Eupatilin Alleviates Acute Myocardial Ischemia‐Reperfusion Injury in Rats via the Akt/GSK‐3β Pathway by Targeting COXs

ABSTRACT

Eupatilin confers cardioprotection against myocardial ischemia‐reperfusion injury (MIRI). Cyclooxygenases (COXs) serve as key rate‐limiting enzymes for prostaglandin biosynthesis. This study investigated the effects of eupatilin on COXs.

H9c2 cells subjected to oxygen‐glucose deprivation/reoxygenation (OGD/R) were treated with graded doses of eupatilin, with aspirin serving as the positive control. Cellular reactive oxygen species (ROS), cell viability, and COX1/COX2 protein expression were subsequently detected. In‐vivo acute MIRI was established in rats by 30‐min ligation of the left anterior descending coronary artery followed by reperfusion. MIRI‐modeled rats received eupatilin or aspirin treatment. Cardiac function was evaluated by echocardiography. Levels of prostaglandin E2 (PGE2), thromboxane A2 (TXA2), and cardiac troponin I (cTnI) were quantified by ELISA. Myocardial infarct size was assessed by TTC staining, and collagen deposition was determined by Masson staining. Immunohistochemistry was performed to detect myocardial COX1 and COX2 expression.

In OGD/R‐injured H9c2 cells, eupatilin dose‐dependently restored cell viability and suppressed cellular ROS overproduction. It suppressed OGD/R‐induced upregulation of COX1 and COX2 and increased phosphorylation of Akt1 (S473) and GSK‐3β (S9). Molecular docking revealed favorable binding affinity between eupatilin and COXs. In MIRI‐model rats, deteriorated cardiac function was accompanied by increased PGE2, TXA2 and cTnI, as well as aggravated myocardial infarction and collagen deposition. Eupatilin administration alleviated myocardial injury by counteracting these pathological alterations. Moreover, eupatilin suppressed MIRI‐driven COX1/COX2 overexpression and enhanced Akt1‐GSK‐3β phosphorylation.

Eupatilin alleviate acute MIRI via COXs inhibition and activation of the Akt/GSK‐3β pathway, providing a theoretical basis for its application against MIRI.

It has been reported that eupatilin exerts cardioprotective effects after myocardial ischemia‐reperfusion injury (MIRI). This study further found that Eupatilin alleviated acute MIRI in rats by specifically inhibiting cyclooxygenase enzymes (COXs) and activating the Akt/GSK‐3β pathway, providing a theoretical basis for the application of eupatilin in MIRI.

1. Introduction

Acute myocardial infarction (AMI) is mainly associated with an increased cardiac load or the induction of vascular lesions [1]. Its pathogenesis involves a sudden and substantial reduction or interruption of the coronary blood supply, which leads to myocardial ischemia and necrosis [2]. Reperfusion is the core strategy in the treatment of AMI, which can rescue the myocardium on the verge of necrosis by restoring blood flow in the occluded coronary artery [3, 4]. However, damage to vascular endothelium can easily trigger platelet aggregation, leading to the formation of microthrombi after reperfusion, which further exacerbates myocardial ischemia. Therefore, there remains a high risk of infarction after reperfusion. Consequently, long‐term use of antithrombotic drugs, such as aspirin, clopidogrel, ticagrelor, and prasugrel, is necessary [3, 4].

For medications designed to prevent damage following myocardial ischemia‐reperfusion, the core approach centers around three aspects: mitigating oxidative stress, stabilizing cell function, and regulating hemodynamics. As a widely used clinical drug, aspirin has the capacity to mitigate the hypoxia‐reoxygenation injury of cardiomyocytes via multiple pathways [5]. These include anti‐inflammatory actions, the inhibition of platelet activation, and the regulation of apoptosis [6, 7]. The main pharmacological action of aspirin involves the non‐discriminatory inhibition of cyclooxygenase enzymes (COXs), specifically COX‐1 and COX‐2 [6]. Aspirin at low doses selectively inhibits COX‐1. In contrast, aspirin at high doses has the effect of inhibiting both COX‐1 and COX‐2. This inhibition leads to a reduction in the synthesis of prostaglandins (such as PGE2) and thromboxane A2 (TXA2). Both of these substances are potent pro‐inflammatory factors, and they can exacerbate the inflammatory infiltration and oxidative stress‐induced damage in cardiomyocytes [8]. However, long‐term use of aspirin causes gastrointestinal lesions, such as mucosal erosions and ulcers [6, 9]. Therefore, the search for alternative natural products for the development of new drugs has emerged as a current research focus.

