Micheliolide reduces the damage, pyroptosis and oxidative stress of cardiomyocytes caused by OGD/R by regulating the AMPK signaling pathway

Abstract

Myocardial ischemia-reperfusion (I/R) injury remains a devastating clinical problem, contributing substantially to morbidity and mortality worldwide. In the present study, we investigated the potential role of Micheliolide (MCL) in attenuating cardiac I/R injury. H9c2 cardiomyocytes were pretreated with MCL for 24 ‍h and then subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Cellular injury was evaluated by measuring cell viability and lactate dehydrogenase (LDH) release, while cell death was assessed using propidium iodide (PI) staining. Oxidative stress was determined by assessing superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and glutathione peroxidase (GSH-Px) activity, while the expression levels of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), and pyroptosis-related proteins were examined by Western blotting. The results demonstrated that MCL significantly alleviated OGD/R-induced damage in H9c2 cells. Moreover, MCL inhibited OGD/R-induced oxidative stress and pyroptosis while enhancing AMPK pathway activation. Importantly, the protective effect of MCL was attenuated in the presence of the AMPK inhibitor Compound C, indicating that activation of the AMPK signaling pathway is required for MCL-mediated cytoprotection.

Introduction

Acute myocardial infarction (AMI) is one of the most common clinical manifestations of coronary heart disease and represents a major cause of mortality worldwide. It causes extensive death of myocardial cells as a consequence of ischemic injury, followed by the formation of fibrotic scars that impair myocardial electrical conductivity and reduce coronary blood flow in the regions surrounding the infarcted myocardium.^(1,2) The process of restoring blood flow, although essential for tissue survival, often exacerbates myocardial damage through a process known as ischemia-reperfusion (I/R) injury.⁽³⁾ Although the precise mechanisms underlying I/R injury remain incompletely understood, current evidence indicates that oxidative stress, inflammatory responses, and disturbances in energy metabolism play central roles in cardiomyocyte death.⁽⁴⁾ Therefore, elucidating the molecular basis of myocardial I/R injury and developing effective preventive and therapeutic strategies remain essential in cardiovascular research.

Phytochemicals have attracted increasing attention due to their potential cardioprotective properties. Micheliolide (MCL), a derivative extracted from parthenolide, is a compound with anti-inflammatory and antioxidant properties that exerts diverse biological functions in inflammatory disorders and malignancies.⁽⁵⁾ Previous studies have highlighted its therapeutic potential in cardiovascular diseases from multiple perspectives. For example, MCL has been shown to reduce atherosclerosis by inhibiting macrophage ferroptosis through the KEAP1/NRF2 pathway,⁽⁶⁾ while another study reported that MCL protects mice from doxorubicin-induced cardiotoxicity by regulating the PI3K/Akt/NF-κB signaling pathway.⁽⁷⁾ These findings suggest that MCL holds significant promise in preventing and treating cardiac injury, although a deeper understanding of its mechanisms of action is essential to facilitate its translation into clinical applications.

AMP-activated protein kinase (AMPK) is a key regulator of cellular physiology, with roles in energy homeostasis, apoptosis, and cell metabolism.⁽⁸⁾ Its activation has been shown to enhance cellular antioxidant defense systems, resulting in reduced oxidative damage during I/R injury Its activation has been shown to enhance cellular antioxidant defense systems, resulting in reduced oxidative damage during I/R injury.⁽⁹⁾ Notably, MCL has also been reported to regulate the AMPK signaling pathway.⁽⁹⁾ Notably, MCL has also been reported to regulate the AMPK signaling pathway.⁽¹⁰⁾

In our previous work, we demonstrated that MCL exerts a cardioprotective effect in a rat model of I/R injury. Building on these findings, the present study aimed to investigate the molecular mechanisms underlying oxygen-glucose deprivation/reoxygenation (OGD/R)-induced damage in H9c2 cardiomyocytes, focusing on evaluating the potential protective role of MCL.

Materials and Methods

Cell culture

The H9c2 rat cardiomyocyte cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 2 ‍mM glutamine, 100 ‍U/ml penicillin, and 100 ‍μg/ml streptomycin (all from Thermo Fisher Scientific, Inc., Waltham, MA). Cells were maintained at 37°C in a humidified incubator under an atmosphere of 5% CO₂.

