Fig 2: MMF attenuates ferroptosis in AML12 cells during hypoxia-reoxygenation injury (HRI). (a) CCK-8 assay showed the viability of AML12 cells after treatment with various MMF concentrations (5 μM-200 μM) for 24 h. (b) Cell viability of AML12 cells with HRI treated with MMF (100 μM), Lip-1 (100 nM) or Z-VAD-FMK (20 μM) for 1 h. (c, d) the levels of MDA and GSH/GSSG ratio in each group. (e, f) C11-BODIPY581/591 was used to monitor the oxidized and nonoxidized variants of lipid peroxides, observed by confocal microscopy and flow cytometry. (g, h) Western blotting and RT-PCR revealed the changes in the expression levels of GPX4, COX2, NRF2, SLC7A11, and HO-1 in AML12 cells. The results are shown as mean ± SD. *** P <0.001, ** P <0.01, * P <0.05 (analyzed by one-way analysis of variance, ANOVA).

Fig 3: MMF alleviates ferroptosis via the NRF2/SLC7A11/HO-1 axis. AML12 cells were transfected with siRNA against NRF2 under hypoxia for 12 h and reoxygenation for 12 h. (a) NRF2 knockdown by siRNA in AML12 cells was confirmed by western blot analysis. (b) CCK-8 assay showing hypoxia-reoxygenation injury (HRI)-induced NC- or siNRF2- AML12 cell death with or without MMF. (c-f) MDA, the GSH/GSSG ratio, and BODIPY 581/591C11 were detected in each group. (g, h) Western blotting and RT-PCR showed the changes in expression of GPX4, COX2, NRF2, SLC7A11, and HO-1 in AML12 cells. The results are shown as mean ± SD. *** P <0.001, ** P <0.01, * P <0.05, ns: no significance (analyzed by two-way analysis of variance ANOVA).

Fig 4: IL-37 inhibits ferroptosis of macrophages and oxidative stress caused by HG/ox-LDL. The macrophages were divided into three groups: i) CTRL group; ii) HG/ox-LDL group: The macrophages were treated with 100 ng/ml ox-LDL and 25 mM glucose for 24 h; and iii) IL-37 group: 30 µM IL-37 for 0.5 h, 100 ng/ml ox-LDL, 25 mM glucose for 24 h. (A and B) Western blot analysis was used to analyze the protein level of GPX4. (C and D) C11 BODIPY staining was used to analyze lipid peroxidation levels of macrophage cell membranes in different groups. (E) MDA level in different group. (F) GSH level in different group. (G) SOD level in different group. *P<0.05 vs. control group, ***P<0.001 vs. control group and #P<0.05 vs. HG/ox-LDL, ###P<0.001 vs. HG/ox-LDL. Each dot represents a biological repetition. HG, high glucose; GPX4, glutathione peroxidase 4; MDA, malondialdehyde; GSH, reduced glutathione; SOD, superoxide dismutase.

Fig 5: Real-time PCR and protein expression detection results of ferroptosis markers GPX4, SLC7A11 and ACSL4. After PRP intervention, the gene expressions of GPX4 and SLC7A11 in the skin granulation tissue of ulcer of diabetic rats were up-regulated, which were significantly higher than those of diabetes control rats (all P<0.001). In contrast, the expression of ACSL4 in PRP group was significantly lower than that in saline group and diabetes control group (both P<0.001) (A-C). The results were consistent with the protein expression results (D). PCR, polymerase chain reaction; PRP, platelet-rich plasma.