Calycosin protects against post-myocardial infarction heart failure by activating the NRF2/HO-1 signaling pathway

Introduction

Heart failure (HF) is the common end-stage of numerous heart diseases (1). HF that develops after myocardial infarction (MI) has become a leading cause of mortality and morbidity worldwide (2). The proliferation and activation of myocardial fibroblasts after MI are driven by pathological physical stimuli, including postinfarction left ventricular systolic dysfunction and neurohumoral activation, a process that serves to preserve the structural integrity of the infarcted ventricle (3). Excessive and maladaptive repair during recovery from MI leads to the development of myocardial fibrosis, which promotes ventricular remodeling and ultimately leads to HF (4). Research indicates that the development of HF is accompanied by enhanced oxidative stress and increased production of reactive oxygen species (ROS) in the failing myocardium, with mitochondrial electron transport being the source of ROS generation and the target of oxidative damage (5). Despite the considerable efforts exerted in HF research, the available therapeutic strategies effective remain limited. Therefore, there is a pressing need to develop novel therapies that target HF-related pathways to improve clinical management.

Ferroptosis is a newly discovered type of regulated cell death that differs from apoptosis, autophagy, necroptosis, and pyroptosis, and it has garnered significant research attention. Intracellular iron is normally stored in ferritin heavy chain 1 (FTH1), keeping free intracellular iron at a low level to maintain oxidative homeostasis. Ferroptosis is driven by the lethal accumulation of lipid peroxides, resulting from increased production catalyzed by ferrous ions due to iron dysregulation and the decreased elimination caused by impaired glutathione peroxidase 4 (GPX4) activity (6). Solute carrier family 7 member 11 (SLC7A11) supports GPX4 activity and indirectly suppresses ferroptosis (7). Several studies have demonstrated that cardiomyocyte loss and remodeling after MI until the development of HF is closely related to oxidative stress and ferroptosis (8-10). GPX4 inhibition during MI contributes to ferroptosis of cardiomyocytes (11). Therefore, targeting ferroptosis may be a novel therapeutic strategy for treating post-MI HF.

Calycosin (CAL), an isoflavonoid phytoestrogen isolated from Radix astragali, exerts various pharmacological activities, including anticancer, anti-inflammatory, antioxidant, and neuroprotective effects (12). In recent years, there has been growing interest in the pharmacological use of CAL for cardiovascular diseases (13,14). CAL has been reported to attenuate myocardial fibrosis and cardiac dysfunction after MI in mice by inhibiting the transforming growth factor beta receptor 1 (TGFBR1) signaling pathway (15). CAL has also been shown to inhibit inflammation and myocardial fibrosis during post-MI HF by activating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway (16). In our previous work, CAL treatment attenuated doxorubicin-induced cardiotoxicity and cardiomyocyte pyroptosis via the inhibition of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome (17). However, the mechanisms by which CAL reduces mitochondrial oxidative stress have not been elucidated. A previous study indicated that CAL can reduce ferroptosis in cases of diabetic nephropathy through inducing antioxidant effects (18). Multiple pharmacological studies of CAL suggest that CAL targets the nuclear factor erythroid 2-related factor 2 (NRF2)/heme oxygenase-1 (HO-1) signaling pathway (19,20). However, whether the protective effect of CAL against post-MI HF is related to ferroptosis and the NRF2/HO-1 signaling pathway is unclear. This study was conducted to clarify the molecular mechanism by which CAL ameliorates post-MI HF and to potentially identify a therapeutic target for post-MI HF treatment. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1421/rc).

Methods

Reagents and antibodies

CAL (99.86% purity) was purchased from MedChemExpress Inc. (Monmouth Junction, NJ, USA). Ferrostatin-1 (Fer-1) (98.00% purity) was obtained from Yuanye Bio-Technology (Shanghai, China). Dimethyl sulfoxide and a cell iron content assay kit were purchased from Solarbio Inc. (Beijing, China). β-actin (cat. no. ab8227), GPX4 (cat. no. ab125066), HO-1 (cat. no. ab68477), and SLC7A11 (cat. no. ab307601) were purchased from Abcam Inc. (Cambridge, UK). NRF2 (cat. no. A21176) and FTH1 (cat. no. A19544) were purchased from ABclonal Technology Co., Ltd. (Woburn, MA, USA). Goat anti-rabbit IgG heavy and light chain (cat. no. ab205718) secondary antibodies were purchased from Abcam for western blotting and immunohistochemistry. The SPARKeasy Improved Tissue/Cell RNA Kit (cat. no. AC0202) and SPARKscript II RT Plus Kit (cat. no. AG0304) were purchased from Shandong Sparkjade Biotechnology Co., Ltd. (Jinan, China). Masson’s trichrome stain kit (cat. no. G1006) and hematoxylin and eosin (HE) staining kit (cat. no. G1005) were purchased from Wuhan Servicebio Biotechnology Co., Ltd. (Wuhan, China). An enhanced bicinchoninic acid (BCA) protein assay kit (cat. no. P0010S) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China), and radioimmunoprecipitation assay (RIPA) buffer was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China).

Cell culture and treatment

H9c2 cells were purchased from the Shanghai Institute for Biological Sciences of the Chinese Academy of Sciences (Shanghai, China). The cells were seeded at a density of 2×10⁵ cells per well in six-well plates and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were maintained at 37 ℃ in a humidified atmosphere of 95% air and 5% CO₂.

To mimic ischemic injury in vitro, cells were subjected to oxygen and glucose deprivation (OGD). The OGD model and the drug treatment were performed as previously described (21,22). Briefly, the culture medium was replaced with serum- and glucose-free DMEM, and cells were transferred to an incubator containing 94% N₂, 5% CO₂, and 1% O₂ at 37 ℃ for 3 hours. Control group cells were maintained in regular media under typical circumstances. Different doses of CAL (10, 20, and 40 µM) were applied to the medium of cells subjected to OGD.

Cell transfection

H9c2 cells were plated at a density of 5×10⁴ cells/well in six-well plates and incubated. Based on the manufacturer’s protocol, small interfering RNA (siRNA) targeting NRF2 (si-NRF2) and negative control (GenePharma, Suzhou, China) were separately transfected into cells with Lipofectamine 2000. After 48 hours, cells were used for further experiments.