OGD/R cell model and treatment

When H9c2 cells reached 70–80% confluence, the culture medium was replaced with glucose-free DMEM, and the cells were incubated for 4 ‍h at 37°C in an anaerobic chamber (HYQX-III-TZ; Yuejin Medical Equipment Co., Ltd., Shanghai, China) containing 95% N₂ and 5% CO₂ to induce oxygen-glucose deprivation (OGD). Following this period, reperfusion was simulated by replacing the medium with normal DMEM and incubating the cells under normoxic conditions (95% air and 5% CO₂) for 24 ‍h at 37°C. At the onset of reperfusion, untreated cells served as the control group, while MCL (Beyotime, Shanghai, China) was added at concentrations of 1, 5, or 10 ‍μM, and Compound C (Beyotime) was added at 10 ‍μM.

To determine the predominant mode of regulated cell death involved in OGD/R-induced H9c2 injury, specific inhibitors targeting apoptosis, pyroptosis, necroptosis, and ferroptosis were employed. Briefly, the cells were divided into the following groups: (1) control group; (2) OGD/R group; (3) OGD/R + Z-VAD-FMK (apoptosis inhibitor, 20 ‍μM; Selleck Chemicals, Houston, TX); (4) OGD/R + Ac-YVAD-CMK (pyroptosis inhibitor, 20 ‍μM; MedChemExpress, Monmouth Junction, NJ); (5) OGD/R + Necrostatin-1 (necroptosis inhibitor, 10 ‍μM; MedChemExpress); and (6) OGD/R + Ferrostatin-1 (ferroptosis inhibitor, 1 ‍μM; Selleck Chemicals). All inhibitors were added to the culture medium at the beginning of the reoxygenation phase. After 24 ‍h of reoxygenation, cell viability was measured using the CCK-8 assay, and cell damage was assessed by lactate dehydrogenase (LDH) release using a commercial LDH detection kit.

Cell viability assay

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Beyotime) following the manufacturer’s instructions. Briefly, H9c2 cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well. After the designated treatments, 10 ‍μl of CCK-8 solution was added to each well, and the plates were incubated for 2 ‍h at 37°C. Absorbance was then measured at 450 ‍nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA).

Measurement of LDH activity

The release of LDH into the culture supernatant, an indicator of cellular injury, was quantified using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). Briefly, culture supernatants were collected and centrifuged at room temperature for 2 ‍min to remove debris. Subsequently, 100 ‍μl of working solution was added to each well and allowed to react for 30 ‍min at room temperature. The reaction was terminated by adding 50 ‍μl of stop solution, and absorbance was recorded at 450 ‍nm using a microplate reader (Bio-Rad Laboratories, Inc.).

Enzyme-Linked Immunosorbent Assay (ELISA)

Interleukin-1β (IL-1β) levels in culture supernatants were measured using a commercial ELISA kit following the manufacturer’s instructions. Absorbance was determined at 450 ‍nm with a microplate reader (Bio-Rad Laboratories, Inc.).

Hoechst 33342/Propidium Iodide (PI) staining

Cell death was evaluated using Hoechst 33342 and PI staining. After treatment, cells were washed with phosphate-buffered saline (PBS), mixed with 1× detection buffer, and stained with 2 ‍μM Hoechst 33342 and 4.5 ‍μM PI per well for 30 ‍min at 37°C. Fluorescence images were then captured and analyzed using a fluorescence microscope (IX51; Olympus, Hachioji, Japan).

Determination of reactive oxygen species (ROS) production

Intracellular ROS generation was assessed using 2',7'-dichlorofluorescein diacetate (DCFH-DA; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. Briefly, the cells were incubated with 10 ‍μM DCFH-DA for 30 ‍min at 37°C. The fluorescence intensity was then measured using a Carl Zeiss AG fluorescence microplate reader at excitation and emission wavelengths of 485 and 525 ‍nm, respectively.

Measurement of malondialdehyde (MDA) content and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity

The levels of MDA, SOD, and GSH-Px activity were determined using commercial assay kits (Nanjing Jiancheng Bioengineering Institute) following the manufacturer’s instructions. Briefly, H9c2 cells were lysed using RIPA lysis buffer (Beyotime), and the lysates were subjected to analysis using the respective assay kits. The results were quantified using a microplate reader (Bio-Rad Laboratories, Inc.).

