Wogonin attenuates septic cardiomyopathy by suppressing ALOX15-mediated ferroptosis

Abstract

Septic cardiomyopathy (SCM), a severe complication in sepsis, significantly increases the mortality of septic patients. Ferroptosis, an iron-regulated cell death, has been implicated in the development of SCM. Wogonin, a flavonoid from the root of the skullcap, exhibits anti-inflammatory, anti-allergic, and anti-apoptotic activities. In this study, we investigated the effects of wogonin on SCM and associated cardiomyocyte ferroptosis. Cecal ligation and puncture (CLP) surgery was performed in mice to establish a SCM model. Wogonin (20, 40 and 60 mg·kg⁻¹, i.p.) was administered 2 h prior to CLP surgery. We showed that wogonin pretreatment dose-dependently mitigated CLP-induced cardiac dysfunction, myocardial damage, and deranged cardiomyocyte contractility. Furthermore, wogonin pretreatment ameliorated cardiac inflammation, oxidative stress, and mitochondrial dysfunction in CLP-challenged mice. We demonstrated that wogonin exerted the cardioprotective effects through suppressing cardiomyocyte ferroptosis both in vivo and in vitro. We revealed that wogonin directly bound to and inhibited ALOX15 (arachidonic acid 15-lipoxygenase), a lipoxygenase that governed the oxidation of polyunsaturated fatty acids to initiate ferroptosis. Pharmacological inhibition of ALOX15 using a specific inhibitor ML351 (10 mg·kg⁻¹·d⁻¹, i.p. for 7 days prior to CLP surgery) markedly diminished cardiac abnormalities and cardiomyocyte ferroptosis in CLP-challenged mice. In LPS-challenged HL-1 cardiomyocytes, overexpression of ALOX15 or supplement of its downstream metabolite 15-HpETE (1 μM) diminished the anti-ferroptotic effects of wogonin. Our results demonstrate that wogonin protects against SCM through inhibition of ALOX15-meditated ferroptosis.

Introduction

Sepsis, a systemic inflammatory response often triggered by infection, is the most common cause of mortality in critically ill patients [1]. Approximately 70% of patients with sepsis develop septic cardiomyopathy (SCM), a severe complication directly associated with the high sepsis morbidity rate [2, 3]. The development of SCM is highly complicated and involves various pathophysiological factors, including inflammation, mitochondrial dysfunction, oxidative stress, calcium imbalance [4], and multiple forms of programmed cell death, including apoptosis [5], pyroptosis [6] and ferroptosis [7, 8]. Recent evidence increasingly suggests the involvement of ferroptosis in the development of SCM, as inhibiting cardiomyocyte ferroptosis significantly improves cardiac function in the context of SCM [9, 10].

Ferroptosis, a novel type of iron-catalyzed regulated cell death, is triggered by the excessive accumulation of peroxides of polyunsaturated fatty acid (PUFA)-containing phospholipids [11]. Specifically, cell membranes or organelle membranes rich in PUFAs are susceptible to oxidative damage and generate lipid peroxides under the actions of oxygen free radicals [12]. The accumulation of lipid peroxides alters membrane permeability, ultimately compromising membrane integrity and resulting in cell damage or death. Thus, lipid peroxidation serves as the final executor of ferroptosis, which is mediated enzymatically by arachidonic acid lipoxygenase (ALOX) [13]. 15-Hydroperoxyeicosatetraenoic acid (15-HpETE), an ALOX15-catalyzed metabolite downstream of arachidonic acid (AA), is a crucial inducer of cardiomyocyte ferroptosis [14, 15]. To counteract 15-HpETE-mediated damage, glutathione peroxidase (GPX4) transforms 15-HpETE into 15-hydroxyeicosatetraenoic acid (15-HETE) [16]. Thus, inhibiting the ALOX15/15-HpETE pathway may effectively prevent ferroptosis, providing a protective strategy against SCM.

Although various synthetic drugs have been employed to treat SCM, their applications are limited because of insufficient therapeutic efficacy and unexpected side effects in clinical settings [17]. These limitations have driven research efforts to explore alternative effective cardioprotective agents from traditional Chinese medicine. Wogonin (5,7-dihydroxy-8-methoxyflavone), a flavonoid derived from the roots of Scutellaria baicalensis Georgi, is one of the most common active ingredients in traditional Chinese medicine [18]. Wogonin has exhibited various pharmacological properties, such as antioxidant [19], antitumor [20], and anti-inflammatory effects, in various diseases [21]. Recent findings have revealed that wogonin has evident protective effects on cardiovascular diseases, including diabetic cardiomyopathy [22], doxorubicin-induced cardiotoxicity [21], and vascular calcification [23]. However, the role of wogonin in the regulation of sepsis-induced cardiomyopathy remains elusive.

In the present study, we aimed to explore the possible effects of wogonin on SCM and cardiomyocyte ferroptosis associated with SCM. Our findings demonstrate that wogonin protects against SCM by inhibiting cardiomyocyte ferroptosis in an ALOX15-dependent manner. These findings reveal the potential of wogonin as a novel drug for the clinical management of SCM.

Materials and methods

Animals and in vivo experiments

Male and female adult C57BL/6 mice (8 weeks old, 20–25 g) were acquired from GemPharmatech in Nanjing, China. All the animal procedures adhered to the rules outlined in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and were approved by the Animal Welfare and Ethics Committee of Zhongshan Hospital Fudan University (2023–021). The mice were allowed sterilized water and food ad libitum; and were housed with a 12-h day-night circadian cycle. The mice were euthanized via cervical dislocation under general anesthesia with 2.5% isoflurane at the conclusion of the experiment. To investigate the involvement of wogonin in SCM, forty-eight mice were divided into eight groups: the Sham, cecal ligation and puncture (CLP), and Sham-Wogonin and CLP-Wogonin groups administered different dosages of wogonin (20, 40, and 60 mg·kg⁻¹), with 6 mice in each group. In addition, the mouse survival rate was evaluated after CLP with or without wogonin treatment at different dosages (20, 40, or 60 mg·kg⁻¹; 10 mice per group). To determine the involvement of ALOX15 in SCM, twenty-four mice were divided into 4 groups: the Sham; CLP, Sham-ML351, and CLP-ML351 groups, with 6 mice per group.