Animals

Six-week-old male Sprague-Dawley (SD) rats (specific-pathogen free) were purchased from Beijing Charles River Laboratories (certificate No. 2016-0011; Beijing, China) and weighed 200±10 g. Animal experiments were performed under a project license (No. 2021-33) granted by the Experimental Animal Ethics Committee of The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, in compliance with the eighth edition of the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 2011). A protocol was prepared before the study without registration. The rats were confined in a chamber with constant temperature and humidity under a 12-hour light-dark cycle and were provided standard food and free access to water.

Establishment of the post-MI HF model

The rats were randomly divided into a sham-operated group and an MI model group. Left anterior descending artery (LAD) ligation was performed in the model group as previously described to construct a model of post-MI HF (23,24). In brief, SD rats were anesthetized with 2% isoflurane (Harvard apparatus). After left-side thoracotomy and exposure of the heart, the LAD was ligated with a 7.0 Prolene suture approximately 2–3 mm below the left atrial appendage. The presence of MI was assessed according to ST-segment changes in electrocardiography. Sham rats underwent the same procedure without ligation. Cardiac function was assessed 1 week postoperatively with a high-resolution small-animal echocardiography system (Vevo 2100; FUJIFILM VisualSonics, Toronto, Canada) equipped with a 13- to 24-MHz linear array transducer (MS-250), and rats that met the HF criteria [left ventricular ejection fraction (LVEF) <50%] were included in the experiment and randomized into four groups: the HF model group, low-dose CAL (CAL-L) group, high-dose CAL (CAL-H) group, and Fer-1 group. Pharmacotherapy was initiated after the grouping was performed. HF rats in the CAL-L and CAL-H groups were treated with CAL (25 and 50 mg/kg, respectively) by gavage for 4 weeks (25). HF rats were treated with Fer-1 (0.7 mg/kg) by daily intraperitoneal injection for 4 weeks (26). Rats in the sham-operated and model groups were treated with the same volume of 0.9% sodium chloride solution for 4 weeks. Previous studies have demonstrated that Fer-1 can ameliorate the development of HF in rats by inhibiting ferroptosis (27), and therefore, we used the Fer-1 group as a positive control. The overall mortality rate of rats was 30% throughout the procedure; most deaths occurred during or after surgery and were probably due to acute pump failure or fatal arrhythmias.

Echocardiographic evaluation

Cardiac function in anesthetized rats (2% isoflurane) was assessed after 4 weeks of treatment with a high-resolution small-animal echocardiography system (Vevo 2100) equipped with a 13- to 24-MHz linear array transducer (MS-250). The LVEF and left ventricular fractional shortening (LVFS) were calculated. All measurements were conducted by a single investigator who was blinded to the experimental groups.

Serum N-terminal pro-brain natriuretic peptide (NT-pro-BNP) determination

Blood samples were collected from the abdominal aorta and allowed to stand for 2 hours. The serum was collected after centrifugation. An enzyme-linked immunosorbent assay (ELISA) kit (cat. no. SEKR-0097; Solarbio Science & Technology Co., Ltd., Beijing, China) was used to detect the serum concentrations of NT-pro-BNP. The absorbance (optical density) was measured with a microplate reader at 450 nm. The concentration of NT-pro-BNP in the samples was calculated according to a standard curve.

Histological examination

Heart tissues were promptly excised, rinsed with phosphate-buffered saline (PBS), and fixed in 4% paraformaldehyde. After fixation, tissues were dehydrated through a graded ethanol series (70–100%), cleared with xylene, and embedded in paraffin. Sections (5 µm) were cut and stained with HE and Masson’s trichrome. Images were captured with a microscope (Leica, Wetzlar, Germany). The collagen area fraction (%) was calculated as follows: collagen area/total visual area × 100%.

Transmission electron microscopy

Samples (2 mm) from the edge of the incision were immediately fixed in 2.5% glutaraldehyde and 2% paraformaldehyde for 4 hours, fixed in 1% osmium tetroxide for 2 hours, dehydrated through a graded ethanol series, and embedded in epoxy resin. Subsequently, 60- to 80-nm ultrathin sections were cut with an ultramicrotome (PT-XL, FUJIFILM VisualSonics), placed on carbon-coated nickel grids, and imaged with a transmission electron microscope (H-7500, Hitachi, Tokyo , Japan) at 80 kV.

ROS determination

Cells were cultured in a six-well plate and treated as described above. The 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) ROS Assay Kit (cat. no. S0033S; Beyotime Biotechnology Co., Ltd.) was used to detect intracellular ROS generation. ROS levels were measured and analyzed with a fluorescence microscope and a flow cytometer.

Mitochondrial function evaluation

We examined the changes in mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) levels in each group of cells to assess the extent of mitochondrial damage. MMP was evaluated via an enhanced MMP assay kit with 5,5’,6,6’-tetrachloro1,1’,3,3’-tetramethylbenzimidazolylcarbocyanine iodide (JC-1) (cat. no. C2003S; Beyotime Biotechnology Co., Ltd., China). ATP levels were evaluated with an ATP assay kit (cat. no. S0026; Beyotime Biotechnology Co., Ltd., China). H9c2 cells were seeded on normal culture plates and manipulated as mentioned previously. Subsequently, the MMP and ATP levels in each group of cells were assayed. Changes in MMP were measured with a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and ATP levels were measured with a microplate reader.

Intracellular iron level determination

H9c2 cells were cultured and manipulated in a standard manner . The cell supernatant and corresponding reagents from a cell iron levels assay kit (cat. no. BC5315; Solarbio) were sequentially added to the wells of a microplate in accordance with the manufacturer’s instructions. Ferrous iron levels were quantified by measuring the absorbance at 510 nm with a microplate reader.

Malondialdehyde (MDA) determination

An MDA assay kit (cat. no. A003-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was applied to evaluate the MDA level of rat myocardial tissue and H9c2 cells. According to the manufacturer’s instructions, the absorbance was measured at 532 nm with a microplate reader. The MDA level in the samples was calculated according to a standard curve.

