Nicorandil Regulates Ferroptosis and Mitigates Septic Cardiomyopathy via TLR4/SLC7A11 Signaling Pathway

Abstract

This study mainly explored the role of nicorandil in regulating ferroptosis and alleviating septic cardiomyopathy through toll-like receptor (TLR) 4/solute carrier family 7 member 11 (SLC7A11) signaling pathway. Twenty-four male SD rats were randomly divided into control, Nic (nicorandil), LPS (lipopolysaccharide), and LPS + Nic groups and given echocardiography. A detection kit was applied to measure the levels of lactic dehydrogenase (LDH), cardiac troponin I (cTnI), and creatine kinase-MB (CK-MB); HE staining and the levels of glutathione (GSH), malondialdehyde (MDA), total iron, and Fe²⁺ of myocardial tissues were detected. Moreover, the expression of TLR4 and SLC7A11 were measured by qRT-PCR and the proteins regulating ferroptosis (TLR4, SLC7A11, GPX4, ACSL4, DMT1, Fpn, and TfR1) were checked by western blot. Myocardial cells (H9C2) were induced with lipopolysaccharide (LPS) and transfected with si-TLR4 or SLC7A11-OE. Then, the viability, ferroptosis, and TLR4/SLC7A11 signaling pathway of cells were examined. Nicorandil could significantly increase left ventricular (LV) ejection fraction (LVEF) while reduce LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV). Also, it greatly reduced the levels of LDH, cTnI, and CK-MB; alleviated the pathological changes of myocardial injury; notably decreased MDA, total iron, and Fe²⁺ levels in myocardial tissues; and significantly increased GSH level. Besides, nicorandil obviously raised protein levels of GPX4, Fpn, and SLC7A11, and decreased protein levels of ACSL4, DMT1, TfR1, and TLR4. After knockdown of TLR4 or overexpression of SLC7A11, the inhibition effect of nicorandil on ferroptosis was strengthened in LPS-induced H9C2 cells. Therefore, nicorandil may regulate ferroptosis through TLR4/SLC7A11 signaling, thereby alleviating septic cardiomyopathy.

Keywords: ferroptosis; nicorandil; septic cardiomyopathy; toll-like receptor (TLR) 4/solute carrier (SLC) 7A11 signaling pathway..

Fig. 1

The improving effect of nicorandil on cardiac function of rats with septic cardiomyopathy. a Representative images of echocardiography in the control, Nic, LPS, and LPS + Nic groups. The heart rate (b), LVEF (c), LVEDV (d), and LVESV (e) of rats in the control, Nic, LPS, and LPS + Nic groups. **P < 0.01. LVEF, left ventricular ejection fraction; LVEDV, left ventricular end-diastolic volume; LVESV, left ventricular end-systolic volume.

Fig. 2

Nicorandil attenuates myocardial injury in rats with septic cardiomyopathy. Biochemical test was performed to determine the levels of LDH (a), cTnI (b), and CK-MB (c) in rat serum in the control, Nic, LPS, and LPS + Nic groups. d H&E staining of myocardial tissue in the control group, Nic group, LPS group, and LPS + Nic group. The red arrows indicated pathological changes. Scale bar = 100 µm. **P < 0.01. LDH, lactic dehydrogenase; cTnI, cardiac troponin I; CK-MB, creatine kinase-MB.

Fig. 3

Nicorandil attenuates ferroptosis in myocardial tissue of rats with septic cardiomyopathy. a The ultrastructural features of myocardial tissue of rats were observed by transmission electron microscopy. The red arrow manifest mitochondrial membrane rupture, mitochondrial atrophy, and the decrease or disappearance of mitochondrial cristae (scale bar = up: 2 μm/down: 500 nm). Biochemical test was applied to determine the levels of MDA (b), GSH (c), total iron (d), and Fe²⁺ (e) in myocardial tissues in the control group, Nic group, LPS group, and LPS + Nic group. f The protein levels of GPX4, ACSL4, and DMT1 in the myocardial tissues of rats in the control group, Nic group, LPS group, and LPS + Nic group were detected by western blot. **P < 0.01. MDA, malondialdehyde; GSH, glutathione.

Fig. 4

Effects of nicorandil on Toll-like receptor (TLR) 4/solute carrier (SLC) 7A11 signaling pathway in rats with septic cardiomyopathy. The levels of TLR4 (a) and SLC7A11 (b) in myocardial tissues of rats in the control group, Nic group, LPS group, and LPS + Nic group detected by qRT-PCR. c The protein levels of TLR4 and SLC7A11 in myocardial tissues of rats in the control group, Nic group, LPS group, and LPS + Nic group measured by western blot. **P < 0.01. TLR, toll-like receptor; SLC, solute carrier.

Fig. 5

Knockdown of TLR4 strengthens the inhibition effect of nicorandil on ferroptosis in LPS-induced H9C2 cells. a–c qRT-PCR and Western blot to measure the expression of TLR4 and SLC7A11. d MTT to measure the cell proliferation levels of each group. e–h The levels of MDA, GSH, total iron, and Fe²⁺ were detected biochemically. i Western blot to measure GPX4, ACSL4, DMT1, TfR1, and Fpn protein levels in cells of each group. **P < 0.01. **P < 0.01.

Fig. 6

Overexpression of SLC7A11 aids in the inhibition of ferroptosis by nicorandil in LPS-induced H9C2 cells. a–c qRT-PCR and Western blot to measure the expression of SLC7A11 and TLR4, d MTT to measure the cell proliferation levels of each group. e–h The levels of MDA, GSH, total iron, and Fe²⁺ were detected biochemically. i Western blot to measure GPX4, ACSL4, DMT1, TfR1, and Fpn protein levels in cells of each group. **P < 0.01.

Fig. 7

A mechanism diagram of the effect of nicorandil on ferroptosis.