Alpha-lipoic acid upregulates the PPARγ/NRF2/GPX4 signal pathway to inhibit ferroptosis in the pathogenesis of unexplained recurrent pregnancy loss

Abstract

Aim: With unknown etiology and limited treatment options, unexplained recurrent pregnancy loss (URPL) remains a thorny problem. Ferroptosis, a newly identified type of cell death, has been shown to be crucial in the development in reproductive disorders. This study aims to explore the specific mechanism of ferroptosis in URPL and to uncover whether alpha-lipoic acid (ALA) can inhibit ferroptosis, and then exert a protective effect in URPL.

Method: The decidua tissues of URPL and control patients who actively terminated pregnancy were collected. The CBA/J × DBA/2 murine models of URPL were established, and were randomly treated with peroxisome proliferator activated receptor γ (PPARγ) agonists (Rosiglitazone) and ALA. The CBA/J × BALB/c murine models of normal pregnancy were intraperitoneally injected with PPARγ inhibitors (T0070907). Here, we used reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH)/GSSG, and FeRhoNox-1 analysis to detect the level of ferroptosis. We used quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis to evaluate the mRNA level of PPARγ. Besides, western blot and immunofluorescence were utilized to test the expression profile of PPARγ/nuclear factor erythroid 2-related factor 2 (NRF2)/glutathione peroxidase 4 (GPX4).

Results: In this study, we found that iron deposition was increased in the decidual tissue of patients with URPL. Additionally, the changes in cell morphology, the level of ROS, MDA, GSH, and the expression of ferroptosis marker proteins NRF2/GPX4 confirmed activated ferroptosis in URPL. Besides, bioinformatics analysis combined with experiments confirmed that PPARγ was critical in triggering NRF2/GPX4 pathway in URPL. Furthermore, URPL mouse models were established, and the results showed that PPARγ/NRF2/GPX4-mediated ferroptosis was also significantly increased, which could be mitigated by ALA treatment.

Conclusion: Overall, these findings suggest that ferroptosis may play an important role in URPL, and ALA might be a promising therapeutic drug for improving pregnancy outcomes in URPL via targeting the PPARγ/NRF2/GPX4 pathway.

Keywords: PPARγ/NRF2/GPX4; alpha-lipoic acid; ferroptosis; unexplained recurrent pregnancy loss.

Figure 1

Increased ferroptosis in decidual tissue in URPL patients. (a) Fe²⁺ content was assessed by RhoNox-1 staining. Scale bar: 50 μm. (b) Ferroptosis in decidua was observed by TEM. The red arrow shows a decrease in mitochondrial volume and an increase in cavitation. Scale bar: 2 μm (left), 500 nm (right). (c)–(e). ROS, MDA, and GSH levels were observed by GSH/GSSG, MDA, and ROS assays. *P < 0.05 compared with the control group.

Figure 2

Expression of NRF2/GPX4 in decidual tissue of URPL patients decreased. (a) and (b) NRF2 and GPX4 were assessed by immunofluorescence. Scale bar: 50 μm. (c) NRF2 and GPX4 were assessed by western blot. *P < 0.05, **P < 0.01 compared with the control group.

Figure 3

Bioinformatics analysis revealed PPARγ-mediated ferroptosis in URPL through NRF2/GPX4. (a) Venn diagram shows URPL ferroptosis-related genes. (b) PPI analysis of the URPL ferroptosis-related genes. (c) KEGG of URPL ferroptosis-related genes. (d) Venn diagram shows the intersection gene between URPL ferroptosis-related genes and the transcription factor of NRF2.

Figure 4

Expression of PPARγ in decidual tissue of URPL patients decreased. (a) PPARγ was assessed by immunofluorescence. Scale bar: 50 μm. (b) mRNA level of PPARγ was assessed by qRT-PCR. *P < 0.05 compared with the control group.

Figure 5

PPARγ/NRF2/GPX4-mediated ferroptosis in URPL. (a) Fe²⁺ content was assessed by RhoNox-1 staining; the PPARγ, NRF2, and GPX4 were assessed by immunofluorescence. Scale bar: 50 μm. (b)–(d) ROS, MDA, and GSH levels were observed by ROS, MDA, and GSH assays. (e) and (f) Absorption of mouse embryos. *P < 0.05, **P < 0.01 compared with the control group, #P < 0.05 compared with the URPL group.

Figure 6

ALA improves ferroptosis in URPL by activating the PPARγ/NRF2/GPX4 pathway. (a) Fe²⁺ content was assessed by RhoNox-1 staining; the PPARγ, NRF2, and GPX4 were assessed by immunofluorescence. Scale bar: 50 μm. (b) PPARγ, NRF2, and GPX4 were assessed by western blot. (c)–(e) ROS, MDA, and GSH levels were observed by ROS, MDA, and GSH assays. (f) and (g) Absorption of mouse embryos. *P < 0.05, **P < 0.01 compared with the control group, #P < 0.05 compared with the URPL group, ##P < 0.01 compared with the URPL group.