Measurement of mitochondrial membrane potential

Mitochondrial membrane potential was assessed using the JC-1 Mitochondrial Membrane Potential Assay Kit (Beyotime) following the manufacturer’s instructions. Briefly, H9c2 cells were seeded into 6-well plates and subjected to the indicated treatments. After treatment, the cells were incubated with JC-1 staining solution (5 ‍μg/ml) for 20 ‍min at 37°C in the dark, followed by two washes with JC-1 staining buffer. Fluorescence was observed using a fluorescence microscope (Olympus IX51) with excitation/emission wavelengths of 485/530 ‍nm for green fluorescence (monomer form, representing depolarized mitochondria) and 535/590 ‍nm for red fluorescence (aggregate form, representing mitochondria with intact membrane potential). The ratio of red to green fluorescence intensity was calculated to estimate mitochondrial membrane potential.

Western blot analysis

Cells were lysed using RIPA buffer (Beyotime), and protein concentrations were determined with a BCA protein assay kit (Beyotime). Equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk at room temperature for 1 ‍h, the membranes were incubated overnight at 4°C with the respective primary antibodies. This was followed by incubation for 1 ‍h at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:2,000; Cell Signaling Technology, Danvers, MA). Immunoreactive bands were visualized using an enhanced chemiluminescence detection reagent (EMD Millipore, Billerica, MA). Protein band intensities were quantified using ImageJ software, and results were normalized to GAPDH as an internal control. The primary antibodies used were as follows: NLRP3 (1:1,000, ab263899; Abcam, Cambridge, UK), Caspase-1 (1:1,000, ab179515; Abcam), N-GSDMD (1:1,000, ab219800; Abcam), IL-1β (1:1,000, ab283818; Abcam), AMPK (1:1,000, ab32047; Abcam), p-AMPK (1:1,000, ab133448; Abcam), ACC (1:1,000, ab45174; Abcam), p-ACC (1:1,000, ab68191; Abcam), and GAPDH (1:1,000, ab9485; Abcam).

Statistical analysis

All experiments were independently repeated at least three times, and data are expressed as the mean ± SD. Statistical analyses were performed using SPSS ver. 16.0 software (SPSS, Inc., Chicago, IL). Comparisons among groups were conducted using one-way analysis of variance (ANOVA), followed by post hoc analysis with the least significant difference (LSD) test. P<0.05 was considered to indicate statistical significance.

Results

MCL alleviates the decrease in cell viability caused by OGD/R

We first evaluated the potential effect of MCL on the survival of H9c2 cells subjected to OGD/R. The chemical structure of MCL is shown in Fig. 1A. As demonstrated by the CCK-8 assay, exposure to OGD/R markedly decreased H9c2 cell viability compared with the control group, whereas MCL treatment attenuated this reduction in a dose-dependent manner (Fig. 1B). To further assess cell injury, LDH activity, a marker of cardiomyocyte damage, was measured. OGD/R significantly increased LDH activity in H9c2 cells, while MCL treatment substantially reduced this increase (Fig. 1C). Collectively, these results indicate that MCL protects H9c2 cells from OGD/R-induced injury by improving cell viability and reducing cytotoxic damage.

Fig. 1.

MCL alleviates the decrease in cell viability caused by OGD/R. (A) Chemical structure of MCL. (B) Cell viability detected by CCK-8 assay. (C) LDH release levels detected using a commercial kit. Data are presented as mean ± SD. ***p<0.001 vs control group; ^&&p<0.01, ^&&&p<0.001 vs OGD/R group. n = 3.

MCL alleviates OGD/R-induced pyroptosis

To determine the predominant mode of regulated cell death in OGD/R-injured H9c2 cells, specific inhibitors were employed, including Z-VAD-FMK (apoptosis inhibitor), Ac-YVAD-CMK (pyroptosis inhibitor), Necrostatin-1 (necroptosis inhibitor), and Ferrostatin-1 (ferroptosis inhibitor). Among these, Ac-YVAD-CMK exerted the most pronounced protective effect, restoring cell viability to nearly 90% and markedly reducing LDH release. These findings suggest that pyroptosis is the principal form of regulated cell death in OGD/R-induced H9c2 injury (Fig. 2A).

Fig. 2.