Wogonin was purchased from Aladdin Biology Technology Institute (W101155, CAS 632-85-9, Shanghai, China). Lipopolysaccharide (LPS; cat.# L2880) and ML351 (cat.# 847163-28-4) were purchased from Sigma‒Aldrich (Sigma, St. Louis, MO, USA). Wogonin solution (40 mg·kg⁻¹) or an equivalent volume of vehicle was administered via intraperitoneal injection 2 h prior to the CLP procedure. The dosage of wogonin was selected on the basis of a prior study [19]. To study the involvement of ALOX15 in the protective effect of wogonin, adult mice were administered the ALOX15 selective inhibitor ML351 (10 mg·kg⁻¹·d⁻¹) intraperitoneally for 7 consecutive days prior to the CLP procedure [15].

CLP model

Male and female mice received anesthesia via intraperitoneal injection of ketamine (50 mg·kg⁻¹) and xylazine (5 mg·kg⁻¹) before undergoing abdominal depilation. Subsequently, a 1.5-cm incision was made along the length of the lower quadrant of the abdomen of the mice, followed by isolation of the cecum. Sublethal sepsis was induced by ligating the distal three-quarters of the cecum with 4–0 silk sutures and making two punctures in the ligated cecum with a 22-gauge needle. The cecum was subsequently reinserted into the abdominal cavity following the expulsion of feces, and the incision was closed with a 4–0 silk suture. Following the CLP procedure, the mice received a 1 mL dose of sterile saline [24]. At 24 h post-CLP, two researchers determined the sepsis scores and recorded the anal temperature. The animal survival rate was assessed at 2-h intervals over a 7-day period.

Murine sepsis score

The murine sepsis score was calculated to assess the sepsis severity according to previous reports [25, 26]. In brief, the sepsis score was determined using the following seven components: appearance, level of consciousness, activity, response to stimulus, eyes, respiration rate, and respiration quality. Each index was scored from 0 to 4. A higher score denoted more severe sepsis. Two independent researchers determined the murine sepsis score.

Echocardiographic assessment

Echocardiography was performed on the mice utilizing a Vevo 3100 system (VisualSonics, Toronto, ON, Canada) to obtain two-dimensional targeted M-mode images on the short axis at the level of the papillary muscle under isoflurane anesthesia. A heating platform was used to maintain normal body temperature. The left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) were derived from the left ventricular dimensions to quantify the degree of cardiac systolic function as previously described [27, 28].

Histological analysis

Cardiac tissues were preserved in a 4% paraformaldehyde solution and subsequently embedded in paraffin. The tissue samples were then sliced into sections. Immunohistochemical labeling was performed by incubating the tissue sections with the anti-4-HNE (1:100, ab46545, Abcam), anti-CD45 (1:250, ab40763, Abcam), and anti-CD68 (1:100, ab283654, Abcam) antibodies overnight, followed by a 30-min incubation with secondary antibodies at room temperature. Images were acquired using a digital microscope and analyzed with ImageJ software [29].

TUNEL staining

Heart tissues were fixed in paraffin, sectioned into 5-μm slices, and stained with a commercial TUNEL Staining Kit (Abbkine Scientific, Wuhan, China) to evaluate cardiomyocyte apoptosis in accordance with the manufacturer’s protocol. Cardiomyocytes were visualized via confocal fluorescence microscopy [30].

Biochemical detection

Mouse serum was collected, and the circulating levels of cardiac troponin I (cTnI), lactate dehydrogenase (LDH), and creatine kinase isoenzyme (CK-MB) were quantified using a commercial kit from Nanjing Jiancheng Bioengineering Institute following the manufacturer’s instructions. The levels of GSH and GSSG were quantified utilizing a glutathione assay kit (cat. #S0052, Beyotime). Cardiac malondialdehyde (MDA) levels were measured utilizing a lipid peroxidation MDA assay kit (cat. #S0131, Beyotime). The 4-HNE adduct was assessed utilizing a Lipid Peroxidation (4-HNE) kit (ab238538, Abcam).

Transmission electron microscopy

Fresh myocardial samples were cut into small pieces (1 mm³) and placed in 0.1 M sodium phosphate (pH 7.4) containing 2.5% glutaraldehyde for at least 24 h at 4 °C. The tissues were dried with an alcohol gradient, inserted into Epon Araldite, and finally fixed in 1% OsO₄ for 1 h. We made ultrathin sections (75–80 nm) using a Leica ultramicrotome with a Diatome diamond knife. The sections were then stained with uranyl acetate for 10 min and lead citrate for another 5 min. A 40–120 kV transmission electron microscope (Hitachi H600, Hitachi, Japan) was used to observe the samples. A digital micrograph tool was used to obtain images [29]. ImageJ software was used to analyze mitochondrial morphology. Mitochondrial damage was characterized by the fragmentation of cristae and the irregular arrangement of mitochondria. The proportion of damaged mitochondria relative to the total number of mitochondria was then quantified [31].

Cell culture and in vitro treatment

Mouse HL-1 cardiomyocytes were cultivated at 37 °C with 5% CO₂ in Dulbecco’s modified Eagle’s medium (Gibco, Waltham; ME, USA), supplemented with 10% fetal bovine serum albumin (Gibco). To determine the effects of wogonin on LPS-induced cardiotoxicity and ferroptosis in vitro, HL-1 cardiomyocytes were exposed to LPS (10 μM, 24 h), wogonin (10 μM, 24 h), or the ferroptosis inhibitor ferrostatin-1 (Fer-1, 5 μM). To evaluate the impact of 15-HpETE on LPS-induced cardiotoxicity, HL-1 cardiomyocytes were treated with 15-HpETE (1 μM) [15].

Cells were seeded at a density of 4.0 × 10⁶ in 6-well plates and then transfected with either an empty vector (VectorBuilder, Guangzhou, China) or pAAV-CMV-Alox15 3 × Flag using NEOFECT DNA transfection reagent (Beijing, China) to overexpress ALOX15. After 6 h, the medium was replaced, and the mixture was incubated for an additional 24 h. Western blot analysis was used to confirm ALOX15 overexpression [14].

Cell viability assay

Cell viability was assessed using a Cell Counting Kit-8 (C0039, Beyotime). The cells were inoculated in a 96-well plate at a density of 1 × 10⁴ per well. A multiplate reader (Biotek Synergy) was used to measure the absorbance at 450 nm after the indicated treatments, and the percentage of viable cells was determined [32].