Currently, a series of chemical drugs, natural products, and traditional Chinese medicine preparations have been demonstrated to alleviate myocardial ischemia‐reperfusion injury (MIRI) by targeting multiple mechanisms. These mechanisms encompass oxidative stress, calcium overload, mitochondrial dysfunction, inflammatory reaction, and endoplasmic reticulum stress, among others [2]. Eupatilin, a lipophilic flavonoid compound extracted from Artemisia plants, exerts multiple functions, including anti‐inflammatory [10, 11], antioxidant [12], and antitumor activities [13, 14]. Moreover, it has been reported that eupatilin ameliorates doxorubicin‐induced cardiotoxicity in mice by attenuating inflammation, oxidative stress, and apoptosis via activation of the PI3K‐Akt pathway [12]. Eupatilin alleviates sepsis‐induced myocardial injury by modulating Mcl‐1 ubiquitination and activating the PI3K/Akt/Foxo3a pathway [8]. Meanwhile, eupatilin can ameliorate hypoxia‐reoxygenation‐induced injury in H9c2 cardiomyocytes through the Akt/GSK‐3β pathway [15]. These reports suggest that eupatilin may exert cardioprotective effects after myocardial ischemia‐reperfusion.

This study explored the impacts of eupatilin on COXs in oxygen‐glucose deprivation/reoxygenation (OGD/R)‐induced H9c2 cells and MIRI‐induced rats. Meanwhile, the COX‐mediated Akt/GSK‐3β pathway was analyzed to explore the pharmacological activity of eupatilin for MIRI treatment.

2. Materials and Methods

2.1. Cells

Rat cardiomyocytes, H9c2, were purchased from BNCC (#BNCC337726, BNCC, China) and cultured in DMEM (#BNCC338068, BNCC, China) supplemented with 10% FBS and 1% P/S at 37°C in an atmosphere of 5% CO₂.

2.2. OGD/R Cell Model

As previously reported [16, 17, 18], when the H9c2 cells reached 80% confluence, the medium was replaced with low‐glucose DMEM, and the cells were incubated at 95% N₂, 0.1% O₂, and 5% CO₂ for 4 h. Then, the cells were cultured in high‐glucose DMEM at 95% air and 5% CO₂ for 2 h.

2.3. Eupatilin Treatment In Vitro

To analyze the protective effects of eupatilin on OGD/R injury, H9c2 cells were subjected to OGD/R and then treated with an increased dose of eupatilin for 24 h at normal culture conditions. The eupatilin (purity ≥ 98%, #HY‐N0783, MCE, China) was first dissolved in 10% dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution (stored at −20°C), and then diluted with complete medium to the final concentrations (2.5, 5, 10, and 20 µM) with a final DMSO concentration ≤ 0.1%.

To further verify the effects of eupatilin on COXs in OGD/R‐induced H9c2 cells, the cells were allocated into four groups: the control group, the OGD/R group, the OGD/R plus aspirin group, and the OGD/R plus eupatilin group. Except for the control group, cells in the other groups underwent OGD/R. Subsequently, they were treated with 0.1% DMSO, 10 µM aspirin [19], or 10 µM eupatilin for 24 h at normal culture conditions.

2.4. Cell Counting Kit (CCK)−8 Assay

Cells (3 × 10⁵ cells/well) were treated with 10 µL CCK‐8 (#CA1210, Solarbio, China) for 4 h at normal culture conditions. The results were measured at 450 nm.

2.5. Cellular Reactive Oxygen Species (ROS) Detection

Cellular ROS was measured using the DCFH‐DA ROS fluorescent probe (#D6470, Solarbio, China). Cells were collected and suspended in 5 µM CM‐H2DCFDA at a concentration of 2 × 10⁶ cells/mL. Cells were cultured at 37°C for 30 min and washed three times with PBS. After stimulating the cells directly with a ROS‐positive control for 20 min, the cells were collected and analyzed using a flow cytometer (CytoFLEX S, Beckman, USA).

