Notch signaling pathway regulates the progression of fetal growth restriction through mediating immune dysfunction

Abstract

Fetal growth restriction (FGR) is associated with an increased risk of neonatal morbidity and mortality, as well as the development of metabolic syndrome in adulthood. The present study investigated the regulatory mechanisms of Notch signaling in FGR progression. The expression levels of Notch1 and Jagged1 were determined using reverse transcription-quantitative PCR, western blotting, immunofluorescence staining and immunohistochemistry (IHC). ELISA was used to measure the concentrations of IL-10, IL-17 and IL-35 in serum and placental samples. ELISA and western blotting determined the inflammation- and angiogenesis-related cytokine levels. Th17, Treg and macrophage levels were determined using IHC and flow cytometry. Additionally, hematoxylin & eosin staining and TUNEL assay assessed placenta histology and trophoblast cell apoptosis. Significant trophoblast apoptosis was observed in the placenta of FGR pregnancies. The expression of Notch1 and Jagged1 in peripheral blood mononuclear cells and placental tissues of FGR pregnancies was significantly lower than in the control group. The FGR group exhibited a remarkable inflammation, anti-angiogenesis and immune dysfunction. In conclusion, the Notch signaling pathway mediates immune balance to regulate the development of FGR. These findings offer the potential for advancing innovative predictive, diagnostic and therapeutic approaches for FGR.

Keywords: Jagged1; Notch1; fetal growth restriction; immune balance; inflammation.

Figure 1

Placenta histopathology and trophoblast cell apoptosis in patients with FGR. (A) Placenta histopathology of patients with FGR were examined through H&E staining. Scale bar, 100 µm. (B) Trophoblast cell apoptosis was determined using TUNEL assay. ^***P<0.001 vs. control. Scale bar, 100 µm. FGR, fetal growth restriction.

Figure 2

The expression and distribution of Notch1 and Jagged1 is decreased in PBMCs and placenta. (A-E) The expression of Notch1 and Jagged1 in PBMCs and placenta was detected by (A and B) reverse transcription-quantitative PCR, (C) western blotting, (D) immunofluorescence and (E) immunohistochemistry, respectively. ^**P<0.01 and ^***P<0.001 vs. control. Scale bar, 100 µm. PBMCs, peripheral blood mononuclear cells; FGR, fetal growth restriction.

Figure 3

Inflammation- and angiogenesis-factors in serum and placental tissue of FGR pregnancies. (A and B) The levels of TNF-α, IL-6, CXCL1, sFlt-1, VEGF, PLGF and PP13 in serum and placenta of FGR pregnancies were determined via (A) ELISA and (B) western blotting. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. control. FGR, fetal growth restriction; CXCL1, C-X-C motif chemokine ligand 1; sFlt-1, soluble fms-like tyrosine kinase-1; VEGF, vascular endothelial growth factor; PLGF, placental growth factor; PP13, placental protein 13.

Figure 4

Immune dysfunction in patients with FGR. (A) Th17 and Treg frequencies were assessed through flow cytometry. (B) The expression of CD86 (M1 macrophage marker), CD206 (M1 macrophage marker), Foxp3 (Treg marker) and CD3 (Th17 marker) was evaluated using immunohistochemistry. Scale bar, 100 µm. (C) Serum and (D) placental levels of IL-10, IL-17, and IL-35 were measured by ELISA. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. control. FGR, fetal growth restriction; Th17, T helper 17 cells; Treg, regulatory T cells; Foxp3, Forkhead Box protein 3.