Western blot analysis

Myocardial tissues and cells from each group were homogenized and lysed in RIPA lysis buffer, and then the protein concentration was determined with a BCA kit. Proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% skim milk and then incubated overnight with primary antibodies at 4 ℃, which was followed by incubation with the secondary antibody. Finally, each protein band was visualized with an Excellent Chemiluminescent Substrate Detection Kit (Elabscience, Houston, TX, USA). The density of the signal was quantified with ImageJ version 1.8.0 software (US National Institutes of Health, Bethesda, MD, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was purified with the SPARKeasy Improved Tissue/Cell RNA Kit (Sparkjade Biotechnology Co., Ltd., Jinan, China) according to the manufacturer’s instructions. Complement DNA was synthesized with the SPARKscript II RT Plus Kit (SparkJade). Quantitative PCR was performed with the LightCycler 480II Real-Time PCR Detection System (Roche, Basel, Switzerland). β-actin was used to normalize gene expression. The primer sequences for messenger RNA (mRNA) analysis are listed in Table 1.

Statistical analysis

The results were statistically analyzed with GraphPad Prism 8.0 (Dotmatics, Boston, MA, USA) and are expressed as the mean ± standard deviation. Differences between groups were assessed via one-way analysis of variance, with post hoc comparisons conducted with the Tukey test. Differences between groups were considered statistically significant when P<0.05.

Results

CAL suppressed oxidative stress and mitochondrial damage in OGD-induced H9c2 cells

Given that oxidative stress state after MI is an important trigger of myocardial damage (28), we constructed an OGD model in H9c2 cells to simulate this pathological state. The intracellular ROS and MDA levels are the main markers reflecting the intracellular oxidative stress state, and thus we examined intracellular ROS and MDA levels in each group. Flow cytometric analysis revealed that OGD significantly increased intracellular ROS levels compared to the control group (1.00±0.07 vs. 4.42±0.19, P<0.001), while CAL (10, 20, and 40 µM) treatment reduced ROS levels to 3.36±0.30, 2.83±0.12, and 2.44±0.51 relative to the OGD group (P<0.01, P<0.001, and P<0.001 vs. OGD) (Figure 1A,1B). Similarly, MDA content in OGD-treated cells was elevated to 14.75±0.53 nmol/mg protein, compared with 3.29±0.33 nmol/mg protein in the control group, whereas CAL (10, 20, and 40 µM) administration significantly lowered MDA to 12.30±0.52, 8.96±0.55, and 6.24±0.89 nmol/mg protein (P<0.01, P<0.001, and P<0.001 vs. OGD) (Figure 1C). Changes in intracellular ROS levels were further visualized under a microscope (Figure 1D). To estimate the integrity of the mitochondrial membrane, we investigated the changes in MMP in OGD-induced H9c2 cells after CAL treatment using JC-1 staining. The green fluorescence intensity was normalized to the control group. OGD exposure markedly elevated the green fluorescence intensity from 1.00±0.06 in the control group to 3.59±0.15 (P<0.001). Treatment with CAL at 10, 20, and 40 µM dose-dependently reduced the green fluorescence intensity to 3.16±0.23, 2.65±0.07, and 1.89±0.31, respectively (P=0.11, P<0.001, and P<0.001 vs. OGD), demonstrating that CAL effectively attenuated OGD-induced MMP loss in a concentration-dependent manner (Figure 1E,1F). The loss of MMP depletes ATP and leads to cell death, we further measured intracellular ATP levels. OGD treatment reduced ATP levels to 1.32±0.22 nmol/mg protein, compared with 3.92±0.51 nmol/mg protein in control group, while CAL (10, 20, and 40 µM) treatment attenuated this reduction, restoring ATP levels to 2.11±0.36, 2.56±0.40, and 3.59±0.48 nmol/mg protein (P=0.20, P<0.05, and P<0.001) (Figure 1G). These results indicated that CAL could attenuate oxidative stress and mitochondrial damage in OGD-induced H9c2 cells.

Figure 1 CAL suppressed oxidative stress and mitochondrial damage in OGD-induced H9c2 cells. (A,B) ROS generation in H9c2 cells was assayed via DCFH-DA staining. (C) The levels of MDA in the H9c2 cells. (D) Representative images of DCFH-DA probe for ROS in H9c2 cells. (E,F) Mitochondrial membrane potential in H9c2 cells was assayed by JC-1 staining. (G) The levels of ATP in the H9c2 cells. Values are the mean ± SD. ^###, P<0.001 compared with the control group. *, P<0.05; **, P<0.01; ***, P<0.001 compared with the OGD group. ATP, adenosine triphosphate; CAL, calycosin; DCFH-DA, 2,7-dichlorodihydrofluorescein diacetate; MDA, malondialdehyde; OGD, oxygen and glucose deprivation; ROS, reactive oxygen species; SD, standard deviation.

CAL inhibited ferroptosis in OGD-induced H9c2 cells

To determine whether CAL affects ferroptosis in OGD-induced H9c2 cells, we examined intracellular iron levels in each group. As shown in Figure 2A, OGD exposure significantly elevated intracellular Fe²⁺ content to 4.14±0.53 nmol/mg protein compared with 1.84±0.11 nmol/mg protein in the control group (P<0.001), indicating iron accumulation. CAL treatment at 10, 20, and 40 µM dose-dependently reduced Fe²⁺ levels to 3.64±0.20, 3.10±0.30, and 2.63±0.22 nmol/mg protein, respectively (P=0.32, P<0.05, P<0.001 vs. OGD). The NRF2/HO-1 pathway is a classic antioxidant pathway in cardiomyocyte injury, and it has also been identified as a key regulator in the development of ferroptosis (29,30). Moreover, GPX4, FTH1, and SLC7A11 are critical to maintaining intracellular iron homeostasis (31). We performed Western blot and qRT-PCR analysis of the NRF2/HO-1 pathway and ferroptosis-related proteins (NRF2, HO-1, GPX4, FTH1, and SLC7A11), which showed that OGD injury significantly inhibited their expression in H9c2 cells (all P<0.05), while CAL dose-dependently reversed this suppression, with the 40 µM concentration showing the most marked effect (all P<0.05 vs. OGD) (Figure 2B-2L). Collectively, these findings indicated that CAL could inhibit ferroptosis in OGD-induced H9c2 cells.

