Naringenin alleviates myocardial ischemia/reperfusion injury by regulating the nuclear factor-erythroid factor 2-related factor 2 (Nrf2) /System xc-/ glutathione peroxidase 4 (GPX4) axis to inhibit ferroptosis

Abstract

Ferroptosis is an important form of myocardial cell death in myocardial ischemia-reperfusion injury (MIRI). Naringenin (NAR), as a flavonoid, has a significant advantage in improving MIRI. But the regulatory effect and mechanism of NAR on ferroptosis in MIRI have not been reported. After the rats were given NAR and induced to form myocardial ischemia-reperfusion (MI/R) injury, Tetrazolium chloride (TTC) staining was used to detect the myocardial infarction area of rats, and Hematoxylin-eosin (H&E) staining was used to detect myocardial injury. The markers of tissue inflammation were detected by ELISA. Serum creatine kinase Serum creatin kinase (CPK), Lactate dehydrogenase (LDH), and lipid peroxide (LPO) and oxidative stress related levels were measured. In addition, iron detection kits were used to detect total iron and Fe²⁺ levels in cardiac tissues, and western blot was used to detect the expression of ferroptosis-related proteins and the expression of nuclear factor-erythroid factor 2-related factor 2 (Nrf2) and glutathione peroxidase 4 (GPX4). At the cellular level, H9C2 cardiomyocytes were induced by hypoxia/reoxygenation (H/R), and ferroptosis inducer Erastin was administered to detect cell viability, ferroptosis-related indicators, oxidative stress related indicators, and expressions of Nrf2 and GPX4, to explore the mechanisms involved. NAR alleviated MI/R-induced pathological damage, inflammation and lipid peroxidation in myocardial tissue of rats. NAR adjusted the NRF2 /System xc - /GPX4 axis and improved ferroptosis. At the cellular level, ferroptosis inducer Erastin reversed the protective effect of NAR on H/R-induced H9C2 cardiomyocytes. In conclusion, NAR can alleviate MIRI by regulating the Nrf2/System xc-/GPX4 axis to inhibit ferroptosis.

Keywords: Naringenin; Nrf2/System xc-/Gpx4 axis; ferroptosis; myocardial ischemia/reperfusion injury.

Figure 1.

The molecular structure of NAR

Figure 2.

NAR alleviated MI/R induced pathologic damage. A. TTC Staining detected the area of myocardial infarction. B. H&E staining was used to detect myocardial injury. Magnification×200. C. CPK and LDH levels were detected with the kits. ***p < 0.01 vs Sham; ##p < 0.01, ### p < 0.001 vs MI/R

Figure 3.

NAR reduced MI/R-induced inflammation and lipid peroxidation. A. ELISA kits detected the expression of IL-6, IL-1β, TNF-α and MPO. B. The kit was used to detect the level of lipid peroxide LPO in cardiac tissue. C. The kit detected levels of MDA, GSH and SOD in cardiac tissue. ***p < 0.01 vs Sham; #p < 0.05, ##p < 0.01, ### p < 0.001 vs MI/R

Figure 4.

NAR reduced Nrf2/System xc-/GPX4 axis to improve ferroptosis. A. Iron assay kits detected total iron and Fe²⁺ levels in cardiac tissue. B. The expression of ferroptosis related protein was detected by Western blot. ***p < 0.01 vs Sham; #p < 0.05, ##p < 0.01, ### p < 0.001 vs MI/R

Figure 5.

Ferroptosis inducer Erastin reversed the inhibition of NAR on H/ R-induced cell ferroptosis. A. CCK-8 detected cell viability of H2C9 cells. B. CCK-8 detected cell viability of H/R induced H2C9 cells. C. Iron assay kits detected total iron and Fe²⁺ levels in H/R induced H2C9 cells. D. The expression of ferroptosis related protein was detected by Western blot. ***p < 0.01 vs Control; ##p < 0.01, ### p < 0.001 vs H/R; @@p < 0.01, @@@p < 0.001 vs H/R + NAR

Figure 6.

Ferroptosis inducer Erastin reversed the protective effect of NAR on H/R induced H9C2 cardiomyocytes. A. The kit was used to detect the level of lipid peroxide LPO in cells. B. DCFH-DA staining was used to detect ROS levels. C. The kit detected levels of oxidative stress in cells. ***p < 0.01 vs Control; ### p < 0.001 vs H/R; @@@p < 0.001 vs H/R + NAR