Ciprofol Ameliorates Myocardial Ischemia/Reperfusion Injury by Inhibiting Ferroptosis Through Upregulating HIF-1α

Abstract

Purpose: Ciprofol is a novel intravenous anesthetic that has been increasingly used in clinical anesthesia and sedation. Studies suggested that ciprofol reduced oxidative stress and inflammatory responses to alleviate cerebral ischemia/reperfusion (I/R) injury, but whether ciprofol protects the heart against I/R injury and the mechanisms are unknown. Herein, we assessed the effects of ciprofol on ferroptosis during myocardial I/R injury.

Methods: Experimental models of myocardial I/R injury in mice (ischemia for 30 min and reperfusion for 24 h) and hypoxia/reoxygenation (H/R) injury in H9c2 cardiomyocytes (hypoxia for 6 h followed by 6 h of reoxygenation) were established. Ciprofol was used prior to ischemia or hypoxia. Echocardiography, myocardial TTC staining, HE staining, DAB-enhanced Perl's staining, transmission electron microscopy, FerroOrange staining, Liperfluo staining, JC-1 staining, Rhodamine-123 staining, DCFH-DA staining, and Western blot were performed. Cell viability, serum cardiac enzymes, and oxidative- and ferroptosis-related biomarkers were measured. HIF-1α siRNA transfection and the specific inhibitor BAY87-2243 were utilized for mechanistic investigation.

Results: Ciprofol treatment reduced myocardial infarct area and myocardium damage, alleviated oxidative stress and mitochondrial injury, suppressed Fe²⁺ accumulation and ferroptosis, and improved cardiac function in mice with myocardial I/R injury. Ciprofol also increased cell viability, attenuated mitochondrial damage, and reduced intracellular Fe²⁺ and lipid peroxidation in cardiomyocytes with H/R injury. Ciprofol enhanced the protein expression of HIF-1α and GPX4 and reduced the expression of ACSL4. Specifically, the protective effects of ciprofol against I/R or H/R injury were abolished by downregulating the expression of HIF-1α using siRNA transfection or the inhibitor BAY87-2243.

Conclusion: Ciprofol ameliorated myocardial I/R injury in mice and H/R injury in cardiomyocytes by inhibiting ferroptosis via the upregulation of HIF-1α expression.

Keywords: GPX4/ACSL4; HIF-1α; ciprofol; ferroptosis; myocardial ischemia/reperfusion injury; oxidative stress.

Figure 1

Schematic diagram. (A) H9c2 cardiomyocytes underwent H/R injury. First, cells were treated with ciprofol (1, 5, 10, 50, and 100 μM) in the normal condition or before hypoxia. Next, ciprofol (10 μM) was used, and si-HIF-1α or si-NC was transfected 36 h before ciprofol treatment. At the end of reoxygenation, cell culture samples were collected. (B) Mice underwent myocardial I/R injury. Ciprofol (10 mg/kg i.p.) was given 1 h before ischemia. BAY87-2243 (9 mg/kg i.g.) was administered daily for 3 days prior to ischemia. At the end of reperfusion, echocardiography was conducted, and heart and blood samples were collected.

Figure 2

Ciprofol attenuated H/R-induced injury in H9c2 cardiomyocytes. (A) Cell viability after ciprofol (1, 5, 10, 50, and 100 μM) in the normal condition. (B) Cell viability after ciprofol (1, 5, 10, 50, and 100 μM) during H/R. (C and D) Levels of LDH and SOD. Ciprofol (10 μM) was added 1 h before H/R. (E and F) Representative images and fluorescence intensity of Rhodamine 123 staining. Ciprofol (10 μM) was added 1 h before H/R. Scale bar = 50 µm. Data are shown as mean ± SD (n = 5‒6). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Ciprofol alleviated I/R-induced myocardial injury in mice. Ciprofol (10 mg/kg i.p.) was given 1 h prior to ischemia. (A and B) Representative myocardial TTC staining images and quantification of infarction size. (C) Representative HE staining images of myocardium. Scale bar = 50 μm. (D and E) Serum levels of cardiac enzymes (CK-MB and cTnI). (F and G) Serum levels of SOD and LPO. Data are shown as mean ± SD (n = 4‒6). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Ciprofol improved cardiac function in mice with myocardial I/R injury. Ciprofol (10 mg/kg i.p.) was given 1 h prior to ischemia. (A) Representative echocardiography images. (B and C) Values of LVEF and LVFS. Data are shown as mean ± SD (n = 8). **P < 0.01.