Figure 2 CAL inhibited ferroptosis in OGD-induced H9c2 cells. (A) The levels of Fe²⁺ in the H9c2 cells. (B-G) The expression of NRF2, HO-1, GPX4, FTH1, and SLC7A11 in H9c2 cells was examined via Western blotting. (H-L) The mRNA expression levels of NRF2, HO-1, GPX4, FTH1, and SLC7A11. Values are the mean ± SD. ^###, P<0.001 compared with the control group. *, P<0.05; **, P<0.01; ***, P<0.001 compared with the OGD group. CAL, calycosin; FTH1, ferritin heavy chain 1; GPX4, glutathione peroxidase 4; HO-1, heme oxygenase-1; mRNA, messenger RNA; NRF2, nuclear factor erythroid 2-related factor 2; OGD, oxygen and glucose deprivation; SD, standard deviation; SLC7A11, solute carrier family 7 member 11.

Erastin reversed the inhibitory effects of CAL on oxidative toxicity in the myocardium in OGD-induced H9c2 cells

Mitochondrial oxidative damage has been proven to be closely related to ferroptosis (32). As shown in Figure 3A-3G, erastin reversed the protective effects of CAL on oxidative stress and mitochondrial damage. Specifically, compared with the CAL-treated group, co-treatment with erastin increased ROS levels from 2.47±0.16 to 3.81±0.16 (P<0.01) and MDA content from 7.51±0.64 to 10.32±0.77 nmol/mg protein (P<0.01). Moreover, erastin abolished CAL-mediated protection of MMP, as reflected by an elevated green fluorescence intensity from 1.78±0.20 to 2.49±0.14 (P<0.01), and reduced ATP levels from 3.27±0.26 to 2.26±0.18 nmol/mg protein (P<0.05). Our previous experiment demonstrated that CAL could improve and oxidative stress (17).

Figure 3 Erastin reversed the inhibitory effects of CAL on oxidative toxicity in OGD-induced H9c2 cells. (A,C) ROS generation in H9c2 cells was assayed via DCFH-DA staining. (B) Representative images of DCFH-DA probe for ROS in H9c2 cells. (D) The levels of MDA in the H9c2 cells. (E,F) Mitochondrial membrane potential in H9c2 cells was assayed via JC-1 staining. (G) The levels of ATP in the H9c2 cells. Values are the mean ± SD. ^###, P<0.001 compared with the control group. **, P<0.01; ***, P<0.001 compared with the OGD group. ^$, P<0.05; ^$$, P<0.01 compared with the OGD + CAL (40 μM) group. ATP, adenosine triphosphate; CAL, calycosin; DCFH-DA, 2,7-dichlorodihydrofluorescein diacetate; JC-1, 5,5’,6,6’-tetrachloro1,1’,3,3’-tetramethylbenzimidazolylcarbocyanine iodide; MDA, malondialdehyde; OGD, oxygen and glucose deprivation; ROS, reactive oxygen species; SD, standard deviation.

Inhibiting the NRF2/HO-1 pathway reversed the anti-ferroptosis effects of CAL on OGD-induced H9c2 cells

To determine whether CAL affects ferroptosis through the NRF2/HO-1 pathway, we co-administered CAL and NRF2 small interfering RNA (siRNA) in OGD-induced H9c2 cells. NRF2 knockdown decreased the expression of NRF2 and HO-1 in the OGD + CAL (40 µM) + si-NRF2 group, compared with the OGD + CAL (40 µM) + si-NC group (Figure 4A-4C). Compared with the OGD + CAL (40 µM) + si-NC group, silencing NRF2 downregulated the protein expression of GPX4, FTH1 and SLC7A11 (all P<0.05) (Figure 4D-4G). Furthermore, compared with the OGD + CAL (40 µM) + si-NC group, silencing NRF2 upregulated intracellular Fe²⁺ levels 2.44±0.32 to 3.56±0.24 nmol/mg protein (P<0.01) (Figure 4H). These results were confirmed by the ATP, MDA, MMP, and ROS assays of ferroptosis-related mitochondrial oxidative damage. Compared with OGD + CAL (40 µM) + si-NC group, NRF2 knockdown significantly elevated ROS levels from 2.33±0.23 to 3.34±0.30 and MDA content from 7.41±1.04 to 11.26±1.00 nmol/mg protein (P<0.01 and P<0.001), while reducing ATP production from 3.54±0.33 to 2.56±0.31 nmol/mg protein (P<0.01). Additionally, MMP loss was exacerbated, as reflected by an increased green fluorescence intensity from 1.60±0.25 to 2.76±0.10 (P<0.001) (Figure 4I-4O).

Figure 4 Inhibition of the NRF2/HO-1 pathway reversed the anti-ferroptotic effects of CAL on OGD-induced H9c2 cells. (A-C) Protein expression of NRF2 and HO-1 in the H9c2 cells was examined by Western blotting. (D-G) Protein expression of GPX4, FTH1, and SLC7A11 in the H9c2 cells was examined by Western blotting. (H) The levels of intracellular iron in the H9c2 cells. (I) The levels of ATP in the H9c2 cells. (J) The levels of MDA in the H9c2 cells of different groups. (K,N) ROS generation in H9c2 cells was assayed by staining with DCFH-DA. (L) Representative images of DCFH-DA probe for ROS in H9c2 cells. (M,O) Mitochondrial membrane potential in H9c2 cells was assayed by JC-1 staining. Values are the mean ± SD. ^###, P<0.001 compared with the control group. **, P<0.01; ***, P<0.001 compared with the OGD group. ^$, P<0.05; ^$$, P<0.01; ^$$$, P<0.001 compared with the OGD + CAL (40 μM) + si-NC group. ATP, adenosine triphosphate; CAL, calycosin; DCFH-DA,2,7-dichlorodihydrofluorescein diacetate; FTH1, ferritin heavy chain 1; GPX4, glutathione peroxidase 4; HO-1, heme oxygenase-1; JC-1, 5,5’,6,6’-tetrachloro1,1’,3,3’-tetramethylbenzimidazolylcarbocyanine iodide; MDA, malondialdehyde; NRF2, nuclear factor erythroid 2-related factor 2; OGD, oxygen and glucose deprivation; ROS, reactive oxygen species; SD, standard deviation; si-NC, small interfering RNA negative control; SLC7A11, solute carrier family 7 member 11.