Primary adult mouse cardiomyocyte (AMCM) isolation and cell shortening/relengthening

Eight-week-old adult mice were anesthetized with 4% chloral hydrate. The inferior vena cava was severed, and the right ventricle was then rapidly filled with an EDTA buffer. After sequential perfusion with EDTA and perfusion buffers and removal of the hearts from the ascending aorta, type II and type IV collagenase (LS004176 and LS004188; Worthington Biochemical Corporation, Lakewood, NJ, USA) were used for digestion. The digested tissues were carefully sectioned into small fragments using forceps. The cells were then retrieved via sedimentation and the restoration of calcium gradients. The success rates for rod-shaped AMCMs range from 80% to 90% [33]. For further treatment, AMCMs were either cultivated in M199 media (supplemented with 0.1% BSA, 0.1% BDM, 1% ITS, 1% CD lipid, and 1% penicillin‒streptomycin) or resuspended in a contractile buffer.

The mechanical properties of the AMCMs were examined using an Olympus IX-70 microscope and a SoftEdge MyoCam system (IonOptix, Milton, MA, USA). The cells were resuspended in a contractile buffer and subjected to electrical stimuli at 0.5 Hz. An IonOptix SoftEdge apparatus was used to record the isotonic contraction of the AMCMs [34]. Prior to recording mechanical parameters, such as the maximum velocities of shortening/relengthening (±dL/dt), peak shortening (PS), time-to-peak shortening (TPS), and time-to-90% relengthening (TR₉₀), the cardiomyocytes were paced at 0.5 Hz.

Propidium iodide (PI) staining

Cells were inoculated in a 12-well plate with 2.5 × 10⁵ cells per well and administered the specified compounds. After incubation, the media was removed, and propidium iodide (5 μg/mL, P1304MP, Invitrogen) was used to stain the cells for 30 min. Images were obtained using a fluorescence microscope (Nikon, Japan) [29].

Detection of the mitochondrial membrane potential and mitochondrial O₂⁻ content

AMCMs were plated in a glass-bottom dishes containing M199 media supplemented with 0.1% BSA, 0.1% BDM, 1% ITS, 1% CD lipid, and 1% penicillin-streptomycin and incubated overnight at 37 °C in a 5% CO₂ atmosphere. JC-1 working solution (Dojindo Kumamoto, Japan MT09) was then added, and incubated continued for an additional 30 min. The mitochondrial O₂⁻ content was determined using MitoSOX (Invitrogen). Briefly, cardiomyocytes were treated with MitoSOX (1 mM) for 10 min before observation with a laser confocal microscope.

Real-time quantitative PCR

Total RNA was isolated from tissues or cells utilizing TRIzol (Invitrogen, NY, USA), and cDNA was synthesized using a cDNA Synthesis Kit (Takara, Shiga, Japan) as a template. Quantitative PCR was conducted using the ABI PRISM 7900 Sequence Detection System (Applied Biosystems) with ChamQTM Universal SYBR^® qPCR Master Mix (Vazyme, Nanjing, China). Relative gene expression was determined via the 2^−ΔΔCt method after normalization to GAPDH expression. The forward (F) and reverse (R) primer sequences used were as follows: ALOX15, 5′-TGCAGAGCTGGTGTCAAGAG-3′ (F) and 5′-TCGTCGCGTCCTTGGTTTTA-3′ (R); Ptgs2, 5′GCGACATACTCAAGCAGGAGCA-3′ (F) and 5′-AGTGGTAACCGCTCAGGTGTTG-3′ (R); and GAPDH, 5′-CATCACTGCCACCCAGAAGACTG-3′ (F) and 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′ (R).

Assessment of lipid peroxidation

The levels of lipid reactive oxygen species were assessed via the use of C11-BODIPY (581/591) (HY-D1301, MCE). Cells were inoculated in a glass-bottom dish at a density of 1.5 × 10⁵ per well. Following the recommended procedures, the cells were treated with 2 μM C11-BODIPY (581/591) for 30 min at 37 °C in the dark. A confocal laser scanning microscope (Leica SP8, Wetzlar, Germany) was then used to visualize the cells [32].

Cellular thermal shift assay (CETSA)

Cells were resuspended in a lysis buffer and then centrifuged to extract the cell lysates. The lysates were divided into two aliquots, which were treated with wogonin (100 μM) or DMSO for 60 min at room temperature. Both the control and experimental samples were then subjected to thermal treatment at various temperatures ranging from 37 °C to 77 °C for 3 min. Protein analysis was subsequently performed via Western blotting [35].

Drug affinity responsive target stability (DARTS) assay

A total of 600 μL of cold M-PER buffer (Thermo Fisher Scientific, USA, cat.# 78503) supplemented with 1% protease inhibitor was used to lyse ~1 × 10⁷ HL-1 cells. Following collection, the supernatant was diluted with 10× TNC buffer (50 mM Tris-HCl, pH 8.0; 50 mM NaCl; and 10 mM CaCl₂). Following centrifugation at 12,000 × g for 10 min at 4 °C, equal quantities of cell lysates were treated with 100 μM wogonin or DMSO at room temperature for 60 min. The lysates were then digested with pronase (1:2000, Roche, Switzerland, cat.# 10165921001) for 30 min at 37 °C, and was terminated by heating and the addition of loading buffer. The target protein levels were assessed by Western blotting.

Western blotting assay

RIPA lysis solution (P0013; Beyotime Biotechnology, Shanghai, China) containing protease inhibitors was used to lyse cardiac tissues and cells. The protein extracts were then separated via SDS‒PAGE and placed on membranes. The membranes were incubated with primary antibodies overnight at 4 °C and then treated with the appropriate secondary antibodies. A ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA) was used to collect the images, and ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for quantification. The primary antibodies used were as follows: anti-Alox15(1:1000,ab244205,Abcam); anti-4-HNE (1:1000, ab48506, Abcam); anti-GPX4 (1:1000, 52455, Cell Signaling Technology); anti-GAPDH (1:2000, ab9485, Abcam); anti-PTGS2 (1:1000,ab179800, Abcam); anti-Vinculin (1:1000, A14193, ABclonal); anti-β-actin (1:20000, 66009-1-Ig, Proteintech); anti-TNF-α (1:500, abs146482, Absin) ; anti-IL-6 (1:500, abs148146, Absin) ; and anti-IL-1beta (1:1000, abs120224, Absin).

Molecular docking

The compound structure files were obtained from the PubChem website (https://pubchem.ncbi.nlm.nih.gov/), and Open Babel 2.3.2 software was used to convert the SDF files into PDB files. The receptor protein structure was obtained from the UniProt database. PyMOL 2.3.4 software was used to eliminate water molecules and ligands from the receptor protein. AutoDockTools software was used to modify the receptor protein structure, including adding hydrogen atoms and balancing charges. Both the receptor protein and small molecule ligands were subsequently converted into pdbqt format. AutoDock Vina 1.1.2 was used for global molecular docking of the small molecule ligands into the receptor protein, and PLIP was employed to analyze the data. The docking results were visualized with PyMOL [36].