2.6. Molecular Docking

In accordance with a previously reported procedure [20], the three‐dimensional structures of COX1 (pdb_00006y3c) and COX2 (pdb_00005ikr) were retrieved from the Protein Data Bank (https://www.rcsb.org/), while the structure of eupatilin (PubChem CID: 5273755) was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Subsequently, using the PyMOL software, ligands and water molecules that were not relevant to the study were removed. Following this, the molecular docking between COX (either COX‐1 or COX‐2) and eupatilin was predicted using the AutoDock Tool (Chimera 1.18, USA). A binding energy of ≤ −8.0 kcal/mol indicates a very strong binding interaction; a binding energy in the range of −6.0 to −8.0 kcal/mol indicates a relatively good binding interaction.

2.7. Animals

Thirty‐six (18 male and 18 female) Sprague−Dawley rats, 8−10 weeks old and weighing 300−360 g, were procured from Charles River (Beijing, China) and then housed for 1 week with free access to food and water (20°C−25°C, 40%−65% humidity, and a 12‐h light−dark cycle).

2.8. MIRI Model

Referring to previously reported procedures [18, 21, 22], an acute MIRI model was constructed. Briefly, the rats were fasted for 12 h before surgery and anesthetized by intraperitoneal injection of 40 mg/kg pentobarbital sodium. The rats received mechanical ventilation after anesthesia and prior to thoracotomy. Subsequently, the rats were fixed, and a longitudinal cut approximately 1.5 cm in length was made on the skin along the left margin of the sternum. Forceps and hemostats were employed to bluntly separate the pectoral muscles in layers. Swift access to the thoracic cavity was gained via the 3rd or 4th intercostal space. Subsequently, the hemostats were used to open the intercostal space, and the pericardium was carefully dissected. In synchronization with the heartbeat, the left hand was used to gently compress the heart, causing it to project from the incision. At the line connecting the lower edge of the left auricle and the conus arteriosus, the anterior descending branch of the coronary artery was ligated with a 6‐0 suture for 30 min. After 30 min ischemic treatment, the slipknot was released, and the heart was gently returned to the thoracic cavity. Air was expelled by squeezing the thoracic cavity and tightening the sutures at the ligation incision. The electrocardiogram (ECG) was measured following surgical procedures using a small animal ECG monitor (#MouseOx Plus, STRAA life Sciences, USA).

2.9. Animal Group

To verify whether the in‐vitro findings are applicable in vivo, the rats were randomly allocated into six groups (each with n = 6): the sham group, the MIRI group, the MIRI plus aspirin (20 mg/kg) group, and the MIRI plus eupatilin (10, 20, and 30 mg/kg) group. Eupatilin was first dissolved in 10% DMSO, and then diluted with saline with a final DMSO concentration ≤ 0.1%.

Rats in the sham group only underwent thoracotomy without the MIRI procedure. Except for the sham group, rats in the other groups underwent MIRI. After 2‐h reperfusion, animals received intraperitoneal drug (aspirin or eupatilin) or vehicle (0.1% DMSO) every day for 14 days. Rats in the sham group or the MIRI group received 0.1% DMSO. Aspirin was dissolved in saline at 37°C, and its dose was chosen based on the previous study [23]. Eupatilin doses (10, 20, and 30 mg/kg) were selected based on the previous studies [8, 24, 25]. The choice of intraperitoneal injection instead of gavage was mainly to maximize the exposure of the parent drug, avoid first‐pass metabolism and gastrointestinal degradation, minimize local gastrointestinal irritation and non‐specific effects, and ensure the stability and reproducibility of experimental results for easier mechanism analysis. This does not imply that researchers overlook clinical relevance. On the contrary, it is precisely to confirm the true efficacy of the drug under the premise of eliminating unreliable absorption. Subsequently, further translational research will naturally transition to gavage administration to evaluate the actual efficacy and safety of the drug after oral administration.

2.10. Echocardiography Detection

Cardiac function was determined via echocardiography (Vevo 2100 Ultrasound System, VisualSonics, Toronto, Ontario, Canada) by evaluating the left ventricular ejection fraction (LVEF).

2.11. Sample Collection

At the end of the experiment, animals were anesthetized using 40 mg/kg pentobarbital sodium. The blood samples from each rat were obtained using the retro‐orbital puncture technique. Following this, the rats were euthanized through cervical dislocation. The heart was rapidly excised, and the left ventricle was frozen at −80°C and sliced into 6−7 slices.

2.12. Enzyme‐Linked Immunosorbent Assay (ELISA)

The levels of prostaglandin E2 (PGE2, #SEKSM‐0034X, Solarbio, China), thromboxane A2 (TXA2, #SEKSM‐0031, Solarbio, China), and cardiac troponin I (cTnI, #U96‐3497E‐48T, YOBIBIO, China) in blood samples were measured using ELISA kits according to the instructions.