MCL alleviates OGD/R-induced pyroptosis. (A) Cell viability assessed by CCK-8 assay and LDH release levels measured by kit. (B) Western blot analysis of NLRP3, Caspase-1, N-GSDMD, and IL-1β protein expression. (C) PI staining for cell death detection. (D) ELISA measurement of IL-1β secretion. Data are presented as mean ± SD. ***p<0.001 vs control group; ^&p<0.05, ^&&&p<0.001 vs OGD/R group. n = 3.

To further verify whether MCL modulates OGD/R-induced pyroptosis, we examined the expression of pyroptosis-related proteins. As shown in Fig. 2B, OGD/R markedly upregulated the expression of NLRP3, Caspase-1, N-GSDMD, and IL-1β compared with the control group. Pretreatment with MCL significantly reduced the expression levels of these pyroptosis markers. Consistently, MCL treatment decreased OGD/R-induced H9c2 cell death and inhibited IL-1β secretion (Fig. 2C and D). Together, these results demonstrate that MCL exerts a protective effect against OGD/R-induced injury in H9c2 cells by suppressing pyroptosis.

MCL alleviates OGD/R-induced oxidative stress

Oxidative stress is a key consequence of OGD/R. As shown in Fig. 3A and B, OGD/R exposure markedly increased ROS generation in H9c2 cells compared with the control group. Pretreatment with MCL significantly reduced ROS accumulation, thereby attenuating OGD/R-induced oxidative stress. In addition, MCL enhanced the activities of endogenous antioxidant enzymes, including SOD and GSH-Px, while simultaneously decreasing the level of the lipid peroxidation marker MDA in OGD/R-injured H9c2 cells (Fig. 3A and B). These findings suggest that MCL has an antioxidant impact on H9c2 cells that have been injured by OGD/R.

Fig. 3.

MCL alleviates OGD/R-induced oxidative stress. (A) MDA content and activities of SOD and GSH-Px detected by commercial kits. (B) ROS production measured using DCFH-DA probe. (C) Mitochondrial membrane potential assessed using JC-1 staining. Data are presented as mean ± SD. ***p<0.001 vs control group; ^&p<0.05, ^&&p<0.05, ^&&&p<0.001 vs OGD/R group. n = 3.

Considering that mitochondrial dysfunction is a hallmark of oxidative stress-induced cellular injury, we next examined whether the protective effect of MCL was associated with the preservation of mitochondrial membrane potential. Using JC-1 staining, we observed that OGD/R markedly decreased the red/green fluorescence ratio, reflecting mitochondrial depolarization. Pretreatment with MCL significantly attenuated this decline in a dose-dependent manner, indicating that MCL preserves mitochondrial membrane integrity and protects against OGD/R-induced mitochondrial dysfunction (Fig. 3C).

MCL promotes the expression of AMPK pathway proteins

Western blotting experiments revealed that OGD/R significantly reduced the phosphorylation levels of AMPK and ACC, as indicated by decreased p-AMPK/AMPK and p-ACC/ACC ratios. In contrast, MCL treatment restored these phosphorylation levels, leading to significant increases in both p-AMPK/AMPK and p-ACC/ACC compared with the OGD/R group (Fig. 4).

Fig. 4.

MCL promotes the expression of AMPK pathway proteins. Western blot analysis of AMPK, p-AMPK, ACC, and p-ACC protein expression. Data are presented as mean ± SD. ***p<0.001 vs control group; ^&p<0.05, ^&&p<0.05, ^&&&p<0.001 vs OGD/R group. n = 3.

MCL targets AMPK to attenuate OGD/R-induced damage

To further confirm whether the protective effects of MCL are mediated through the AMPK signaling pathway, we inhibited AMPK activity using the specific inhibitor Compound C. As shown in Fig. 5A, Compound C effectively suppressed AMPK activation. Importantly, the presence of Compound C reversed the protective effects of MCL on OGD/R-induced injury, as evidenced by reduced improvements in cell viability and increased pyroptosis and oxidative stress markers (Fig. 5B–G). These findings demonstrate that MCL exerts its protective effects against OGD/R-induced H9c2 cell injury primarily through activation of the AMPK signaling pathway, thereby inhibiting pyroptosis and oxidative stress.

Fig. 5.