15-HpETE assay

The 15-HpETE assay was conducted using ultrahigh-performance liquid chromatography‒mass spectrometry. 15-HpETE was isolated from 50 mg of cardiac tissue or 1 × 10⁷ cells using methanol and ethyl acetate (containing 0.01 mol/L butylated hydroxytoluene) after which 1 μL of the internal standard 5-HETE-d₈ (5 ng) was added according to a previously reported metabolomic method for extracting eicosanoids [15].

Statistical analysis

All the quantitative data are presented as the means ± standard errors (SEMs). The findings were analyzed using Prism 9.5.1 software (GraphPad, San Diego, CA, USA). Two-tailed Student’s t-tests were used to compare the results from two groups. One-way analysis of variance was employed to compare the results from several groups with the Tukey test for post hoc analysis. Kaplan–Meier survival curves were plotted to determine survival rates, which were analyzed for significant differences using the log-rank test. A value of P < 0.05 was considered to indicate statistical significance.

Results

Wogonin protects against CLP-induced SCM

To evaluate the effects of wogonin on SCM, we established a mouse model of CLP and administered different doses (20, 40, and 60 mg·kg⁻¹) of wogonin intraperitoneally 2 h prior to the CLP procedure (Fig. 1a). Notably, 40 mg·kg⁻¹ wogonin resulted in the most pronounced reduction in the mortality rate following CLP (Fig. 1b). Additionally, CLP led to an elevated sepsis score and a reduced anal temperature, both of which were prevented by wogonin administration (Fig. 1c, d). Echocardiographic assessment revealed substantial cardiac dysfunction following CLP, as indicated by reductions in the LVEF and LVFS. Notably, these echocardiographic alterations were significantly alleviated by wogonin (Fig. 1e–g). Furthermore, wogonin significantly decreased the percentage of TUNEL-positive cells following CLP surgery (Fig. 1h, i). Wogonin administration also markedly decreased the CLP-induced increases in the levels of myocardial injury biomarkers, including plasma cTnI, CK-MB, and LDH (Fig. 1j–l). Notably, 40 and 60 mg·kg⁻¹ wogonin demonstrated the most significant attenuation of SCM. These results suggest that wogonin provides protection against CLP-induced SCM.

Fig. 1. Wogonin protects against CLP-induced SCM.

a Scheme of wogonin treatment and CLP in mice and time points at which functional assessments were performed. b Seven-day survival rates of mice after CLP, n = 10. c Anal temperatures (n = 6 per group). d Sepsis score (n = 6 per group). e Representative M-mode echocardiographic images. f, g Echocardiographic measurements of the left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) (n = 6 per group). h Representative TUNEL staining micrographs depicting apoptosis (blue, DAPI staining; red, cTnT staining; green: TUNEL) (scale bar = 100 μm; n = 6 per group). i Pooled TUNEL staining images. j–l Serum troponin I (TnI), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase (LDH) levels (n = 6 per group). The data are presented as the means ± SEMs. P values were determined using one-way ANOVA followed by Tukey’s post hoc test. Kaplan‒Meier survival curves were plotted to examine survival rates, followed by the log-rank test to evaluate the differences. **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns indicates no significant difference.

Wogonin attenuates CLP-induced mechanical abnormalities in cardiomyocytes

To assess the impact of wogonin on cardiomyocyte function, we isolated primary AMCMs and examined their mechanical properties. CLP markedly decreased the PS and ±dL/dt, and prolonged the TR₉₀. However, CLP did not affect the resting cell length or TPS of cardiomyocytes. While wogonin alone had no significant effect on the mechanical properties of cardiomyocytes, it effectively prevented CLP-induced abnormalities in cardiomyocytes (Fig. 2a–g).

Fig. 2. Wogonin attenuates CLP-induced mechanical abnormalities in cardiomyocytes.

a Representative traces of cardiomyocyte shortening. b Resting cell lengths. c Peak shortening. d Maximum velocity of shortening (+dL/dt). e Maximum velocity of relengthening (−dL/dt). f Time-to-peak 90% shortening (TPS) and g time-to-90% relengthening (TR₉₀). The data are presented as the means ± SEMs; n = 40 cells per group. P values were determined using one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Wogonin alleviates inflammation, oxidative stress, and mitochondrial injury in CLP-induced SCM

The initiation of the inflammatory response is a critical pathophysiological feature of SCM [37]. To determine whether wogonin exerts anti-inflammatory effects in SCM, we evaluated the protein concentrations of inflammatory markers in the myocardium of CLP-treated mice. Our results revealed that CLP led to significant increases in the protein levels of IL-6, TNF-α, and IL-1β, which were alleviated by wogonin administration (Fig. 3a–d). Immunohistochemical staining also demonstrated that wogonin attenuated the infiltration of CD45-positive leukocytes and CD68-positive macrophages induced by CLP (Fig. 3e–g). Oxidative stress is another key feature in cardiomyocytes during sepsis [38]. DHE staining was therefore used to evaluate the impact of wogonin on CLP-induced myocardial oxidative stress. Our findings indicated that CLP caused significant oxidative stress in the myocardium; which was notably prevented by wogonin administration (Fig. 3h, i). Additionally, CLP markedly reduced the mitochondrial membrane potential, which was attenuated by wogonin treatment (Fig. 3j, k). Transmission electron microscopy revealed substantial localized injury to the mitochondria, as indicated by decreased mitochondrial size, reduced cristae density, cristae distortion, and localized myofiber disorganization, all of which were prevented by wogonin administration (Fig. 3l, m). Furthermore, CLP increased mitochondrial reactive oxygen species production, as shown by MitoSOX staining, which was alleviated by wogonin (Fig. 3n, o). Taken together, these findings indicate that wogonin mitigates CLP-induced inflammation, oxidative stress, and mitochondrial dysfunction in cardiomyocytes.

Fig. 3. Wogonin alleviates inflammation, oxidative stress, and mitochondrial injury in CLP-induced SCM model mice.

