Daphnetin alleviates unexplained recurrent spontaneous abortion by regulating the NR4A1/BACH2 axis in mice.

BACKGROUND: Daphnetin has demonstrated various pharmacological activities. The current study evaluated the potential of daphnetin in alleviating unexplained recurrent spontaneous abortion (URSA) and explored underlying mechanisms. METHODS: Mice with URSA were gavaged with 1 mg/kg, 10 mg/kg, and 20 mg/kg of daphnetin, or infected with adeno-associated viruses harboring knockdown of NR4A1 or overexpression of BACH2 before modeling. Human peripheral blood T lymphocytes were induced into CD4(+) T cells, followed by lentivirus infection and daphnetin treatment. The influence of daphnetin on CD4(+) T cell viability and Treg and Th17 cell differentiation in cells was analyzed. The concentrations of Treg cells-associated cytokines (TGF-β, IL-10) and Th17 cells-associated cytokines (IL-17, IL-23) in the supernatants of CD4(+) T cells were assessed. The regulation of NR4A1 on BACH2 was analyzed by ChIP and dual-luciferase assays. RESULTS: Daphnetin resulted in fewer immature, resorbed, or dead embryos in mice with URSA, with the most pronounced therapeutic effect of 10 mg/kg. Daphnetin attenuated decidual hemorrhage, with a gain in the percentage/number of Treg cells and a loss of the percentage/number of Th17 cells in the spleen and decidual tissues. Daphnetin enhanced the expression of FoxP3, TGF-β, and IL-10, and suppressed the expression of RORγt, IL-17, IL-23, and the contents of TNF-α, IL-6, and IL-1β in CD4(+) T cells. Overexpression of BACH2 further alleviated URSA deterioration caused by NR4A1 knockdown. Daphnetin mediated the transcriptional activation of BACH2 by upregulating NR4A1. CONCLUSIONS: Upregulation of NR4A1 by daphnetin mediates BACH2 transcription and Th17/Treg cell homeostasis to improve URSA.