2.13. 2,3,5‐triphenyltetrazolium Chloride (TTC) Staining

The frozen heart slices (2 mm) were incubated in Tris buffer (pH 7.4) containing 1% TTC (#G3002, Solarbio, China) at 37°C for 15 min in a dark room, and then fixed with 4% formalin for 2 h. The infarcted area is not stained by TTC. The infarct area was evaluated using ImageJ software. The infarct size was calculated by the ratio of the infarct area to the total area.

2.14. Masson Staining

The heart slices were fixed with 4% paraformaldehyde, then embedded in paraffin and sectioned into sections with a thickness of 5 μm. The sections were deparaffinized and hydrated, and then stained with Masson's trichrome stain kit (#G1340, Solarbio, China) according to the manufacturer's instructions. The images were collected, and the collagen deposition was calculated as the ratio of the collagen deposition area to the total myocardial area.

2.15. Immunohistochemistry

According to the reported immunohistochemistry procedure [26], the fixed heart slices were embedded in paraffin and sectioned into sections with a thickness of 5 μm; then the cardiac sections (5 μm) were deparaffinized with xylene, hydrated with gradient ethanol, subjected to antigen retrieval with Proteinase K and 3% H₂O₂, and then incubated with specific antibodies (Table 1) at 4°C overnight. After washing with PBS, the sections were incubated with the second antibody for 90 min at 37°C. Then, the washed sections were stained with diaminobenzidine and hematoxylin. Finally, the sections were dehydrated with xylene, cleared with gradient ethanol, and mounted with neutral gum. The images were collected, and the positive expression was analyzed using ImageJ software.

Table 1.

Antibodies in this study.

Name	Product no.	Dilution	Source
COX‐1	A7531	WB: 1:800 IHC: 1:100	Abclonal
COX‐2	A3560	WB: 1:3000 IHC: 1:1000	Abclonal
Phospho‐Akt1 (S473)	AP0140	WB: 1:800	Abclonal
Akt1	A17909	WB: 1:3000	Abclonal
Phospho‐GSK‐3β‐S9	AP0039	WB: 1:300	Abclonal
GSK‐3β	A2081	WB: 1:1000	Abclonal
β‐actin	AC038	WB: 1:40000	Abclonal
HRP‐conjugated Goat anti‐Rat IgG (H + L)	AS028	WB: 1:7000 IHC: 1:1000	Abclonal

Abbreviations: IHC, immunohistochemistry; WB, Western blotting.

2.16. Western Blotting

In accordance with a reported Western blotting procedure [27], proteins were extracted from cells and tissues using a RIPA lysis buffer. They were then separated by 10% gel electrophoresis. Initially, a voltage of 150 V was applied. Once the leading edge of the sample had entered the separating gel by approximately 1 cm, the voltage was increased to 180 V. After separation, the proteins were transferred onto membranes. The membranes were blocked with 5% milk for 1.5 h at 37°C, and subsequently immunodetected with specific antibodies at 4°C overnight. After washing with PBST twice, the membranes were incubated with the second antibody at 37°C for 2 h. The details regarding the antibodies are presented in Table 1. After washing with PBST, the bands were made visible through the use of a chemiluminescent imaging system (ChemiScope 6000, Clinx, China). ImageJ software was utilized to analyze the gray values of these bands.

2.17. Statistical Analysis

Statistical analyses were performed using GraphPad Prism 8.0.1 software (La Jolla, San Diego, CA, USA). The results were expressed as the mean ± standard deviation (SD). One‐way analysis of variance followed by post hoc comparison (Tukey's test) was used to analyze the differences among groups. A p value less than 0.05 was considered statistically significant.

3. Results

3.1. Eupatilin Improved OGD/R Injury and Regulated the COX‐Mediated AKT/GSK Pathway

To analyze the protective effects of eupatilin on OGD/R injury in cardiomyocytes, the OGD/R‐induced H9c2 cells were treated with different doses of eupatilin (2.5, 5, 10, and 20 µM) for 24 h (Figure 1A). The results showed that eupatilin treatment significantly suppressed cellular ROS (Figure 1B) and increased cell viability (Figure 1C) in OGD/R‐induced cells (p < 0.05). Moreover, the protective effects were enhanced as the dose of eupatilin increased.