Wogonin was administered at a dose of 40 mg·kg⁻¹. a–d Representative Western blots of IL-6, TNF-α, and IL-1β and quantitative analysis of protein levels in cardiac tissues (n = 6 per group). e–g Representative immunohistochemical staining images of CD45 and CD68 and quantitative analysis of the CD45- and CD68-positive cells (n = 6 per group). h, i Representative DHE staining images and quantitative analysis of intracellular ROS levels in CLP-injured cardiomyocytes (n = 8 per group). j, k Representative images and quantitative analysis of adult mouse cardiomyocytes treated with JC-1 showing aggregates (red), monomers (green), and the merged fluorescence signals to evaluate the mitochondrial membrane potential (scale bar = 100 μm; n = 8 fields per group). l, m Representative transmission electron microscopy images and quantitative analysis of mitochondrial morphology (scale bar = 2 µm or 400 nm; n = 6 fields per group). n, o Representative images and quantitative analysis of adult mouse cardiomyocytes treated with MitoSOX (scale bar = 100 μm; n = 16 fields per group). The data are presented as the means ± SEMs. P values were determined using one-way ANOVA followed by Tukey’s post hoc test. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Wogonin ameliorates CLP-induced cardiac ferroptosis in vivo

Recent evidence indicates that ferroptosis contributes to the pathophysiology of SCM [7, 30, 39, 40]. Consequently, we evaluated the effects of wogonin on cardiac ferroptosis in a sepsis model. CLP led to a substantial reduction in cardiac GPX4 protein levels and increases in the PTGS2 protein and mRNA levels, but these alterations were attenuated by wogonin therapy (Fig. 4a–d). We subsequently analyzed the concentrations of 4-hydroxynonenal (4-HNE), an important biomarker of lipid peroxidation and ferroptosis. The findings revealed a significant increase in 4-HNE levels in the hearts of CLP-treated mice, which was reduced by wogonin treatment (Fig. 4e–i). Similarly, CLP increased the levels of MDA, another lipid peroxidation byproduct, in the heart and plasma, and these effects were mitigated by wogonin treatment (Fig. 4j, k). Moreover, CLP caused a significant reduction in the myocardial GSH/GSSG ratio, which was increased after wogonin therapy (Fig. 4l). These findings demonstrate that wogonin protects against cardiac ferroptosis during sepsis.

Fig. 4. Wogonin ameliorates sepsis-induced myocardial injury in CLP mice by regulating ferroptosis.

a–c Representative immunoblotting images of GPX4 and PTGS2 and quantitative analysis of their protein levels in cardiac tissues (n = 6 per group). d Quantification of the relative Ptgs2 mRNA levels in heart tissues (n = 6 per group). e, f Representative immunohistochemical staining images of 4-HNE and quantitative analysis of the 4-HNE-positive area (scale bars = 100 µm; n = 6 per group). g, h Representative immunoblotting images of 4-HNE and quantitative analysis of 4-HNE levels in mouse hearts (n = 6 per group). i Quantification of 4-HNE adducts in mouse hearts by ELISA (n = 6 per group). j Malondialdehyde (MDA) levels in heart tissues (n = 6 per group). k Malondialdehyde (MDA) levels in plasma (n = 6 per group). l GSH/GSSG ratio in cardiac tissues (n = 6 per group). The data are presented as the means ± SEMs. P values were determined using one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Wogonin inhibits LPS-induced ferroptosis in cardiomyocytes in vitro

To examine the function of wogonin in cardiomyocyte ferroptosis during sepsis, we examined ferroptosis-related indices in LPS-treated HL-1 cardiomyocytes. First, a CCK8 assay was used to assess the cytotoxic effects of wogonin and determine a suitable dose. Wogonin had little effect on HL-1 cell viability at concentrations lower than 60 μM (Fig. 5a). LPS challenge provoked a significant reduction in cell viability, and treatment with 10 and 20 μM wogonin resulted in the most pronounced restoration of cell viability (Fig. 5b). In addition, LPS treatment resulted in a significant number of PI-positive HL-1 cells (an indicator of cell death), which was markedly mitigated by wogonin (Fig. 5c, d). LPS challenge resulted in the downregulation of GPX4 and the upregulation of PTGS2 in HL-1 cardiomyocytes, with these effects being attenuated by wogonin (Fig. 5e, g). Furthermore, C11-BODIPY staining revealed that wogonin markedly reduced LPS-induced lipid peroxidation (Fig. 5h, i). LPS treatment led to significant MDA accumulation and a decrease in the GSH/GSSG ratio in HL-1 cardiomyocytes, which was consistent with the in vivo findings. These effects were mitigated by wogonin (Fig. 5j, k). Like wogonin, the ferroptosis inhibitor Fer-1 also reversed LPS-induced cell death and lipid peroxidation and decreased the GSH/GSSG ratio (Fig. 5b–d, h–k). These findings further indicate that wogonin suppresses cardiomyocyte ferroptosis in SCM.

Fig. 5. Wogonin inhibits LPS-induced cardiomyocyte ferroptosis in vitro.

a, b The effects of wogonin on the HL-1 cell viability with or without LPS challenge (10 µM) or ferrostatin-1 treatment (Fer-1, 5 µM) were determined by CCK8 assays. c, d Representative images of propidium iodide (PI) staining of HL-1 cells treated with LPS, wogonin, or Fer-1 and quantitative analysis (scale bar = 300 μm; n = 6 per group). e–g Representative immunoblotting images of GPX4 and PTGS2 and quantitative analysis of their protein levels in cardiac tissues (n = 6 per group). h, i Lipid ROS production in adult mouse cardiomyocytes treated with LPS, wogonin, or Fer-1 assessed by C11-BODIPY^581/591 fluorescent staining (scale bar = 100 μm; n = 6 per group). j Malondialdehyde (MDA) levels in HL-1 cardiomyocytes treated with LPS, wogonin, or Fer-1 (n = 6 per group). k GSH/GSSG ratios in HL-1 cardiomyocytes treated with LPS, wogonin, or Fer-1 (n = 6 per group). The data are presented as the means ± SEMs. P values were determined using one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns indicates no significant difference.

Wogonin interacts with and inhibits the activity of ALOX15

Next, we explored the possible mechanisms by which wogonin regulates ferroptosis in SCM. We intersected the differentially expressed genes obtained after RNA-seq analysis of SCM tissues (GSE44363 and GSE142615), the possible target genes of wogonin obtained from the Swiss Target Prediction (http://www.swisstargetprediction.ch/) and SEA databases (https://sea.bkslab.org/) [41], and the ferroptosis-related genes obtained from FerrDB (http://www.zhounan.org/ferrdb/current/) [42]. ALOX15, a lipoxygenase that catalyzes lipid peroxidation, was identified as a potential target gene of wogonin (Fig. 6a). Molecular docking analysis suggested a potential interaction between wogonin and ALOX15, with a binding energy of −7.9 kcal·mol⁻¹ (Fig. 6b). Drug affinity responsive target stability (DARTS) assays revealed that wogonin markedly decreased the protease susceptibility of ALOX15 in HL-1 cell lysates (Fig. 6c). Using a cell thermal shift assay (CETSA), we found that wogonin decreased ALOX15 degradation with increasing temperature (Fig. 6d, e) and greatly hindered ALOX15 activity (Fig. 6f). These results suggest that wogonin directly interacts with ALOX15 and inhibits its activity.

