Fig 2: ML385 inhibits the neuroprotective effect of AS-IV at 24 h post-SAH. (A–C) Modified Garcia scores, beam balance scores, and brain water content 24 h after SAH in rats (n = 8). (D,E) Representative images and quantitative analysis of FJB staining in right temporal cerebral cortex of rats (n = 6, scale bar = 50 µm). (F) Quantification of iron concentrations in rats after SAH (n = 8). (G–I) Quantitative analysis of lipid ROS, MDA, and GSH levels (n = 8). (J) Representative Western blot images of SLC7A11 and GPX4. (K) Quantitative analysis of SLC7A11 and GPX4 (n = 6). Data are represented as mean ± SD. *p < 0.05, NS: not statistically significant; DMSO: dimethyl sulfoxide; FJB, Fluoro-Jade B.

Fig 3: AS-IV and Fer-1 treatment attenuated ferroptosis 24 h after SAH. (A) Quantitative analysis of iron concentrations after SAH (n = 8). (B–D) Quantitative analysis of lipid ROS, MDA, and GSH levels (n = 8). (E–G) Western blot analysis of protein expression levels of SLC7A11 and GPX4 (n = 6). (H) Typical double immunofluorescence images of GPX4 and NeuN (scale bar = 50 µm). Data are represented as mean ± SD. *p < 0.05, NS: not statistically significant.

Fig 4: NRF2-mediated autophagy dysfunction and ferroptosis are involved in foam cell formation induced by HUA. THP-1 and RAW264.7 cells were treated with oxLDL (100 µg/ml) or coincubated with UA (15 mg/dl) for 24 h. (a and b) qPCR was used to detect the transcription of lipid metabolism-related genes (CD36, ABCA1, and ABCG1) and ferroptosis-related genes (NFE2L2, SLC7A11, and GPX4) in macrophage-derived foam cells. (c and d) Representative western blot images of the protein level of the NRF2/SLC7A11/GPX4 signaling pathway and autophagy-related proteins (LC3B and p62). (e and f) Quantification of NRF2, SLC7A11, GPX4, LC3B, and p62 protein levels. Data are means ± SD, n = 3. ABCA1: ATP-binding cassette transporter A1; ABCG1: ATP-binding cassette transporter G1; HUA: high level of uric acid; oxLDL: oxidized low-density lipoprotein; qPCR: quantitative PCR. ns indicates no significance. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: HUA inhibits the protein level of the NRF2/SLC7A11/GPX4 signaling pathway in atherosclerotic plaque from HFD-treated ApoE-/- mice. The protein level of the NRF2/SLC7A11/GPX4 signaling pathway was assessed by immunofluorescence staining. (a–c) Representative immunostaining of NRF2, SLC7A11, and GPX4 in aortic atherosclerotic lesions in HUA+HFD versus HFD group. (d–f) Quantification of the mean fluorescence intensity for NRF2, SLC7A11, and GPX4. Data are means ± SD, 3 cross-sections/mouse, n = 3. HFD: high fat diet; HUA: high level of uric acid; HUA+HFD: high level of uric acid and high fat diet group. **P < 0.01 and ***P < 0.001.