Figure 1.

Effects of eupatilin on cell viability and cellular ROS in OGD/R‐induced H9c2 cells. The H9c2 cells were induced by OGD/R and treated with different doses of eupatilin (5 μΜ, 10 μΜ, 20 μΜ) for 24 h. (A) Flowchart of cellular experiments. (B) Cellular ROS was measured by the DCFH‐DA ROS fluorescent probe. (C) The results of cellular ROS were analyzed using flow cytometry. (D) Cell viability was measured by CCK‐8. n = 6. *p < 0.05, **p < 0.01.

To explore the possible mechanism of eupatilin in the OGD/R injury, the levels of COX1, COX2, phosphorylated Akt1 (S473), and phosphorylated GSK‐3β (S9) proteins were measured by western blotting (Figures 2A,B,C). It showed that the levels of COX1 and COX2 proteins were significantly inhibited, while the ratios of p‐Akt1 (S473)/Akt1 and p‐GSK‐3β (S9)/GSK‐3β were significantly elevated increasing doses of eupatilin (p < 0.05). To further confirm the interaction between eupatilin and COXs, the molecular docking was used to predict their binding activity (Figure 2D). It showed that eupatilin had good binding with COX1 (score = −8.3 kcal/mol) or COX2 (score = −8.8 kcal/mol).

Figure 2.

Effects of eupatilin on the COX‐mediated Akt/GSK pathway in OGD/R‐induced H9c2 cells. The levels of COX1 (A), COX2 (B), phosphorylated Akt1 (S473), and phosphorylated GSK‐3β (S9) proteins (C) were measured by Western blotting. (D) The interaction between eupatilin and COXs was predicted using molecular docking. n = 6, *p < 0.05, **p < 0.01.

3.2. Eupatilin Improved OGD/R Injury in H9c2 Cells by Targeting COXs

To verify that the protective mechanism of eupatilin targets COXs, aspirin was selected as a positive control in this study (Figure 3A). The results showed that eupatilin or aspirin treatment significantly suppressed cellular ROS (Figure 3B) and promoted cell viability (Figure 3D) in the OGD/R‐induced H9c2 cells (p < 0.01). Meanwhile, the eupatilin or aspirin treatment significantly suppressed the PGE2 content in the OGD/R‐induced cells (p < 0.01).

Figure 3.

Eupatilin treatment improved the OGD/R‐induced injury via COXs. Aspirin was used as a positive control. (A) Flowchart of cellular experiments. (B) Cellular ROS was measured by the DCFH‐DA ROS fluorescent probe. The results of cellular ROS were analyzed using flow cytometry. (C) Cell viability was measured by CCK‐8. (D) PGE2 contents were measured by an ELISA kit. n = 6, *p < 0.05, **p < 0.01.

Moreover, the levels of COX1 (Figure 4A) and COX2 (Figure 4B) were significantly decreased after eupatilin or aspirin treatment in the OGD/R‐induced H9c2 cells (p < 0.01). The ratios of p‐Akt1 (S473)/Akt1 and p‐GSK‐3β (S9)/GSK‐3β proteins (Figure 4C) were significantly upregulated after eupatilin or aspirin treatment in the OGD/R‐induced H9c2 cells (p < 0.05).

Figure 4.

Eupatilin treatment upregulated the Akt/GSK pathway via COXs in OGD/R‐induced H9c2 cells. Levels of COX1 (A), COX2 (B), p‐Akt1 (S473)/Akt1, and p‐GSK‐3β (S9)/GSK‐3β (C) proteins were measured by western blotting. n = 6, *p < 0.05, **p < 0.01.

3.3. Eupatilin Improved Myocardial Injury in MIRI‐Induced Rats

To further verify that eupatilin improves MIRI in rats by acting on COX, the MIRI‐induced rats were treated with different doses of eupatilin (10, 20, and 30 mg/kg) or aspirin (20 mg/kg) every day for 14 days (Figure 5A). In the sham group, the waveforms of each cardiac cycle were regular, and the ST‐segment basically adhered to the isoelectric line. In the MIRI group, a significant upward convex elevation of the ST‐segment was observed, which confirmed the successful establishment of the MIRI model (Figure 5B). The echocardiography results showed that rats with MIRI displayed significantly compromised systolic function as evidenced by the decline in LVEF compared with the sham group (Figure 5C, p < 0.01). Eupatilin or aspirin treatment significantly upregulated the LVEF, thereby improving systolic function (p < 0.01).