Fig. 6. Wogonin attenuates SCM by suppressing ferroptosis via its interaction with ALOX15.

a Venn diagram of the differentially expressed genes identified from the GSE142615 and GSE44363 datasets and wogonin targets obtained from known databases and FerrDb. b Molecular docking of wogonin to ALOX15. c Effect of wogonin on the pronase-induced proteolysis of ALOX15 analyzed by a DARTS assay (n = 3 per group). d, e Binding affinity of wogonin for ALOX15 was determined using CETSAs (n = 3 per group). f Rate of ALOX15 enzymatic activity inhibition by wogonin (n = 3 per group). g GSE44363 contains data regarding the ALOX15 expression levels in mouse hearts treated for 24 h with either saline or lipopolysaccharide. h GSE142615 contains data regarding the ALOX15 expression levels in mouse hearts treated for 6 h with either saline or lipopolysaccharide. i qPCR detection of ALOX15 gene expression in mouse hearts after CLP (n = 3 per group). j qPCR detection of ALOX15 gene expression in HL-1 cells treated with LPS (n = 3 per group). k, l Representative immunoblotting images of the ALOX15 protein in HL-1 cells treated with LPS and quantitative analysis (n = 6 per group). m, n Representative immunoblotting images of the ALOX15 protein in mouse hearts after CLP and quantitative analysis (n = 6 per group). o Relative enzymatic activity of ALOX15 in HL-1 cells (n = 3 per group). p, q Evaluation of several oxylipins associated with ALOX15 catabolism, including 15-HETE and 15-HpETE (n = 6 per group). The data are presented as the means ± SEMs. P values were determined using two-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns indicates no significant difference.

We further examined whether wogonin affects ALOX15 expression in SCM. RNA-seq analysis (GSE44363 and GSE142615) revealed elevated ALOX15 expression in SCM heart tissues (Fig. 6g, h), a finding corroborated by PCR (Fig. 6i, j). Western blot analysis also revealed that LPS challenge or CLP increased ALOX15 protein expression in HL-1 cells (Fig. 6k, l) and mouse cardiac tissues (Fig. 6m, n), which were alleviated by wogonin treatment. Wogonin also lowered ALOX15 activity in a dose-dependent manner (Fig. 6o). AA is normally converted to 15-hydroperoxyeicosatetraenoic acid (15-HpETE) by ALOX15, and 15-hydroxyeicosatetraenoic acid (15-HETE) is produced via additional metabolic processes [43]. We then examined these metabolites in CLP-challenged hearts to determine ALOX15 activity. CLP increased the levels of ALOX15 metabolites, including 15-HpETE and 15-HETE, in heart tissues, but the levels of these metabolites were reduced by wogonin therapy (Fig. 6p, q). These data indicate the possible involvement of ALOX15 in wogonin-regulated ferroptosis.

Pharmacological inhibition of ALOX15 attenuates CLP-induced ferroptosis and SCM

To determine the roles of ALOX15 in cardiac dysfunction and ferroptosis during sepsis, the selective ALOX15 inhibitor ML351 (10 mg·kg⁻¹·d⁻¹) was administered for a 7-day period prior to CLP. Our results revealed that ML351 ameliorated the CLP-induced reductions in LVEF and LVFS (Fig. 7a–c). ML351 also decreased the levels of myocardial injury indicators, such as cTnI, CK-MB, and LDH (Fig. 7d–f). In addition, the levels of ALOX15 metabolites, including 15-HpETE and 15-HETE, were also decreased upon ML351 administration in CLP-treated mice (Fig. 7g, h). Western blot analysis revealed that ML351 markedly restored GPX4 levels and decreased PTGS2 levels in the hearts of mice after CLP (Fig. 7i, k). Moreover, ML351 markedly attenuated lipid peroxidation and alleviated the imbalance in GSH in the SCM model, as indicated by reduced 4-HNE levels (Fig. 7l–n), decreased cardiac MDA levels (Fig. 7o) and a restored GSH/GSSG ratio (Fig. 7p). These results indicate that ALOX15 participates in cardiomyocyte lipid peroxidation and ferroptosis during sepsis and that suppressing ALOX15 activity protects against SCM.

Fig. 7. Pharmacological inhibition of ALOX15 inhibits ferroptosis and attenuates CLP-induced myocardial damage.

a Representative M-mode echocardiographic images. b, c Quantitative analysis of the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) (n = 6 per group). d–f Serum troponin I (TnI), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase (LDH) levels (n = 6 per group). g, h Evaluation of several oxylipins associated with ALOX15 catabolism; including 15-HETE and 15-HpETE (n = 6 per group). i–k Representative immunoblotting images of the PTGS2 and GPX4 proteins in mouse hearts subjected to the indicated treatments and quantitative analysis (n = 6 per group). l, m Representative immunohistochemical staining images of 4-HNE and quantitative analysis of the 4-HNE-positive area (scale bars = 100 µm; n = 6 per group). n Quantification of the 4-HNE adducts in mouse hearts by ELISA (n = 6 per group). o Malondialdehyde (MDA) levels in heart tissues (n = 6 per group). p GSH/GSSG ratios in cardiac tissues (n = 6 per group). The data are presented as the means ± SEMs. P values were determined using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

ALOX15 is involved in the protective effects of wogonin against ferroptosis in cardiomyocytes

ALOX15 was overexpressed in HL-1 cardiomyocytes prior to exposure to wogonin or LPS (Fig. 8a). The overexpression of ALOX15 significantly counteracted the wogonin-induced decrease and increase in PTGS2 and GPX4 levels, respectively, in LPS-treated cardiomyocytes (Fig. 8a–c). Wogonin mitigated LPS-induced ferroptosis, as demonstrated by increased cell viability (Fig. 8d), reduced cell death (Fig. 8e, f), decreased MDA levels (Fig. 8g), an elevated GSH/GSSG ratio (Fig. 8h), and decreased lipid peroxidation in AMCMs (Fig. 8i, j). In LPS-treated cardiomyocytes, the anti-ferroptotic effects of wogonin were mitigated by the overexpression of ALOX15 (Fig. 8a–j). Similar to ALOX15 overexpression, treatment with 15-HpETE (an intermediate metabolite of ALOX15 that triggers ferroptosis in cardiomyocytes) counteracted the anti-ferroptotic effects of wogonin, as evidenced by elevated PTGS2 levels, reduced GPX4 levels, reduced cell viability, an increased percentage of PI-positive stained cells, increased MDA levels, a decreased GSH/GSSG ratio, and increased lipid peroxidation (Fig. 8k–t). Taken together, our findings demonstrate that the anti-ferroptotic effects of wogonin are mediated in an ALOX15/15-HpETE-dependent manner.