CAL improved cardiac function and reduced fibrosis in post-MI HF rats

Given the effects of CAL in vitro, we further examined the effect of CAL on MI in vivo. A LAD ligation was performed in rats to investigate the effects of CAL on cardiac fibrosis and ventricular dysfunction post-MI. Following a 4-week administration of CAL (25 and 50 mg/kg) and Fer-1 (0.7 mg/kg), echocardiography was performed to assess cardiac function. No significant differences were observed between the control and sham groups. In contrast, the model group showed significantly impaired cardiac function with LVEF from 79.87%±2.77% in the sham group to 33.83%±3.57% (P<0.001) and LVFS from 42.93%±2.05% to 13.76%±1.57% (P<0.01). CAL treatment produced a dose-dependent protective effect, with LVEF values of 46.6%±4.57% and 57.4%±5.40% and LVFS values of 20.03%±2.15% and 26.40%±3.34% in the low- and high-dose groups, respectively (all P<0.05 vs. model) (Figure 5A-5C). An elevated serum NT-pro-BNP level is a key indicator of impaired cardiac function, and we measured its levels in experimental rat groups using ELISA. Serum NT-pro-BNP levels were significantly higher in the model group (360.28±22.34 pg/mL) than in the sham group (103.67±17.44 pg/mL) (P<0.001). CAL administration dose-dependently reduced NT-pro-BNP to 268.73±26.93 and 182.64±33.56 pg/mL, respectively (P<0.01 and P<0.001 vs. model) (Figure 5D). Subsequently, heart tissues were collected for histological examination with HE staining and Masson trichrome staining. As shown in Figure 5E, the myocardial tissues in the control and sham groups were well arranged, with normal cell morphology appearing under HE staining. Myocardial tissue from model rats exhibited extensive scar tissue formation and was accompanied by inflammatory cell infiltration and the disorganization of cardiomyocytes. However, CAL dose-dependently reduced the areas of scar tissue and attenuated the influx of inflammatory cells, and myocardial tissue regularity and cell integrity were significantly improved. Masson’s trichrome staining revealed that the model group had significantly increased collagen deposition compared with the sham group (47.53%±2.87% vs. 9.99%±1.34%, P<0.001). CAL at 25 and 50 mg/kg dose-dependently reduced the fibrotic area to 32.86%±2.43% and 20.51%±2.38%, respectively (both P<0.001 vs. model) (Figure 5F,5G). Fer-1 had similar effects to those of CAL. Overall, these data suggest that CAL can reduce myocardial fibrosis and improve cardiac function in rats after post-MI HF.

Figure 5 CAL attenuates myocardial fibrosis and improves cardiac function in rats with post-myocardial infarction heart failure. (A-C) Echocardiography measurement of the cardiac function of rats. (D) Comparison of serum NT-pro-BNP levels of rats in each group. (E) Representative images of HE staining. Scale bar, 50 μm. (F) Representative images of Masson trichrome staining. Scale bar, 50 μm. (G) Collagen volume fraction. Values are the mean ± SD. ^##, P<0.01; ^###, P<0.001 compared with the sham group. *, P<0.05; **, P<0.01; ***, P<0.001 compared with the model group. CAL, calycosin; Fer-1, ferrostatin-1; H, high; HE, hematoxylin and eosin; L, low; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; NT-pro-BNP, N-terminal pro-brain natriuretic peptide; SD, standard deviation.

CAL inhibited NRF2/HO-1 pathway-mediated cardiomyocyte ferroptosis in post-MI HF rats.

Based on our prior in vitro evidence indicating that CAL inhibits ferroptosis to ameliorate cardiotoxicity, we next examined its efficacy against cardiomyocyte ferroptosis in an in vivo rat model of MI-induced HF. To verify whether the anti-ferroptotic effect of CAL was related to the amelioration of mitochondrial oxidative damage, we used transmission electron microscopy. This showed that the myocardial cells in the control and sham groups were clearly structured, with neat and orderly arrangement of thick and thin myofilaments, regular nuclei, and relatively homogeneous mitochondrial size. Compared with those in the sham group, the myofilaments in the model group were disorganized, which was accompanied by myofilament fracture and lysis, mitochondrial swelling, and disorganized mitochondrial arrangement, and most mitochondrial cristae were blurred or even broken. The myofilaments in the CAL group were more neatly arranged than were those in the model group, and the mitochondrial cristae were less damaged. Only some cristae were broken, the swelling was relatively reduced, with the improvement being more pronounced in the CAL-H group. Fer-1 had similar effects as those of CAL (Figure 6A). As shown in Figure 6B, MDA content was significantly elevated in the model group (2.55±0.32 nmol/mg protein) compared with the sham group (0.73±0.22 nmol/mg protein, P<0.001). CAL treatment at 25 and 50 mg/kg dose-dependently reduced MDA levels to 1.81±0.18 and 0.99±0.23 nmol/mg protein, respectively (P<0.01 and P<0.001 vs. model), while Fer-1 similarly decreased MDA to 0.89±0.09 nmol/mg protein (P<0.001 vs. model).

Figure 6 CAL inhibited NRF2/HO-1 pathway-mediated cardiomyocyte ferroptosis in post-MI HF rats. (A) Transmission electron microscopy of rat myocardial tissue (scale bar, 2 μm). (B) The levels of MDA in the rat myocardial tissues of the different groups. (C-H) Protein expression of NRF2, HO-1, GPX4, FTH1, and SLC7A11 in the rat myocardial tissues as examined by Western blotting. (I-M) The mRNA expression levels of NRF2, HO-1, GPX4, FTH1, and SLC7A11 according to qRT-PCR. Values are the mean ± SD. ^###, P<0.001 compared with the sham group. *, P<0.05; **, P<0.01; ***, P<0.001 compared with the model group. CAL, calycosin; Fer-1, ferrostatin-1; FTH1, ferritin heavy chain 1; GPX4, glutathione peroxidase 4; H, high; HF, heart failure; HO-1, heme oxygenase-1; L, low; MDA, malondialdehyde; MI, myocardial infarction; mRNA, messenger RNA; NRF2, nuclear factor erythroid 2-related factor 2; qRT-PCR, quantitative real-time polymerase chain reaction; SD, standard deviation; SLC7A11, solute carrier family 7 member 11.

