Dapagliflozin inhibits ferroptosis to improve chronic heart failure by regulating Nrf2/HO-1/GPX4 signaling pathway

Abstract

Objective: To study the effect of Dapagliflozin on ferroptosis in rabbits with chronic heart failure and to reveal its possible mechanism.

Methods: Nine healthy adult male New Zealand white rabbits were randomly divided into Sham group (only thorax opening was performed in Sham group, no ascending aorta circumferential ligation was performed), Heart failure group (HF group, ascending aorta circumferential ligation was performed in HF group to establish the animal model of heart failure), and Dapagliflozin group (DAPA group, after the rabbit chronic heart failure model was successfully made in DAPA group). Dapagliflozin was given by force-feeding method. Echocardiography was used to assess cardiac function, HE staining to evaluate pathological changes in the heart, Prussia blue staining to observe iron ions in myocardial tissue, and enzyme-linked immunosorbent assay (ELISA) to determine serum levels of the inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) at the end of week 12 and/or the end of week 16. The oxidative stress related indexes of malondialdehyde (MDA), superoxide dismutase (SOD) and superoxide dismutase (GSH-Px) in serum were quantitatively analyzed by colorimetry. Protein expression levels of nuclear factor E2-related factor 2(Nrf2), heme oxygenase-1(HO-1), glutathione peroxidase 4(Gpx4) were detected by Western blot.

Results: In animals with chronic heart failure, Dapagliflozin improved cardiomyocyte hypertrophy, degeneration and necrosis. Dapagliflozin increased serum GSH-Px and SOD levels and decreased IL-1β, IL-6, TNF-α and MDA levels (P < 0.05) in a rabbit model of heart failure. Dapagliflozin also decreased cardiac iron ion levels and increased Nrf2, HO-1 and GPX4 protein expression.

Conclusion: Dapagliflozin can improve heart failure by inhibiting oxidative stress and ferroptosis, and its mechanism may be related to the regulation of Nrf2/HO-1/GPX4 signaling pathway.

Fig 1. Flowchart of the entire experimental process.

Fig 2. Echocardiography results of each group of rabbits.

Note: (A). LVEF; (B). LVESD; (C). LVEDD; (D). IVS; (E). LVPW; *Compared with the Sham group, *P<0.05, **P<0.01, ***P<0.001; ^#compared with the HF group, ^#P<0.05, ^##P<0.01, ^###P<0.001.

Fig 3. Heart rate changes in each group of rabbits.

Note: **Compared with the Sham group, **P<0.01;##compared with the HF group, ##P<0.01.

Fig 4. Morphological changes of myocardial tissue in each group of experimental rabbits (HE staining, ×200).

Fig 5. Levels of serum Fe²⁺ content in rabbits in each group as measured by iron kit.

Note: *Compared with the Sham group, *P<0.05; ns indicates no statistical significance.

Fig 6. The levels of inflammatory factors were detected by ELISA.

Note: (A). IL-1β; (B). IL-6; (C). TNF-α; *Compared with the Sham group, *P<0.05, **P<0.01, ***P<0.001; #compared with the HF group, #P<0.05, ##P<0.01, ###P<0.001.

Fig 7. The levels of GSH-Px, SOD and MDA levels in each group.

Note: (A). GSH-Px; (B). SOD; (C). MDA; *Compared with the Sham group, *P<0.05; #compared with the HF group, #P<0.05; ns indicates no statistical significance.

Fig 8. The protein expressions of Nrf2, HO-1 and GPX4 in myocardial tissue of rabbits were detected by WB.

Fig 9. Protein expression levels of Nrf2, HO-1 and GPX4 in each group.

Note: (A). GPX4; (B). HO-1; (C). Nrf2; *Compared with the Sham group, *P<0.05, **P<0.01, ***P<0.001; #compared with the HF group, #P<0.05, ##P<0.01, ###P<0.001.