Fig. 8. ALOX15 overexpression or 15-HpETE supplementation attenuated the protection against LPS-induced cardiomyocyte ferroptosis offered by wogonin.

a–c Representative immunoblotting images of the ALOX15, PTGS2, and GPX4 proteins in HL-1 cells with or without ALOX15 overexpression and quantitative analysis (n = 6 per group). d Viability of HL-1 cells with or without ALOX15 overexpression (n = 6 per group). e, f Representative images of propidium iodide (PI) staining of HL-1 cells with or without ALOX15 overexpression and quantitative analysis (scale bar = 300 μm; n = 6 per group). g, h MDA levels and GSH/GSSG ratios in HL-1 cells with or without ALOX15 overexpression (n = 6 per group). i, j Lipid ROS production in adult mouse cardiomyocytes with or without ALOX15 overexpression assessed by C11-BODIPY 581/591 fluorescence (scale bar = 100 µm; n = 6 per group). k–m Representative immunoblotting images of the PTGS2 and GPX4 proteins in HL-1 cells with or without 15-HpETE treatment and quantitative analysis (n = 6 per group). n Viability of HL-1 cells with or without 15-HpETE treatment (n = 6 per group). o, p Representative images of HL-1 cells stained with propidium iodide (PI) with or without 15-HpETE treatment and quantitative analysis (scale bar = 300 μm; n = 6 per group). q, r MDA contents and GSH/GSSG ratios in HL-1 cells with or without 15-HpETE treatment (n = 6 per group). s, t Lipid ROS production in adult mouse cardiomyocytes with or without 15-HpETE treatment assessed by C11-BODIPY 581/591 fluorescence (scale bar = 100 μm; n = 6 per group). The data are presented as the means ± SEMs. P values were determined using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Discussion

The findings of our current study revealed that wogonin, a flavonoid derived from S. baicalensis root extracts, regulates cardiomyocyte ferroptosis and protects against SCM. Our results showed that wogonin exhibited cardioprotective and anti-ferroptotic effects both in vivo and in vitro. Moreover, wogonin not only interacted with ALOX15 but also inhibited ALOX15 activity, thereby lowering 15-HpETE production and attenuating lipid peroxidation. Moreover, the wogonin-mediated protection against cardiomyocyte ferroptosis was significantly diminished by ALOX15 overexpression or 15-HpETE supplementation. Collectively, our findings underscore that wogonin inhibits SCM, likely by suppressing ALOX15/15-HpETE-mediated ferroptosis (Fig. 9).

Fig. 9. Proposed model of the cardioprotective effects of wogonin in SCM.

Sepsis upregulates ALOX15, and ALOX15 catalyzes the conversion of PL-PUFAs into PL-PUFAs-OOH, thereby promoting membrane-mediated phospholipid peroxidation and triggering cardiomyocyte ferroptosis, resulting in impaired cardiac function. Wogonin appears to be a particularly promising active compound that can effectively inhibit the activity of ALOX15 and reduce phospholipid peroxidation in cardiomyocytes to prevent ferroptosis in SCM. This figure was created with BioRender.com.

SCM is a devastating acute cardiac syndrome in patients with sepsis. SCM has a high incidence rate and significantly increases the mortality rate in patients with sepsis [44]. The pathogenesis of SCM involves multiple types of cardiomyocyte cell death, including apoptosis [5], pyroptosis [6], and ferroptosis [7, 8]. In addition, mitochondrial injury is a cardinal mechanism in the onset and development of SCM [45–48]. In particular, the oxidation and nitrosation reactions that occur within mitochondria lead to the excessive production of ROS. Oxidative stress subsequently triggers cardiomyocyte death and disrupts myocardial homeostasis, resulting in functional and structural damage to the heart [49]. The roots of S. baicalensis Georgi contain many forms of bioactive compounds, including wogonin, baicalein, and baicalin [50]. Notably, the protective effects of baicalein and baicalin on SCM and other types of septic organ damage have been widely examined [51–54]. However, it remains unknown whether wogonin has any benefits in the context of SCM. Importantly, wogonin has been proven to possess important pharmacological properties, including anti-inflammatory, anti-apoptotic, and antioxidant effects [18, 21, 55, 56]. Additionally, wogonin reduces the release of cytochrome c by altering mitochondrial permeability, exhibits anti-apoptotic effects, and alleviates doxorubicin-induced cardiotoxicity [21]. Therefore, we suspected that wogonin may also affect SCM. Consistent with previous results [19, 21, 57], our findings demonstrated that wogonin effectively protects cardiac function and reduces inflammatory responses, oxidative stress, and mitochondrial damage in the context of SCM. In addition, wogonin improved the contractile function of cardiomyocytes during sepsis. The levels of several oxidative stress markers (such as 4-HNE and MDA) were also significantly reduced following wogonin treatment. Taken together, these data suggest that wogonin alleviates SCM by regulating mechanisms related to oxidative stress.

Considering the close relationship between oxidative stress and ferroptosis in SCM [7], we speculated that ferroptosis may be involved in the wogonin-mediated beneficial responses. Ferroptosis is a regulated type of oxidative cell death characterized by the iron-dependent depletion of GSH and subsequent lipid peroxidation [58]. Prior studies have demonstrated the role of ferroptosis in several types of cardiovascular diseases, including SCM [59–62]. In 2020, Li and colleagues were the first to reveal the participation of ferritinophagy-mediated ferroptosis in sepsis-induced cardiac injury [7]. Another independent study from our group demonstrated that the cardiac-specific overexpression of catalase mitigated LPS-induced cardiomyopathy by modulating autophagy and ferroptosis [40]. To this end, pharmacological inhibition of ferroptosis is anticipated to be an innovative approach for addressing conditions related to sepsis-related organ damage, including SCM [63]. In recent years, several natural compounds have been shown to have excellent anti-ferroptosis properties and have contributed to the treatment of cardiovascular diseases [32, 64]. Our findings indicated that sepsis provoked substantial changes in ferroptosis-related indicators, including the levels of 4-HNE, MDA, GSH/GSSG, GPX4, and PTGS2. Notably, wogonin therapy mitigated these alterations both in vivo and in vitro. Therefore, wogonin can be used to effectively treat SCM by suppressing cardiomyocyte ferroptosis.

