Abstract
Miscarriage is a common condition during pregnancy and its mechanisms remain largely unknown. Extravillous trophoblast (EVT) cell invasion is required to maintain normal pregnancy and its malfunction has been proposed as a major cause for miscarriage. Homeostasis of matrix metalloproteinase 9 (MMP9) is a key to regulate EVT cell invasion. Total flavonoids from Semen Cuscutae (TFSC) have been applied clinically used for preventing or treating miscarriage in the past. Given its potential clinical benefit on preventing miscarriage, this study aims at examining the therapeutic effect of TFSC in the prevention of premature birth by upregulating MMP9 and promote EVT cell invasion. HTR-8 cells migration and invasion functions were analyzed using wound healing and transwell assays. The regulatory effect of TFSC on MMP9 expression and relevant signaling pathways were analyzed by Western Blot. The results show compared to control group, TFSC significantly promoted the migration of EVT cells in a dose and time-dependent manner. The migration and invasion of EVT cells were maximized at the highest dosage of 5 μg/ml of TFSC. The expression of MMP9 in EVT cells was significantly increased after TFSC treatment. Furthermore, cells treated with TFSC significantly upregulated protein expressions in Notch, AKT and p38/MAPK signaling pathways. We believe TFSC can promote the migration and invasion of EVT cells by increasing MMP9 expression, and prevent miscarriage by activating Notch, AKT, and MAPK signaling pathways.
Highlights
During embryogenesis, the synergistic functions of proliferation, migration and invasion of Extravillous trophoblast (EVT) cells play essential role to ensure nutrient intake of embryos and normal pregnancy[1]
In order to better understand the pharmacodynamics of total flavonoids from Semen Cuscutae (TFSC), in this study we investigated how TFSC regulate the migration and invasion abilities of HTR-8 EVT cells, and its role in matrix metalloproteinase 9 (MMP9) expression and downstream signaling cascades such as Notch, AKT and MAPK pathways involved
The results showed that the proliferation rate of EVT cells were similar in all treatment groups and TFSC did not affect the viability of EVT cells indicating that TFSC had no significant toxicity effect on EVT cells (Fig. 2)
Summary
The synergistic functions of proliferation, migration and invasion of EVT cells play essential role to ensure nutrient intake of embryos and normal pregnancy[1]. Semen Cuscutae is widely used in China, and total flavonoids from Semen Cuscutae (TFSC) is a main estrogenic active constituent of the plant which include hyperoside, quercetin, kaempferol and rutin, isorhamnetin[9,10] It could improve liver and kidney function, treat impotence and seminal, improve testosterone level (AR level) and androgen receptor gene expression, reverse the kidney-yang deficiency and prevent miscarriage[9,11]. It has been reported that PIM2 upregulated COX-2 and MMP9 expression via PI3K signaling pathway, which was in turn mediated by Notch[118] In spite of these evidences, studies on the relationship between MMP9 and the regulating effects of Notch, PI3K/AKT and MAPK signaling pathways in EVT cell invasion remain scarce and more in-depth analysis is needed to uncover the underlying molecular mechanism
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