MCL targets AMPK to improve OGD/R-induced cell damage. (A) Western blot analysis of AMPK, p-AMPK, ACC, and p-ACC protein expression. (B) Cell viability assessed using CCK-8 assay. (C) LDH release levels measured. (D) Western blot analysis of NLRP3, Caspase-1, N-GSDMD, and IL-1β protein expression. (E) PI staining for cell death detection. (F) MDA content and activities of SOD and GSH-Px were detected. (G) ROS production measured using DCFH-DA probe. Data are presented as mean ± SD. ***p<0.001 vs control group; ^&&p<0.01, ^&&&p<0.001 vs OGD/R group; ^##p<0.01, ^###p<0.001 vs OGD/R + 10μM MCL group. n = 3.

Discussion

There are currently few effective treatments for post-acute myocardial infarction heart failure, and their benefits remain limited. Therefore, preventing cardiomyocyte death is an essential strategy because it reduces myocardial injury and helps preserve long-term cardiac function.⁽¹¹⁾ MCL has been shown to play a role in cardiovascular diseases owing to its diverse biological activities^(6,7); however, its specific mechanism of action in reperfusion injury after myocardial infarction has not been fully clarified. In the present study, we treated OGD/R cell models with MCL and found that MCL therapy significantly improved cardiomyocyte viability while reducing oxidative stress and pyroptosis. Notably, we also observed that MCL activated the AMPK signaling pathway, and this activation contributed to the protective effect of MCL against OGD/R-induced cardiomyocyte injury.

Myocardial cell death or damage is the primary pathological event underlying myocardial I/R injury. Since cardiomyocytes constitutively express LDH, which is released upon cell injury or death, the level of LDH activity in the culture medium provides an indicator of OGD/R-induced H9c2 cell damage.⁽¹²⁾ In our study, OGD/R exposure significantly reduced cell viability and increased LDH release, whereas treatment with MCL effectively protected against OGD/R-induced injury.

Oxidative stress is also a hallmark of cardiomyocyte injury during I/R. Previous studies have shown that oxidative stress is elevated during I/R and contributes to cardiomyocyte death, while reducing oxidative stress can attenuate I/R-induced cardiac injury.⁽¹²⁾ Among the biomarkers of oxidative stress, MDA reflects lipid peroxidation, whereas antioxidant enzymes such as SOD and GSH-Px serve as compensatory mechanisms against oxidative damage.⁽¹³⁾ It has been demonstrated that MCL alleviates sepsis-induced acute lung injury by inhibiting oxidative stress.⁽¹⁴⁾ Consistent with these findings, our OGD/R model revealed that MCL reduced oxidative stress, as evidenced by decreased ROS generation and MDA content, together with enhanced activity of SOD and GSH-Px.

Inflammasome activation leads to pyroptosis, a form of programmed cell death characterized by inflammatory responses.⁽¹⁵⁾ Pyroptosis has been shown to play an important role in the progression of AMI, and its inhibition can markedly improve myocardial injury Pyroptosis has been shown to play an important role in the progression of AMI, and its inhibition can markedly improve myocardial injury.⁽¹⁶⁾ Among the inflammasomes, the NLRP3 inflammasome is considered a key activator of pyroptosis. Once assembled, the mature NLRP3 inflammasome cleaves caspase-1, which in turn cleaves proteins of the GSDM family, thereby triggering pyroptotic cell death. Several studies have reported that MCL exerts protective effects in various diseases through its ability to inhibit pyroptosis.⁽¹⁶⁾ Among the inflammasomes, the NLRP3 inflammasome is considered a key activator of pyroptosis. Once assembled, the mature NLRP3 inflammasome cleaves caspase-1, which in turn cleaves proteins of the GSDM family, thereby triggering pyroptotic cell death.⁽¹⁷⁾ Several studies have reported that MCL exerts protective effects in various diseases through its ability to inhibit pyroptosis.^(18,19) In line with this, our findings demonstrated that OGD/R injury activated the NLRP3 inflammasome in H9c2 cells, and this was accompanied by an increase in the N-terminal fragment of GSDMD, confirming the occurrence of pyroptosis. Importantly, MCL treatment during OGD/R injury suppressed pyroptosis, thereby mitigating cell damage.

An interesting finding in our study was that although the caspase-1 inhibitor Ac-YVAD-CMK had the strongest protective effect against OGD/R-induced cell death, inhibitors of ferroptosis and necroptosis also had significant protective effects. This observation suggests that under OGD/R stress, multiple regulated cell death pathways, including pyroptosis, ferroptosis, and necroptosis, may be activated simultaneously or exhibit crosstalk, which is consistent with previous reports revealing interactions between these pathways.^(20,21) Therefore, pyroptosis may not be the sole process responsible for the observed phenotypes. MCL effectively inhibits pyroptosis, and its protective effects may also indirectly affect other cell death pathways. Nevertheless, our data clearly demonstrate that inhibition of NLRP3/Caspase-1/GSDMD-mediated pyroptosis is an important mechanism of MCL’s cytoprotective effects.

