FIGURE 5.

SNP (NO donor) pretreatment attenuated oxidative stress induced by H₂O₂ and upregulated NRF2 in vitro, while ML385 (NRF2 inhibitor) reversed the protective effect of SNP on H₂O₂-stimulated AML12 cells. (A) SNP or SNP + ML385 was administered 12 h before the 12 h stimulation of H₂O₂. (B) Morphology of AML12 cells in Normal and Normal + ML385 groups. (C) Relative MFI of ROS, Annexin V, in Normal and Normal + ML385 groups. (D) Morphology of AML12 cells in Normal and Normal + SNP groups. (E) Relative MFI of ROS, Annexin V, in Normal and Normal + SNP groups. (F) Morphology of AML12 cells in Normal, H₂O₂, SNP + H₂O₂, and ML385 + SNP + H₂O₂ groups. (G) The level of NO in Normal, H₂O₂, SNP + H₂O₂, and ML385 + SNP + H₂O₂ groups. (H) Representative flow cytometric results of ROS, Annexin V, and (I) statistical analyses. (J) Relative mRNA level of Bcl-2 and Bcl-xL. (K) Relative mRNA level of Nrf2, Keap1, Nqo1, Ho1, TrxR, GSTp1, CAT, and GPX1 in Normal, H₂O₂, SNP + H₂O₂, and ML385 + SNP + H₂O₂ groups. (L) Mechanisms by which dietary nitrate protects HIRI. Dietary nitrate is converted to NO, which is transported through the cell membrane and disintegrates KEAP1 and NRF2. NRF2 is transferred into the nucleus, wherein it activates downstream genes and transcribes related proteins to modulate oxidative stress. Data are expressed as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, and NS denotes no significance.