Figure 5.

Eupatilin treatment improved cardiac function in rats with MIRI. SD rats were performed by MIRI and then treated with aspirin or eupatilin every day for 14 days. (A) Flowchart of animal experiments. (B) Representative ECG images of control and MIRI. (C) Representative echocardiographs. (D) Statistical analysis of left ventricular ejection fraction (LVEF). n = 6, **p < 0.01.

Moreover, the levels of PGE2, TXA2, and cTnI in the blood were significantly upregulated after MIRI compared the sham group (Figure 6A, p < 0.01). Treatment with eupatilin or aspirin clearly downregulated the levels of PGE2, TXA2, and cTnI in the rats with MIRI (p < 0.05). Meanwhile, the myocardial infarction was measured by TTC staining (Figure 6B), and collagen deposition was measured by Masson staining (Figure 6C). The results showed that MIRI surgery induced myocardial infarction and collagen deposition compared with sham rats (p < 0.01). Treatment with eupatilin or aspirin significantly alleviated the MIRI‐induced myocardial infarction and collagen deposition (p < 0.05). Moreover, the ameliorative effect of eupatilin was enhanced with an increase in its dosage.

Figure 6.

Eupatilin treatment improved myocardial injury in rats with MIRI. (A) Levels of PGE2, TXA2, and cTnI1 in blood were measured by ELISA kits. (B) Myocardial infarction was analyzed by TTC staining. Red area: normal myocardium; White area: infarct area. (C) Myocardial collagen deposition was analyzed by Masson staining. Blue‐green: collagen deposition. n = 6, *p < 0.05, **p < 0.01.

3.4. Eupatilin Inhibited COXs and the AKT/GSK Pathway in MIRI‐Induced Rats

The expression of COX1 (Figure 7A) and COX2 (Figure 7B) was measured in myocardial tissues using immunohistochemistry, and the results revealed that treatment with eupatilin or aspirin significantly downregulated the MIRI‐induced high levels of COX1 and COX2 in the myocardial tissues compared with the MIRI group (p < 0.05). In parallel, the protein levels of COX1 (Figure 8A), COX2 (Figure 8B), p‐Akt1 (S473)/Akt1, and p‐GSK‐3β (S9)/GSK‐3β (Figure 8C) proteins were measured in myocardial tissues using western blotting. In line with the in vitro results, the MIRI‐induced high levels of COX1 and COX2 proteins were significantly suppressed after eupatilin or aspirin treatment (Figures 8A,B, P < 0.05). The ratios of p‐Akt1 (S473)/Akt1 and p‐GSK‐3β (S9)/GSK‐3β proteins were further upregulated after eupatilin or aspirin treatment compared with the MIRI group (Figure 8C, p < 0.05). The above results were enhanced with the increase in the concentration of eupatilin.

Figure 7.

Eupatilin inhibited the expression of COX1 and COX2 in MIRI‐induced rats. The expressions of COX1 (A) and COX2 (B) were measured by immunohistochemistry. n = 6, *p < 0.05, **p < 0.01.

Figure 8.

Eupatilin inhibited the COXs and upregulated the Akt/GSK pathway in MIRI‐induced rats. The levels of COX1 (A), COX2 (B), p‐Akt1 (S473)/Akt1, and p‐GSK‐3β (S9)/GSK‐3β (C) proteins were measured by Western blotting. n = 6, *p < 0.05, **p < 0.01.

4. Discussion

As a natural bioactive flavone, eupatilin has been applied to the treatment of various diseases, such as pancreatic cancer [13], subarachnoid hemorrhage [10], hyperlipidemia [28], and pancreatitis [29]. Meanwhile, a variety of action mechanisms of eupatilin have been discovered [30], such as anti‐inflammatory, anti‐oxidant, anti‐cancer, and neuroprotective effects. Here, based on previous studies [8, 12, 15], this study found that eupatilin ameliorated acute MIRI in rats. Moreover, it was further found that its protective mechanism was associated with the COX‐mediated Akt/GSK‐3β pathway.