We further used Western blotting and qRT-PCR to examine the effects of CAL on the NRF2/HO-1 signaling pathway and ferroptosis-related proteins, including NRF2, HO-1, GPX4, FTH1, and SLC7A11, to identify the mechanisms through which CAL attenuated cardiac fibrosis and improved cardiac function in post-MI HF rats. NRF2, HO-1, GPX4, FTH1, and SLC7A11 levels were downregulated in the model group compared with the sham group (all P<0.05), and CAL administration upregulated the expression of these proteins, with the most significant effects at 50 mg/kg (all P<0.05 vs. model) (Figure 6C-6M). Fer-1 treatment produced similar upregulation of these proteins compared with the model group (all P<0.05 vs. model) (Figure 6C-6M).

Discussion

This study investigated the protective effect of CAL against post-MI HF, along with its molecular mechanism, through in vivo and in vitro experiments (Figure 7). The principal findings were as follows: (I) CAL improved cardiac function in HF rats by inhibiting myocardial tissue injury and ferroptosis. (II) CAL inhibited OGD-induced ferroptosis in H9c2 cells and improved cellular oxidative damage and mitochondrial injury. (III) The effect of CAL on ferroptosis may be mediated by the NRF2/HO-1 signaling pathway.

Figure 7 Calycosin protects against post-myocardial infarction heart failure via a NRF2/HO-1-dependent ferroptosis pathway. ATP, adenosine triphosphate; FTH1, ferritin heavy chain 1; GPX4, glutathione peroxidase 4; HO-1, heme oxygenase-1; MDA, malondialdehyde; NRF2, nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species; SLC7A11, solute carrier family 7 member 11.

CAL is an extract of Radix astragali, and previous studies on CAL in cardiac injury have focused on the anti-inflammatory and autophagy-activating properties of CAL (15-17,33). Moreover, the antioxidant effect of CAL has been confirmed in several experiments in other disease models (18,20). In our study, we found that CAL treatment significantly improved cardiac dysfunction and reduced ferroptosis and fibrosis in post-MI HF rats. Moreover, CAL treatment reduced damage and ferroptosis in OGD-induced H9c2 cells and alleviated mitochondrial oxidative damage of H9c2 cells.

Ferroptosis is a novel type of regulated cell death that has been shown to be associated with the progression of HF (34-37); therefore, targeting ferroptosis holds substantial potential as a novel therapeutic intervention for HF. Iron that is ingested generally enters the cell via transferrin and is stored in ferritins to maintain low intracellular iron levels (38). Abnormal breakdown of intracellular ferritins increases intracellular iron levels, and high iron levels lead to the generation of ROS through the Fenton reaction and increase the activity of iron-requiring oxidases, leading to the onset of intracellular lipid peroxidation (39-41). GPX4, a specific inhibitor of lipid peroxidation, can inhibit the onset of ferroptosis by reducing oxidized lipids (42,43). MDA is a known marker for the onset of lipid peroxidation, and therefore, an increase in MDA expression and a decrease in GPX4 expression are often collectively used as a marker for the onset of ferroptosis. In our study, ferroptosis was elevated in a rat model of post-MI HF, as evidenced by decreased GPX4, FTH1, and SLC7A11 expressions and increased MDA levels and mitochondrial damage. CAL treatment inhibited ferroptosis and concurrently reduced mitochondrial damage in cardiomyocytes in a post-MI HF model. Ferroptosis involves oxidative damage, predominantly mitochondrial damage, due to the intracellular membrane damage caused by the excessive accumulation of iron-dependent lipid peroxidation products (44). Mitochondria play an important role in maintaining cardiac energy homeostasis and redox balance, and there is increasing evidence suggesting that the redox disorders and energy metabolism disorders caused by mitochondrial dysfunction are associated with the progression of HF (45,46). In this study, CAL exerted its cardioprotective effect by ameliorating mitochondrial damage and inhibiting ferroptosis.

NRF2 is a major transcriptional activator of the antioxidant response, controlling numerous genes that mitigate cellular redox imbalance. Thus, the NRF2/HO-1 pathway is recognized as a critical upstream signaling pathway that regulates ferroptosis (47). Several studies have demonstrated that NRF2/HO-1 pathway activation can inhibit the occurrence of ferroptosis through its antioxidant capacity (48,49). Elevated NRF2 and HO-1 activity are adaptions to the postinfarction hypoxic ischemia in cardiomyocytes and inhibit oxidative damage in ventricular remodeling (27,50-52). Our results showed that CAL upregulated NRF2 and HO-1. To further confirm the mechanism by which CAL affects the NRF2/HO-1 signaling pathway, siRNA targeting NRF2 was administered to OGD-induced H9c2 cells. The results showed that CAL significantly reduced OGD-induced H9c2 ferroptosis and mitochondrial damage. Crucially, these protective effects of CAL were abrogated by NRF2 siRNA knockdown, indicating that the mechanism of the cardioprotective effect of CAL is mainly mediated by the NRF2/HO-1 pathway.

Conclusions

In summary, CAL exerts a protective effect in the treatment of post-MI HF. CAL activates the expression of the NRF2/HO-1 signaling pathway in cardiomyocytes to inhibit the onset of ferroptosis. We believe that CAL may be a promising candidate for the treatment of post-MI HF.

Certain limitations to our study should be acknowledged. For example, the specific dose-response relationship of CAL in the treatment of MI was not explored. Moreover, the long-term effects and potential side-effects of CAL in clinical applications remain unclear. Future research could include large-scale clinical trials to validate the efficacy and safety of CAL. Moreover, investigating the interaction between CAL and other existing MI treatment drugs may lead to the development of more comprehensive treatment strategies. By addressing these aspects, we can advance the application of CAL, moving from preclinical research to real-world clinical practice, and better aid patients with post-MI HF.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1421/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1421/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1421/prf

