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. 2024 May;24(5):481-498.
doi: 10.1007/s12012-024-09852-7. Epub 2024 Apr 22.

N-acetylcysteine Protects Against Myocardial Ischemia-Reperfusion Injury Through Anti-ferroptosis in Type 1 Diabetic Mice

Dongcheng Zhou #  1 , Yuhui Yang #  1 , Jiajia Chen #  1 , Jiaqi Zhou  1 , Jianfeng He  1 , Danyong Liu  1 , Anyuan Zhang  1 , Bixian Yuan  1 , Yuxin Jiang  1 , Weiyi Xia  1 , Ronghui Han  1 , Zhengyuan Xia  2   3
Affiliations

N-acetylcysteine Protects Against Myocardial Ischemia-Reperfusion Injury Through Anti-ferroptosis in Type 1 Diabetic Mice

Dongcheng Zhou et al. Cardiovasc Toxicol. 2024 May.

Abstract

The hearts of subjects with diabetes are vulnerable to ischemia-reperfusion injury (IRI). In contrast, experimentally rodent hearts have been shown to be more resistant to IRI at the very early stages of diabetes induction than the heart of the non-diabetic control mice, and the mechanism is largely unclear. Ferroptosis has recently been shown to play an important role in myocardial IRI including that in diabetes, while the specific mechanisms are still unclear. Non-diabetic control (NC) and streptozotocin-induced diabetic (DM) mice were treated with the antioxidant N-acetylcysteine (NAC) in drinking water for 4 week starting at 1 week after diabetes induction. Mice were subjected to myocardial IRI induced by occluding the coronary artery for 30 min followed by 2 h of reperfusion, subsequently at 1, 2, and 5 week of diabetes induction. The post-ischemic myocardial infarct size in the DM mice was smaller than that in NC mice at 1 week of diabetes but greater than that in the NC mice at 2 and 5 week of diabetes, which were associated with a significant increase of ferroptosis at 2 and 5 week but a significant reduction of ferroptosis at 1 week of diabetes. NAC significantly attenuated post-ischemic ferroptosis as well as oxidative stress and reduced infarct size at 2 and 5 week of diabetes. Application of erastin, a ferroptosis inducer, reversed the cardioprotective effects of NAC. It is concluded that increased oxidative stress and ferroptosis are the major factors attributable to the increased vulnerability to myocardial IRI in diabetes and that attenuation of ferroptosis represents a major mechanism whereby NAC confers cardioprotection against myocardial IRI in diabetes.

Keywords: Diabetes mellitus; Ferroptosis; Lipid peroxidation; Myocardial ischemia reperfusion injury; NAC.

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Conflict of interest statement

The authors have declared that no competing interest exists.

