleiotropic cytokine with pro-inflammatory and anti-inflammatory functions. Its pro-angiogenesis effect has long been reported . However, these findings are inconsistent with the pathophysiological characteristics of angiogenesis inhibition in preeclampsia, which suggests that the participation of IL-6 in the pathogenesis of preeclampsia does not represent a simple negative regulation; its role in promoting angiogenesis may also be one of the factors influencing the protection of trophoblast cell function. The TMT-based proteomics analysis showed that after IL-6 stimulation, the protein encoded by U2AF2 in the supernatant was significantly downregulated. sFLT-1 (soluble Fms-like tyrosine kinase 1) is a negative regulator of angiogenesis; it plays an important role in the pathogenesis of preeclampsia. U2AF2 can splice FLT-1 into sFLT-1, inhibiting angiogenesis, leading to endothelial dysfunction.The role of IL-6 in promoting angiogenesis may be related to the downstream pathways inhibiting the expression of U2AF2 and reducing the production of sFLT-1.
In conclusion, we found that IL-6 has a dual role in regulating gestational trophoblast cell function, involving multi-protein and multi-pathway interactions. Further, proteomics screening represents a potential macroscopic measure for studying the molecular mechanisms and prognostic targets associated with the pathogenesis of preeclampsia and subsequently, treatment strategies. However, this study is only an in vitro cytology study; the specific mechanism underlying the participation of IL-6 in preeclampsia needs to be further explored and verified. In future research, we can also establish animal models of preeclampsia to further explore the regulation of trophoblast cell function by IL-6 in vivo and to verify the role of FLNB, DKK1, and U2AF2 and other proteins from different pathways in preeclampsia.