Figure 5

Ciprofol suppressed H/R-induced ferroptosis and increased HIF-1α expression in H9c2 cardiomyocytes. Ciprofol (10 μM) was added 1 h before H/R. (A and B) Representative images and fluorescence intensity of FerroOrange staining. Scale bar = 50 µm. (C and D) Representative images and fluorescence intensity of Liperfluo staining. Scale bar = 50 µm. (E and F) Levels of Fe²⁺ and MDA. (G–J) Western blot bands and quantification of HIF-1α, ACSL4, and GPX4 protein expression. Data are shown as mean ± SD (n = 4‒6). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Ciprofol reduced Fe²⁺ accumulation and mitochondrial damage in mice with myocardial I/R injury. Ciprofol (10 mg/kg i.p.) was given 1 h prior to ischemia. (A) Representative Perl’s staining images of myocardium. Scale bar = 100 µm. (B) Representative mitochondrial ultrastructure images under transmission electron microscopy. Scale bar = 500 nm. (C‒E) Serum levels of Fe²⁺, MDA, and GSH. Data are shown as mean ± SD (n = 4‒5). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Knockdown of HIF-1α abolished the protective effects of ciprofol against H/R-induced injury in H9c2 cardiomyocytes. si-HIF-1α or si-NC was transfected 36 h before ciprofol (10μM) treatment. (A–C) Levels of LDH, SOD, and LPO. (D and E) Representative DCFH-DA staining images and fluorescence intensity of intracellular ROS. Scale bar = 50 µm. Data are shown as mean ± SD (n = 5‒6). **P < 0.01, ***P < 0.001.

Figure 8

Knockdown of HIF-1α reversed the protective effects of ciprofol on mitochondrial function in H9c2 cardiomyocytes subjected to H/R injury. si-HIF-1α or si-NC was transfected 36 h before ciprofol (10μM) treatment. (A) Representative JC‐1 staining images showing mitochondrial membrane potential. Scale bar = 50 µm. (B and C) Fluorescence intensity of JC-1 monomers (green) and aggregates (red). Data are shown as mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 9

Ciprofol inhibited H/R-induced ferroptosis in H9c2 cardiomyocytes by upregulating HIF-1α. si-HIF-1α or si-NC was transfected 36 h before ciprofol (10μM) treatment. (A–C) Levels of Fe²⁺, MDA, and GSH. (D and E) Representative images and fluorescence intensity of FerroOrange staining. Scale bar = 50 µm. (F and G) Representative images and fluorescence intensity of Liperfluo staining. Scale bar = 50 µm. (H‒K) Western blot bands and quantification of HIF-1α, ACSL4, and GPX4 protein expression. Data are shown as mean ± SD (n = 4‒6). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 10

Inhibiting HIF-1α reversed the protective effect of ciprofol on I/R-induced myocardial infarction in mice. BAY87-2243 (9 mg/kg i.g.) was administered daily for 3 days prior to ischemia. (A) Representative myocardial TTC staining images. (B) Quantification of myocardial infarction size. Data are shown as mean ± SD (n = 6). **P < 0.01, ***P < 0.001.

Figure 11

Inhibiting HIF-1α reversed the protective effects of ciprofol on Fe²⁺ accumulation and mitochondrial damage in mice with myocardial I/R injury. BAY87-2243 (9 mg/kg i.g.) was administered daily for 3 days prior to ischemia. (A and B) Serum levels of Fe²⁺ and GSH. (C) Representative Perl’s staining in myocardial tissues. Scale bar = 50 µm. (D) Representative mitochondrial ultrastructure images under transmission electron microscopy. Scale bar = 2 µm. Data are shown as mean ± SD (n = 4‒5). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 12

Ciprofol inhibited I/R-induced myocardial ferroptosis in mice by upregulating HIF-1α. BAY87-2243 (9 mg/kg i.g.) was administered daily for 3 days prior to ischemia. (A and B) Representative images and fluorescence intensity of HIF-1α expression in myocardium. Scale bar = 50 µm. (C‒F) Western blot bands and quantification of HIF-1α, ACSL4, and GPX4 protein expression. Data are shown as mean ± SD (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 13

Schematic mechanism for the cardioprotection of ciprofol against I/R-induced myocardial ferroptosis in mice through upregulating HIF-1α.