Figures

Fig. 1
Fig. 1
Diabetic heart’s time-dependent changes in Ferroptosis. A 8w mice were injected with STZ for at 50 mg/kg/d for 5 consecutive days to induce T1DM. DM mice were either untreated or started to receive NAC treatment at 1 week of T1DM for a duration of 1 week (D2w with NAC 1w) or for 4 week (D5w with NAC 4w) before being subjected to ischemia–reperfusion (IR) achieved by 30 min of coronary ligation followed by 2 h of reperfusion. B Expression of Gpx4 and Slc7a11 in mice with DM, assessed using Western blotting. Quantification of western blots was performed using Image J. C Heart tissue lipid peroxidation in DM mice, assessed by observing the changes in MDA levels. D Heart tissue labile iron levels in DM mice, assessed using the Iron Colorimetric Assay Kit. E Alterations of heart tissue GSH levels, assessed using the Glutathione Fluorometric Assay Kit. F H&E staining of myocardium, myofibrils with vacuolar degeneration, loose and lightly stained cytoplasm, inconspicuous transverse lines were observed in D5w. The magnifications is 40 times. Date are expressed as mean ± SD, n = 8 mice per group. *p < 0.05, **p < 0.01, versus NC group; #p < 0.05, ##p < 0.01, versus D1w group; ^p < 0.05, ^^p < 0.01, versus D2w group. There is no significant statistical difference between groups without annotation symbols. D1w Diabetes for 1 week, D2w Diabetes for 2 week, D5w Diabetes for 5 week
Fig. 2
Fig. 2
The MIRI in the first week of diabetic mice. C and A Expression of Gpx4 and Slc7a11 in mice with DM, assessed using Western blotting. B Levels of serum CK-MB, measured after reperfusion using the CK-MB ELISA kit. C and D Lipid peroxidation in DM mice, assessed by observing the changes in MDA and 15-F2t-IsoP levels. E Labile iron levels in DM mice, assessed using the Iron Colorimetric Assay Kit. F Alterations of GSH levels, assessed using the Glutathione Fluorometric Assay Kit. G Prussian blue stain showed abnormal iron deposition (brown) in myocardium, with the iron deposition increasing after IR, but little difference between D1w and control group. H Expression and the integrated optical density (IOD) of Ferritin assessed using IHC in myocardium (brown, arrow head) and, showing no difference between D1w + IR and NC + IR group. The magnifications is 40 times. Date are expressed as mean ± SD, n = 8 mice per group. *p < 0.05, **p < 0.01, versus NC group; #p < 0.05, ##p < 0.01, versus D1w group; ^p < 0.05, ^^p < 0.01, versus NC + IR group. There is no significant statistical difference between groups without annotation symbols. D1w Diabetes for 1 week
Fig. 3
Fig. 3
The MIRI with or without NAC treatment in second week of diabetic mice. A Expression of Gpx4 and Slc7a11 in mice with DM, assessed using Western blotting. B Levels of serum CK-MB, measured after reperfusion using the CK-MB ELISA kit. C and D Lipid peroxidation in DM mice, assessed by observing the changes in MDA and 15-F2t-IsoP levels. E Labile iron levels in DM mice, assessed using the Iron Colorimetric Assay Kit. F Alterations of GSH levels, assessed using the Glutathione Fluorometric Assay Kit. G Prussian blue stain showed abnormal iron (brown) deposition in myocardium. Iron infiltration was significantly increased after IRI in D2w mice, but there was no significant improvement after NAC treatment for 1 week. H Expression and the integrated optical density (IOD) of Ferritin assessed using IHC in myocardium (brown, arrowhead). Ferritin in D2w mice decreased significantly after IR, but it could still be detectable with IHC. Meanwhile, after NAC treatment, the ferritin levels were increased after ischemia by in D2w. The magnifications is 40 times. Data are expressed as mean ± SD, n = 8 mice per group. *p < 0.05, **p < 0.01, versus NC group; #p < 0.05, ##p < 0.01, versus D2w group; ^p < 0.05, ^^p < 0.01, versus NC + IR group; $p < 0.05, $$p < 0.01, versus D2w + IR group. There is no significant statistical difference between groups without annotation symbols (p > 0.05). D2w Diabetes for 2 week, D2w + NAC Diabetes for 2 week and NAC treatment for 1 week
Fig. 4
Fig. 4
The MIRI with or without NAC treatment in the fifth week of diabetic mice. A Expression of Gpx4 and Slc7a11 in mice with DM, assessed using Western blotting. B Levels of serum CK-MB, measured after reperfusion using the CK-MB ELISA kit. C and D Lipid peroxidation in DM mice, assessed by observing the changes in MDA and 15-F2t-IsoP levels. E Labile iron levels in DM mice, assessed using the Iron Colorimetric Assay Kit. F Alterations of GSH levels, assessed using the Glutathione Fluorometric Assay Kit. G Prussian blue stain showed abnormal iron (brown) deposition in myocardium. Further increase in iron deposition was observed in D5w + IR group, while this enhancement of iron deposition was significantly reduced after 4 week of NAC treatment. H Expression of Ferritin assessed using IHC in myocardium (brown, arrowhead), Positive of Ferritin could hardly be detected after IR in D5w mice, but the degradation of Ferritin was significantly inhibited by NAC treatment for 4 week. The magnifications is 40 times. Date are shown as mean ± SD, n = 8 mice per group. *p < 0.05, **p < 0.01, versus NC group; #p < 0.05, ##p < 0.01, versus D5w group; ^p < 0.05, ^^p < 0.01, versus NC + IR group; $p < 0.05, $$p < 0.01, versus D5w + IR group. There is no significant statistical difference between groups without annotation symbols (p > 0.05). D5w Diabetes for 5 week; D5w + NAC Diabetes for 5 week and NAC treatment for 4 week
Fig. 5
Fig. 5
The effects of NAC on postischemic myocardial infract size and cardiac ultrastructure in diabetic mice by regulating Ferroptosis. A Infarct size (IS) is expressed as a percentage of the area at risk (AAR). Ischemia reperfusion (I/R) was achieved by 30-min ischemia followed by 2-h reperfusion in diabetic mices with or without NAC. B Cardiac ultrasound and changes of EF after IR. C Level of CK-MB showing the extent of myocardial damage. Date are expressed as mean ± SD, n = 8 mice per group. *p < 0.05, **p < 0.01, versus NC group; #p < 0.05, ##p < 0.01, versus NC + IR group; ^p < 0.05, ^^p < 0.01, versus D1w + IR group; $p < 0.05, $$p < 0.01, versus D2w + IR group; %p < 0.05, %%p < 0.01, versus D5w + IR group; @p < 0.05, @@p < 0.01, versus D5w + IR + NAC group. There is no significant statistical difference between groups without annotation symbols (p > 0.05). D1w Diabetes for a week, D2w Diabetes for 2 week, D5w Diabetes for 5 week; D2w + NAC Diabetes for 2 week and NAC treatment for 1 week, D5w + NAC Diabetes for 5 week and NAC treatment for 4 week
Fig. 6
Fig. 6
The effect of NAC on cardiac ultrastructure in diabetic mice with IR. A Typical morphological changes of ferroptosis in cardiomyocytes were observed using transmission electron microscopy. With the development of diabetes, ferroptosis-related injury during IR was gradually aggravated, which manifested as the increase and oxidation of lipid droplets in cardiac tissue, the disappearance of mitochondrial crest and the destruction of mitochondrial membrane increased. After 4 week of NAC treatment, mitochondrial damage was significantly alleviated. Erastin reverses the protective effect of NAC on cardiac ultrastructure. D1w Diabetes for a week, D2w Diabetes for 2 week, D5w Diabetes for 5 week, D2w + NAC Diabetes for 2 week and NAC treatment for 1 week, D5w + NAC Diabetes for 5 week with NAC treatment for 4 week
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