Cyclooxygenases (COX1 and COX2) are enzymes that catalyze the rate‐limiting step of prostaglandin synthesis [31]. There is a 63% amino‐acid structural similarity between COX1 and COX2. However, the active site of COX2 is more spacious and can accommodate bulkier ligands because isoleucine 523 of COX1 is replaced by valine 509 in COX2 [31]. In normal conditions, COX1 is highly constitutively expressed in all tissues and certain cell types. However, in acute and chronic neurological disorders, COX1 expression is related to the production of pro‐inflammatory mediators [32]. During injury, infection, and in response to bacterial toxins, the expression of COX2 is preferentially observed. The COX2 expression leads to the overproduction of PGE2, which in turn triggers inflammation and pain [31]. Non‐steroidal anti‐inflammatory drugs (NSAIDs) are the classical non‐selective COX inhibitors, including aspirin, indomethacin, mefenamic acid, ibuprofen, and oxicams. In cardiovascular‐related diseases, aspirin is commonly used to prevent atherothrombosis [6]. The mechanism of action of aspirin is such that low‐dose aspirin selectively inhibits COX1, while high‐dose aspirin inhibits both COX1 and COX2. In this study, aspirin was used as a positive drug, and it was found that eupatilin shared the same action mechanism as aspirin in MIRI.

It is well‐known that MIRI induces high‐level expression of COX, which in turn triggers inflammation and oxidative stress. Oxidative stress and apoptotic responses in MIRI have been reported to involve the AKT/GSK‐3β pathway [15]. Impaired protein kinase B (AKT) signaling exacerbates oxidative stress, inflammation, and apoptosis. Meanwhile, AKT regulates glycogen synthase kinase 3‐beta (GSK‐3β) through phosphorylation at Ser9 [33], and the phosphorylated GSK‐3β promotes nuclear factor erythroid 2‐related factor 2 (Nrf2) expression in the nucleus and antioxidant enzyme production, thereby exerting a protective effect [34]. In this study, we found that eupatilin suppressed ROS and apoptosis by inhibiting COX in OGD/R‐induced cardiomyocytes, and further promoted the phosphorylated AKT and phosphorylated GSK‐3β. This finding suggests that eupatilin is an anti‐inflammatory drug for MIRI.

In in‐vivo experiments, we also found that eupatilin improved cardiac function in rats with MIRI. Additionally, myocardial infarction and collagen deposition were alleviated. This was associated with a decrease in COXs, along with an increase in phosphorylated AKT and phosphorylated GSK‐3β. In the MIRI model, the development of collagen deposition and fibrosis typically spans from weeks to months [35, 36]. In this study, measuring myocardial fibrosis at the 14‐day time‐point might reflect acute inflammatory infiltration instead of well‐established fibrotic remodeling.

However, there are limitations in this study. The effect of phosphorylation of GSK‐3β on the nuclear accumulation of Nrf2 in MIRI remains to be further explored. Other molecular regulatory mechanisms of eupatilin on MIRI need to be explored. Moreover, it is necessary to determine whether long‐term administration of eupatilin may cause related side effects, such as gastric ulcers, kidney injury, and liver injury. Importantly, the current sample size is inadequate to detect gender differences (usually, a sample size of > 10−15 per group). Consequently, gender‐stratified analysis was not performed. Therefore, all results only represent the average effect after combining both genders, and gender‐specific responses cannot be excluded. It has been demonstrated that there are significant differences between male and female individuals in the pathological mechanisms of ischemic heart injury, inflammatory responses, apoptotic pathways, and repair capabilities [37, 38]. Thus, independent gender‐based analysis is proposed for future research. Meanwhile, H9c2 cells, derived from rat embryonic myoblasts, exhibits a combination of skeletal and cardiac muscle characteristics. It lacks the electrophysiological and metabolic features typical of mature cardiomyocytes. For future investigations, neonatal rat ventricular cardiomyocytes (NRVCMs) will be employed for validation purposes, thereby enhancing the robustness of the in‐vitro experimental argumentation.

5. Conclusion

This study showed that eupatilin alleviated acute MIRI in rats by inhibiting COX and activating the Akt/GSK‐3β pathway. This study provides a theoretical basis for the application of eupatilin in MIRI.

Author Contributions

Min Guo: conceptualization, writing − original draft, project administration, formal analysis, data curation, methodology. Qianqian Li: software, supervision, writing − review and editing, investigation, methodology, visualization. Lili Ji: writing − review and editing, conceptualization, supervision, investigation.

Funding

The authors have nothing to report.

Ethics Statement

This study was approved by the Ethics Committee of Shandong Healthcare Group Zibo Hospital (Approval No. 2024‐L091). All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.