Funding: This work was financially supported in part by the Grants from the National Natural Science Foundation of China (No. 81804045), the Natural Science Foundation of Shandong Province (No. ZR2021LZY038), the Shandong Traditional Chinese Medicine Science and Technology Development Program (No. 2021M180), Shandong Province Traditional Chinese Medicine Characteristic Therapies (No. 3700020699), the Shandong Geriatrics Society 2021 Science and Technology Research Project (No. LKJGG2021W106), and the Qilu School of Chinese Medicine Academic School Inheritance Project (No. Ip2022-17).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1421/coif). X.W. reports that this work was financially supported in part by the Grants from the National Natural Science Foundation of China (No. 81804045), the Natural Science Foundation of Shandong Province (No. ZR2021LZY038), the Shandong Traditional Chinese Medicine Science and Technology Development Program (No. 2021M180), Shandong Province Traditional Chinese Medicine Characteristic Therapies (No. 3700020699), the Shandong Geriatrics Society 2021 Science and Technology Research Project (No. LKJGG2021W106), and the Qilu School of Chinese Medicine Academic School Inheritance Project (No. Ip2022-17). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Animal experiments were performed under a project license (No. 2021-33) granted by the Experimental Animal Ethics Committee of The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, in compliance with the eighth edition of the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 2011).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Udell JA, Bahit MC, Campbell P, et al. Prevention of heart failure after acute myocardial infarction. Lancet 2025;406:1154-70. [Crossref] [PubMed]
2. Roger VL. Epidemiology of heart failure. Circ Res 2013;113:646-59. [Crossref] [PubMed]
3. Frangogiannis NG. Cardiac fibrosis. Cardiovasc Res 2021;117:1450-88. [Crossref] [PubMed]
4. Frangogiannis NG. Cardiac fibrosis: Cell biological mechanisms, molecular pathways and therapeutic opportunities. Mol Aspects Med 2019;65:70-99. [Crossref] [PubMed]
5. van der Pol A, van Gilst WH, Voors AA, et al. Treating oxidative stress in heart failure: past, present and future. Eur J Heart Fail 2019;21:425-35. [Crossref] [PubMed]
6. Miotto G, Rossetto M, Di Paolo ML, et al. Insight into the mechanism of ferroptosis inhibition by ferrostatin-1. Redox Biol 2020;28:101328. [Crossref] [PubMed]
7. Stockwell BR, Friedmann Angeli JP, Bayir H, et al. Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell 2017;171:273-85. [Crossref] [PubMed]
8. Wang Z, Liu X, Wu Y, et al. Trimetazidine alleviates heart failure after myocardial infarction by promoting PINK1/parkin-mediated mitophagy and suppressing GPX4-dependent ferroptosis. Eur J Pharmacol 2025;1008:178376. [Crossref] [PubMed]
9. Piamsiri C, Maneechote C, Jinawong K, et al. Pharmacological inhibition of apoptosis, necroptosis, and ferroptosis confers effective cardioprotection in post-myocardial infarction in rats. Sci Rep 2025;15:31140. [Crossref] [PubMed]
10. Chen X, Yu X, Zhong S, et al. ALDH2/eIF3E Interaction Modulates Protein Translation Critical for Cardiomyocyte Ferroptosis in Acute Myocardial Ischemia Injury. Circulation 2026;153:164-84. [Crossref] [PubMed]
11. Park TJ, Park JH, Lee GS, et al. Quantitative proteomic analyses reveal that GPX4 downregulation during myocardial infarction contributes to ferroptosis in cardiomyocytes. Cell Death Dis 2019;10:835. [Crossref] [PubMed]
12. Deng M, Chen H, Long J, et al. Calycosin: a Review of its Pharmacological Effects and Application Prospects. Expert Rev Anti Infect Ther 2021;19:911-25. [Crossref] [PubMed]
13. Zhou Y, Song W, Wang Y, et al. Calycosin regulates gut microbiota-bile acid-FXR axis to protect rats from cerebral ischemia-reperfusion injury. Eur J Pharmacol 2025;1000:177707. [Crossref] [PubMed]
14. Yuan HJ, Han QC, Yu H, et al. Calycosin treats acute myocardial infarction via NLRP3 inflammasome: Bioinformatics, network pharmacology and experimental validation. Eur J Pharmacol 2025;997:177621. [Crossref] [PubMed]
15. Chen G, Xu H, Xu T, et al. Calycosin reduces myocardial fibrosis and improves cardiac function in post-myocardial infarction mice by suppressing TGFBR1 signaling pathways. Phytomedicine 2022;104:154277. [Crossref] [PubMed]
16. Wang X, Li W, Zhang Y, et al. Calycosin as a Novel PI3K Activator Reduces Inflammation and Fibrosis in Heart Failure Through AKT-IKK/STAT3 Axis. Front Pharmacol 2022;13:828061. [Crossref] [PubMed]
17. Zhang L, Fan C, Jiao HC, et al. Calycosin Alleviates Doxorubicin-Induced Cardiotoxicity and Pyroptosis by Inhibiting NLRP3 Inflammasome Activation. Oxid Med Cell Longev 2022;2022:1733834. [Crossref] [PubMed]
18. Huang D, Shen P, Wang C, et al. Calycosin plays a protective role in diabetic kidney disease through the regulation of ferroptosis. Pharm Biol 2022;60:990-6. [Crossref] [PubMed]
19. Su X, Huang Q, Chen J, et al. Calycosin suppresses expression of pro-inflammatory cytokines via the activation of p62/Nrf2-linked heme oxygenase 1 in rheumatoid arthritis synovial fibroblasts. Pharmacol Res 2016;113:695-704. [Crossref] [PubMed]
20. Lu CY, Day CH, Kuo CH, et al. Calycosin alleviates H(2) O(2) -induced astrocyte injury by restricting oxidative stress through the Akt/Nrf2/HO-1 signaling pathway. Environ Toxicol 2022;37:858-67. [Crossref] [PubMed]
