Baicalein Attenuated Recurrent Pregnancy Loss by Inhibiting Ferroptosis via Activation of Nrf2/GPx4 Axis

Abstract

Objective: To verify effect of baicalein on recurrent pregnancy loss (RPL) and explore its mechanism via inhibition of ferroptosis.

Methods: In vivo, CBA/J (n=60) mated DBA/2 (n=50) mice were established as RPL group, while CBA/J mated BALB/c (n=10) mice were regarded as control group. Baicalein (10 and 40 mg/kg), ferroptosis inhibitor (ferrostatin-1, 5 mg/kg) and iron chelator (deferoxamine, 1 mg/kg) were administered in the RPL mice model (n=10 per group) from embryonic day 0.5-12.5 (E0.5-E12.5). Pregnancy outcomes and ferroptosis related markers were detected. Lipid peroxidation was assessed by malondialdehyde (MDA) and antioxidant system was determined by glutathione (GSH), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity. The Fe²⁺ concentration was tested to analyze iron accumulation and Western blotting was performed to detect key protein expressions. In vitro, different concentrations of baicalein (0.1, 0.2, and 0.4 µmol/L) were supplemented under the exposure of erastin in HTR-8/SVneo cells. Moreover, RSL3 and si-RNA were used to suppress the expression of glutathione peroxidase 4 (GPx4) or nuclear factor erythroid 2-related factor 2 (Nrf2), respectively, in HTR-8/SVneo cells. Cell viability, cytotoxicity, ferroptosis related markers, protein expressions of Nrf2 and GPX4 were detected in HTR-8/SVneo cells to determine the signaling pathway of baicalein against ferroptosis.

Results: Baicalein significantly attenuated fetal loss (P<0.01) and placental damage in RPL mice. Besides, baicalein reduced placental ferroptosis which manifested decreased MDA, iron content, Acyl-CoA synthetase long-chain family protein expression and enhanced GSH and GPx levels (P<0.01) as well as increased protein expressions that were resistant to ferroptosis (GPx4, SLC7A11 and Nrf2, P<0.01 or P<0.05). In vitro, different concentrations of baicalein restored a ferroptosis inducer-erastin induced HTR-8/SVneo cells damage and lipid peroxidation, but GPx4 inhibition diminished the protective effect of baicalein (P<0.01). Additionally, Nrf2 silencing notably decreased GPx4 expression (P<0.01) and abolished baicalein-mediated anti-ferroptosis effect in HTR-8/SVneo cells.

Conclusion: Baicalein has a protective effect on RPL via inhibiting ferroptosis through Nrf2/GPx4 signaling axis.

Keywords: Nrf2/GPx4; baicalein; ferroptosis; pregnancy loss; trophoblasts.