Journal: Zoological Research

Article Title: Rescuing fertility: C-Phycocyanin prevents ovarian damage through NRF2-mediated ferroptosis pathways in polycystic ovary syndrome models

doi: 10.24272/j.issn.2095-8137.2025.032

Figure Lengend Snippet: C-PC suppresses DHEA-induced ferroptosis in granulosa cells (GCs) GCs were exposed to varying concentrations of C-PC. A: Cell viability following exposure to C-PC (0–50 μmol/L) ( n =6). B, C: Total and nuclear GC lysates were extracted, and protein levels of HO-1, GPX4, xCT, and NRF2 were detected ( n =3). D, E: Confocal fluorescent staining was used to evaluate NRF2 and GPX4 expression levels. F, G: Quantification of mean fluorescence intensity of NRF2 and GPX4 ( n =6). H: Expression of genes related to ferroptosis ( ACSL4 and TFR1 ) determined by RT-qPCR ( n =3). I: Intracellular reactive oxygen species (ROS) and lipid ROS levels visualized using dihydroethidium (DHE) and BODIPY 493/591 fluorescent probes, respectively. J: Quantification of ROS and lipid ROS fluorescence intensity ( n =3). K: Intracellular ROS levels measured by flow cytometry ( n =3). L: Levels of MDA, SOD, and GSH determined by ELISA ( n =3). All experiments were independently repeated at least three times. Data are expressed as mean±SD. * : P <0.05; ** : P <0.01; *** : P <0.001 vs. Control group; # : P <0.05; ## : P <0.01; ### : P <0.001 vs. DHEA group.

Article Snippet: Various antibodies were purchased, including antibodies against 4-HNE (bs-6313R; Bioss, China), histone H3 (4499; Cell Signaling Technology, USA), β-actin (66009-1; Proteintech, China), xCT/SLC7A11 (26864-1; Proteintech, China), HO-1 (10701-1; Proteintech, China), GPX4 (YM8430; Immunoway, USA), and NRF2 (GTX103322; GeneTex, USA).

Techniques: Staining, Expressing, Fluorescence, Quantitative RT-PCR, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control

Journal: Zoological Research

Article Title: Rescuing fertility: C-Phycocyanin prevents ovarian damage through NRF2-mediated ferroptosis pathways in polycystic ovary syndrome models

doi: 10.24272/j.issn.2095-8137.2025.032

Figure Lengend Snippet: C-PC inhibits ferroptosis by modulating oxidative stress in DHEA-treated GCs A–D: GCs were preincubated with either ferrostatin-1 (Fer-1), a ferroptosis inhibitor (A, B), or deferoxamine (DFO), an iron chelator, for 1 h prior to supplementation with C-PC (2 h) and subsequent exposure to DHEA (10 μmol/L, 48 h) in DMEM. (C, D) Expression levels of GPX4, xCT, HO-1, and 4-HNE in GCs were analyzed by western blotting and quantified. E–H: Effects of N-acetylcysteine (NAC) and H 2 O 2 on DHEA-induced ferroptosis-related protein expression (E, G). Expression levels of GPX4, xCT, HO-1, and 4-HNE in GCs were analyzed using western blotting and quantified (F, H) ( n =3). Data are expressed as mean±SD. * : P <0.05; ** : P <0.01; *** : P <0.001 vs. Control group; # : P <0.05; ## : P <0.01; ### : P <0.001 vs. DHEA group; & : P <0.05 ; && : P <0.01 vs. DHEA+Fer-1/DFO/NAC/H 2 O 2 group.

Article Snippet: Various antibodies were purchased, including antibodies against 4-HNE (bs-6313R; Bioss, China), histone H3 (4499; Cell Signaling Technology, USA), β-actin (66009-1; Proteintech, China), xCT/SLC7A11 (26864-1; Proteintech, China), HO-1 (10701-1; Proteintech, China), GPX4 (YM8430; Immunoway, USA), and NRF2 (GTX103322; GeneTex, USA).

Techniques: Expressing, Western Blot, Control

Journal: Zoological Research

Article Title: Rescuing fertility: C-Phycocyanin prevents ovarian damage through NRF2-mediated ferroptosis pathways in polycystic ovary syndrome models

doi: 10.24272/j.issn.2095-8137.2025.032

Figure Lengend Snippet: NRF2 is a predicted key target of C-PC in ferroptosis regulation relevant to PCOS treatment A: Molecular docking model showing interaction between C-PC and NRF2 protein. B: Protein stability of NRF2 assessed by CETSA. C: NRF2 protein expression in GCs. D: ChIP-qPCR analysis of NRF2 enrichment on GPX4 and xCT promoter regions ( n =3). GCs were preincubated with C-PC (5 μmol/L, 2 h) prior to supplementation with ML385 (5 μmol/L, 1 h) and subsequent exposure to DHEA (10 μmol/L, 48 h) in DMEM. E: Western blotting was performed to assess expression levels of NRF2, HO-1, GPX4, xCT, and 4-HNE in GCs. F: Immunofluorescence images of GPX4 expression. Sca$le bar: 50 μm. G: Intracellular ROS and lipid ROS visualized using DHE and BODIPY 493/591 fluorescent probes, respectively. Scale bar: 20 μm. Values are expressed as mean±SD. *** : P <0.001 vs. Control group; ### : P <0.001 vs. DHEA group.

Article Snippet: Various antibodies were purchased, including antibodies against 4-HNE (bs-6313R; Bioss, China), histone H3 (4499; Cell Signaling Technology, USA), β-actin (66009-1; Proteintech, China), xCT/SLC7A11 (26864-1; Proteintech, China), HO-1 (10701-1; Proteintech, China), GPX4 (YM8430; Immunoway, USA), and NRF2 (GTX103322; GeneTex, USA).

Techniques: Expressing, ChIP-qPCR, Western Blot, Immunofluorescence, Control