Fig. 4

MMA triggers ferroptosis through oxidative stress. In vitro, AC16 cells were treated with MMA (17.5 mM) alone for 3 h or with NAC (5 mM) for 2 h followed by MMA (17.5 mM) for 3 h. In vivo, mice were assigned to the sham operation or myocardial I/R injury group. MMA (400 mg/kg/d) was administered seven days before the I/R injury. Meanwhile, NAC (300 mg/kg) was administered three times before the I/R model (on day 2, 4, and 6 of the administration process). a Pathway Enrichment showed a ferroptosis pathway associated with MMA treatment. b TEM analysis of mitochondria in AC16 cells. Yellow arrows indicate mitochondria (Scale bar: 20 μm). c, d Representative photomicrographs and averaged data of GPX4 and SLC7A11 expression in mice heart tissue (n = 4/group) (Scale bar: 100 μm). e, f Western blot analyzed the protein level of GPX4 and SLC7A11 in AC16 cells (n = 3–4/group). g, h Western blot analyzed the protein level of GPX4 and SLC7A11 in mice heart tissue (n = 3/group). i GSH was relieved by NAC in mice heart tissue. Data are expressed as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and n.s, not significant