Numerous essential physiological processes, including energy consumption, cell division, survival, growth, apoptosis, and autophagy, are mediated by AMPK.⁽²²⁾ Previous studies have shown that AMPK activation enhances cell survival under stress conditions, including OGD/R-induced injury. Previous studies have shown that AMPK activation enhances cell survival under stress conditions, including OGD/R-induced injury.⁽²³⁾ For example, activation of AMPK by GSK621 was reported to alleviate cardiomyocyte injury caused by OGD/R,⁽²⁴⁾ while gypenoside A protected against I/R injury through suppression of miR-143-3p via AMPK pathway activation.⁽²⁵⁾ In addition to these cytoprotective effects, AMPK activation has been closely associated with antioxidant defense, as forced AMPK activation can reduce oxidative stress and limit cellular damage. In addition to these cytoprotective effects, AMPK activation has been closely associated with antioxidant defense, as forced AMPK activation can reduce oxidative stress and limit cellular damage.⁽²⁶⁾ Furthermore, active AMPK has been linked to NLRP3 regulation and mitochondrial homeostasis, as it not only controls energy utilization but also suppresses pyroptosis. Specifically, AMPK has been shown to prevent GSDME-mediated pyroptosis by phosphorylating GSDME at threonine 6.^(27,28) Consistent with these findings, our results demonstrate that MCL exerts its protective effects in OGD/R-injured cardiomyocytes by activating AMPK signaling, thereby inhibiting pyroptosis and oxidative stress and preserving cardiomyocyte viability.

Our study demonstrated that MCL alleviates OGD/R-induced oxidative stress and pyroptosis primarily through activation of the AMPK signaling pathway. Nonetheless, it should be acknowledged that additional mechanisms may also contribute to the protective effects of MCL. Previous studies have reported that MCL can activate the NRF2 pathway,⁽⁶⁾ a key regulator of the antioxidant response. Furthermore, MCL has been shown to stimulate autophagy⁽¹⁹⁾ and reduce mitochondrial ROS production,⁽²⁹⁾ both of which may play complementary roles in attenuating OGD/R-induced cellular injury. Although these mechanisms were not directly examined in the present study, they represent important directions for future investigation. Therefore, elucidating the relative contributions of these pathways could be essential for advancing the therapeutic potential of MCL in the treatment of myocardial I/R injury.

While this study provides evidence supporting a mechanism by which MCL protects against OGD/R-induced cardiomyocyte injury through AMPK activation, several limitations should be considered. First, all experiments were conducted in vitro using H9c2 cells, and the protective effects of MCL have not yet been validated in vivo. Thus, further studies employing animal models are required to evaluate both the efficacy and systemic effects of MCL under physiological conditions. Second, the role of AMPK was assessed using the pharmacological inhibitor Compound C; however, this compound has been reported to exert off-target effects, which may confound interpretation of the results. To more definitively establish the involvement of AMPK in the protective mechanism of MCL, future work should incorporate genetic approaches such as AMPK knockdown or knockout models.

In conclusion, the findings of this study demonstrate that MCL protects H9c2 cells from OGD/R-induced injury by preventing oxidative stress and pyroptosis, and mechanistic analysis indicates that this protective effect is mediated through activation of the AMPK signaling pathway (Fig. 6).

Fig. 6.

The graphical abstract of this paper. OGD/R stimulates cardiomyocyte pyroptosis and oxidative damage, which is manifested by increased expression of NLRP3, N-GSDMD, Caspase-1, and IL-1β; increased levels of SOD, MDA, and ROS, and decreased levels of GSH-Px, leading to cell damage. Micheliolide can alleviate OGD/R-induced pyroptosis and oxidative stress through the AMPK signaling pathway.

Author Contributions

JY designed the study, completed the experiment and supervised the data collection, QJ analyzed the data, interpreted the data, ZL prepare the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript.

Availability of Data and Materials

All data generated or analyzed during this study are included in this published article. The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Conflict of Interest

No potential conflicts of interest were disclosed.