Our subsequent investigations aimed to elucidate the mechanism by which wogonin attenuates ferroptosis in sepsis. Lipoxygenase (LOX) is involved in lipid peroxidation and directly initiates ferroptosis [65]. Mammalian LOX primarily facilitates the dual oxidation of intracellular free fatty acids [66], whereas the eicosanoic acid formed from AA by way of LOX serves as an important lipid signaling mediator [67]. In the AA metabolic cascade, AA is first enzymatically transformed into hydroperoxyeicosatetraenoic acid (HpETE) by lipoxygenases (LOXs), which is followed by other metabolic processes that generate hydroxyeicosatetraenoic acid (HETE), leukotrienes, and lipids [67]. Early data have demonstrated that inflammation and oxidative stress caused by cardiac ALOX15 are linked to the progression of diabetic cardiomyopathy [68]. 15-HpETE induces ferroptosis in cardiomyocytes and exacerbates cardiac ischemia-reperfusion injury [15]. The ALOX15-specific inhibitor ML351 may inhibit ferroptosis in cardiomyocytes, safeguard the injured myocardium; and enhance cardiac functional recovery [15]. These findings suggest that modulating cardiac ALOX15 levels may be a feasible therapeutic strategy for both the prevention and treatment of SCM. The results of our target prediction analysis indicated that ALOX15 is a possible target of wogonin because of its anti-ferroptotic effects. DARTS assays indicated that wogonin prevented ALOX15 proteolysis by pronase, suggesting a direct interaction between wogonin and ALOX15, which was confirmed by CETSAs. In addition, our findings revealed increased levels of ALOX15 in cardiac tissues and model-cultured cardiomyocytes of sepsis-induced stress. Interestingly, wogonin also decreased the protein levels of ALOX15, suggesting that other pharmacological effects may exist beyond the direct binding of wogonin to ALOX15. Wogonin may alter ALOX15 expression through transcriptional or posttranslational regulation mechanisms. For example, a recent study showed that ALOX15 transcription was suppressed by ERK1/2 activation in hepatic ischemia-reperfusion injury [69]. Intriguingly, wogonin has been shown to activate ERK signaling in several cell types and exert protective effects on multiple human diseases, such as breast cancer [70], hepatocellular carcinoma [71], and osteoarthritis [18]. Therefore, wogonin likely suppresses ALOX15-mediated cardiomyocyte ferroptosis by activating ERK signaling in SCM. Further studies are warranted to decipher the upstream regulators of ALOX15 and the possible nonphysical mechanisms by which wogonin regulates ALOX15 expression. Our findings indicate that the therapeutic effects of wogonin in SCM may be partially attributed to the anti-ferroptotic effects of wogonin mediated by inhibition of the ALOX15/15-HpETE axis.

Our research further substantiates the clinical translational potential of targeting ALOX15 for the treatment of SCM. ML351, a selective ALOX15 inhibitor, has been used in many preclinical models of metabolic disorders [15, 72, 73]. In a preclinical study on diabetes, ML351 suppressed beta-cell oxidative stress, beta-cell malfunction, and hyperglycemia in mice [73]. Moreover, in a cardiac ischemia-reperfusion injury model, ML351 was shown to increase Pgc1α protein levels, resulting in reduced cardiomyocyte apoptosis and myocardial injury and improved cardiac functional recovery [15]. In addition to ML351, various ALOX15 inhibitors, including baicalin, PD146176, N-benzyl-N-hydroxy-5-benzamidine (BHPP), nordihydroguaiaretic acid (NDGA), and ML355, have been identified and shown to have therapeutic efficacy in the treatment of diseases [74]. However, the clinical use of ALOX15 inhibitors has several limitations, including isomer specificity, homologous specificity, and off-target effects [74]. Compared with chemically synthesized compounds (e.g., ML351), wogonin is derived from the roots of S. baicalensis Georgi, which increases its therapeutic safety. Moreover, wogonin exerts multiple protective effects by inhibiting inflammation and oxidative stress [50]. Our study also revealed that ML351 markedly decreased CLP-induced ferroptosis in cardiomyocytes, improved cardiac function, reduced the lipid peroxidation associated with ALOX15 metabolites, and mitigated myocardial injury. Conversely, ALOX15 overexpression or 15-HpETE supplementation diminished the beneficial effects of wogonin on LPS-induced ferroptosis in cardiomyocytes. Taken together, our findings indicate that the anti-ferroptotic effects of wogonin are dependent on ALOX15.

Although wogonin has been indicated to have the potential to alleviate SCM [22], our current study still has several limitations. First, we did not examine the precise molecular basis by which wogonin regulates ALOX15 expression or its subcellular localization. Recent evidence has suggested that other types of regulated cell death (e.g., pyroptosis) may also be involved in the onset and development of SCM [45, 46, 75]. Early findings have indicated that wogonin may also suppress apoptosis during the onset and progression of various pathological conditions [21, 55]. Our study did not explore the potential involvement of other forms of cell death (e.g., apoptosis, pyroptosis, and necroptosis) in wogonin-mediated cardioprotection in the context of sepsis. Finally, our study did not compare the efficacy and safety of wogonin with those of classical ALOX15 inhibitors in the SCM model. More research is needed to explore the advantages and disadvantages of wogonin treatment in comparison with other ALOX15 inhibitors in the context of SCM.

In summary, our results demonstrate that wogonin protects against SCM by inhibiting ALOX15-mediated lipid peroxidation and ferroptosis. Consequently, wogonin represents a potential therapeutic agent for the clinical management of SCM.

Author contributions

HY: writing—original draft, review and editing; conceptualization, methodology, and investigation. LW: methodology, investigation, writing—review and editing. YML: methodology, investigation, writing—review and editing. JXZ: writing—review and editing. HTH: writing—review and editing. MLD: writing—review and editing, supervision, methodology, investigation. JR: writing—review and editing, supervision, funding acquisition, conceptualization.

Funding

This work was supported in part by the Ganzhou City Science and Technology Plan Project (No. 2023LNS17411), the National Natural Science Foundation of China (No. 81571895), and the Natural Science Foundation of Guangdong Province, China (No. 2023A1515010261).

Data availability

Data will be made available on request.

Competing interests

The authors declare no competing interests.