21. Chen L, Li S, Zhu J, et al. Mangiferin prevents myocardial infarction-induced apoptosis and heart failure in mice by activating the Sirt1/FoxO3a pathway. J Cell Mol Med 2021;25:2944-55. [Crossref] [PubMed]
22. Wang K, Li Z, Li Y, et al. Cardioprotection of Klotho against myocardial infarction-induced heart failure through inducing autophagy. Mech Ageing Dev 2022;207:111714. [Crossref] [PubMed]
23. Goldman S, Raya TE. Rat infarct model of myocardial infarction and heart failure. J Card Fail 1995;1:169-77. [Crossref] [PubMed]
24. Zhang X, Wang Q, Wang X, et al. Tanshinone IIA protects against heart failure post-myocardial infarction via AMPKs/mTOR-dependent autophagy pathway. Biomed Pharmacother 2019;112:108599. [Crossref] [PubMed]
25. Li D, Zhao L, Li Y, et al. Gastro-Protective Effects of Calycosin Against Precancerous Lesions of Gastric Carcinoma in Rats. Drug Des Devel Ther 2020;14:2207-19. [Crossref] [PubMed]
26. Ye Y, Chen A, Li L, et al. Repression of the antiporter SLC7A11/glutathione/glutathione peroxidase 4 axis drives ferroptosis of vascular smooth muscle cells to facilitate vascular calcification. Kidney Int 2022;102:1259-75. [Crossref] [PubMed]
27. Fang X, Wang H, Han D, et al. Ferroptosis as a target for protection against cardiomyopathy. Proc Natl Acad Sci U S A 2019;116:2672-80. [Crossref] [PubMed]
28. Hori M, Nishida K. Oxidative stress and left ventricular remodelling after myocardial infarction. Cardiovasc Res 2009;81:457-64. [Crossref] [PubMed]
29. Zhang Q, Wang L, Wang S, et al. Signaling pathways and targeted therapy for myocardial infarction. Signal Transduct Target Ther 2022;7:78. [Crossref] [PubMed]
30. Li J, Lu K, Sun F, et al. Panaxydol attenuates ferroptosis against LPS-induced acute lung injury in mice by Keap1-Nrf2/HO-1 pathway. J Transl Med 2021;19:96. [Crossref] [PubMed]
31. Hu K, Jiang P, Hu J, et al. Dapagliflozin attenuates LPS-induced myocardial injury by reducing ferroptosis. J Bioenerg Biomembr 2024;56:361-71. [Crossref] [PubMed]
32. Gao M, Yi J, Zhu J, et al. Role of Mitochondria in Ferroptosis. Mol Cell 2019;73:354-363.e3. [Crossref] [PubMed]
33. Lu X, Lu L, Gao L, et al. Calycosin attenuates doxorubicin-induced cardiotoxicity via autophagy regulation in zebrafish models. Biomed Pharmacother 2021;137:111375. [Crossref] [PubMed]
34. Zheng H, Shi L, Tong C, et al. circSnx12 Is Involved in Ferroptosis During Heart Failure by Targeting miR-224-5p. Front Cardiovasc Med 2021;8:656093. [Crossref] [PubMed]
35. Zhang X, Zheng C, Gao Z, et al. SLC7A11/xCT Prevents Cardiac Hypertrophy by Inhibiting Ferroptosis. Cardiovasc Drugs Ther 2022;36:437-47. [Crossref] [PubMed]
36. Ning D, Yang X, Wang T, et al. Atorvastatin treatment ameliorates cardiac function and remodeling induced by isoproterenol attack through mitigation of ferroptosis. Biochem Biophys Res Commun 2021;574:39-47. [Crossref] [PubMed]
37. Liu K, Chen S, Lu R. Identification of important genes related to ferroptosis and hypoxia in acute myocardial infarction based on WGCNA. Bioengineered 2021;12:7950-63. [Crossref] [PubMed]
38. Masaldan S, Bush AI, Devos D, et al. Striking while the iron is hot: Iron metabolism and ferroptosis in neurodegeneration. Free Radic Biol Med 2019;133:221-33. [Crossref] [PubMed]
39. Galaris D, Barbouti A, Pantopoulos K. Iron homeostasis and oxidative stress: An intimate relationship. Biochim Biophys Acta Mol Cell Res 2019;1866:118535. [Crossref] [PubMed]
40. Chen X, Yu C, Kang R, et al. Iron Metabolism in Ferroptosis. Front Cell Dev Biol 2020;8:590226. [Crossref] [PubMed]
41. Dixon SJ, Lemberg KM, Lamprecht MR, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 2012;149:1060-72. [Crossref] [PubMed]
42. Ursini F, Maiorino M. Lipid peroxidation and ferroptosis: The role of GSH and GPx4. Free Radic Biol Med 2020;152:175-85. [Crossref] [PubMed]
43. Forcina GC, Dixon SJ. GPX4 at the Crossroads of Lipid Homeostasis and Ferroptosis. Proteomics 2019;19:e1800311. [Crossref] [PubMed]
44. Wang H, Liu C, Zhao Y, et al. Mitochondria regulation in ferroptosis. Eur J Cell Biol 2020;99:151058. [Crossref] [PubMed]
45. Zhou B, Tian R. Mitochondrial dysfunction in pathophysiology of heart failure. J Clin Invest 2018;128:3716-26. [Crossref] [PubMed]
46. Ramachandra CJA, Hernandez-Resendiz S, Crespo-Avilan GE, et al. Mitochondria in acute myocardial infarction and cardioprotection. EBioMedicine 2020;57:102884. [Crossref] [PubMed]
47. Dodson M, de la Vega MR, Cholanians AB, et al. Modulating NRF2 in Disease: Timing Is Everything. Annu Rev Pharmacol Toxicol 2019;59:555-75. [Crossref] [PubMed]
48. Lv Z, Wang F, Zhang X, et al. Etomidate Attenuates the Ferroptosis in Myocardial Ischemia/Reperfusion Rat Model via Nrf2/HO-1 Pathway. Shock 2021;56:440-9. [Crossref] [PubMed]
49. Liu XJ, Lv YF, Cui WZ, et al. Icariin inhibits hypoxia/reoxygenation-induced ferroptosis of cardiomyocytes via regulation of the Nrf2/HO-1 signaling pathway. FEBS Open Bio 2021;11:2966-76. [Crossref] [PubMed]
50. Oppedisano F, Mollace R, Tavernese A, et al. PUFA Supplementation and Heart Failure: Effects on Fibrosis and Cardiac Remodeling. Nutrients 2021;13:2965. [Crossref] [PubMed]
51. Tian C, Gao L, Rudebush TL, et al. Extracellular Vesicles Regulate Sympatho-Excitation by Nrf2 in Heart Failure. Circ Res 2022;131:687-700. [Crossref] [PubMed]
52. Mayoral-González I, Calderón-Sánchez EM, Galeano-Otero I, et al. Cardiac protection induced by urocortin-2 enables the regulation of apoptosis and fibrosis after ischemia and reperfusion involving miR-29a modulation. Mol Ther Nucleic Acids 2022;27:838-53. [Crossref] [PubMed]

(English Language Editor: J. Gray)