Cannabinoid Receptor-2 agonist AM1241 Attenuates Myocardial Ischemia-Reperfusion-Induced Oxidative Stress in Rats via Nrf2/HO-1 Pathway

Ischemic heart disease represents a prevalent cardiovascular condition posing a significant threat to human health [1]. The primary therapeutic strategy involves restoring blood supply to the ischemic myocardium to reduce mortality rates. However, reperfusion of ischemic myocardium can paradoxically lead to myocardial ischemia-reperfusion injury (MIRI) [1, 2].

The pathogenesis of MIRI is multifaceted, involving factors such as oxidative stress, endoplasmic reticulum stress, and inflammatory responses [2]. Studies indicate that excessive generation of reactive oxygen species (ROS) during myocardial reperfusion enhances oxidative stress, contributing to harmful damage in patients [2]. MIRI and oxidative stress not only impact treatment outcomes but also constrain the development and application of novel medical technologies. Therefore, in-depth exploration of MIRI, oxidative stress mechanisms, and preventive measures is crucial for managing ischemic heart disease. Increased expression of heme oxygenase-1 (HO-1), a stress-response protein, has been shown to mitigate cell damage induced by MIRI [3]. Nrf2, a redox-sensitive transcription factor, interacts with antioxidant response elements, playing a pivotal role in activating HO-1 transcription. Recently, it has been proposed that upregulation of HO-1 expression induced by Nrf2 activation can protect the heart from oxidative stress [4].

The cannabinoid type II receptor, also known as the CB2 receptor, is a G protein-coupled receptor with seven transmembrane domains that are predominantly expressed in the cells of the immune system [5]. The endocannabinoid system, particularly the CB2 receptor, is moderately expressed in several peripheral tissues, including cardiovascular tissues [5, 6]. However, its activation following exposure to harmful stimuli has recently been demonstrated to confer cardioprotective effects in ischemic heart disease [5]. Simultaneously, CB2 receptors and endogenous cannabinoids exert protective effects in diabetes, diabetic cardiomyopathy, atherosclerosis, and various forms of I/R injury (such as hepatic, cardiac, and stroke), mediated through antioxidative stress and anti-inflammatory responses [7‒10]. Meanwhile, the activated CB2 receptor has been shown to have great potential in antioxidant stress and anti-inflammatory responses in various disease models [11, 12]. Wang et al. [13] found that the CB2 receptor agonist AM1241 activates the PI3K/Akt/Nrf2 signaling pathway to reduce excessive oxidative stress and inflammation in ischemic hearts, eliciting endogenous myocardial regeneration. Zhang et al. [14] showed that AM1241 protects skeletal muscle against ischemia-reperfusion injury via the Nrf2 signal transduction pathway. To date, the role of the CB2 receptor in MIRI-related oxidative stress and its relevant mechanism have not been fully elucidated. This study aims to investigate the role of the CB2 receptor agonist AM1241 in MIRI-induced oxidative stress and its underlying molecular basis, thereby providing evidence for its clinical use.

Forty male Sprague-Dawley (SD) rats, weighing 200–250 g, were obtained from SPF (Beijing) Biotechnology Co., LTD. AM1241 (Target Mol, USA) was dissolved in 10% DMSO and distilled water for administration, while Nrf2 inhibitor ML385 was procured from Medchem Express (MCE Co. Ltd., Shanghai, China).

All SD rats (n = 40) were randomly divided into Sham group (only threading without ligation, n = 10); IR group (IR with saline injection, n = 10); IR+AM1241 group (IR with intraperitoneal injection of AM1241 6 mg/kg, n = 10); and IR+AM1241+ML385 group (IR with intraperitoneal injection of AM1241 6 mg/kg and ML385 30 mg/kg, n = 10). AM1241 and/or ML385 were administered 1 h prior to the modeling. Rats were anesthetized with thiopental sodium (40 mg/kg, i.p.), the thoracic cavity was opened at the 3rd and 4th left intercostal space to reveal the heart. Thereafter, the needle with 6-0 noninvasive sutures was then inserted into the left edge of pulmonary conus and the lower edge of left atrial appendage. After 10 min of stabilization, a double-layer plastic cannula was inserted, and ligation was then performed. The presence of myocardial infarction as evidenced by either ST segment elevation or T-wave peak, as well as local cyanosis in the heart on electrocardiogram indicated successful modeling of the MIRI. After 30 min of ischemia, the inner cannula was drawn out for reperfusion for 2 h. All experimental protocols were approved by the Animal Experimentation Ethics Committee.

Left ventricular performance was evaluated at the 2 h mark following MIRI utilizing the Vevo 770 high-resolution imaging platform. The left ventricular fractional shortening and ejection fraction were automatically derived via echocardiographic software. Each metric was assessed by computing the mean value across five consecutive cardiac cycles.

The blood sample was taken at the end of reperfusion and centrifuged at 1,000 rpm for 15 min. Thereafter, the serum was collected and then subjected to enzyme-linked immunosorbent assay (ELISA) to assess cardiac injury markers, which encompass cardiac troponin I, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase isoenzyme MB (CK-MB). Results were quantified in units per liter. Evaluation and analysis were performed employing a spectrophotometer (Beijing Shiji Kexin Scientific Instrument Co., Ltd., Beijing, China).

At the end of reperfusion, the rats were killed, and their hearts were collected for subsequent analysis. The heart was frozen at −80°C for 20 min, cut into 7–9 transverse slices and then incubated with 1% triphenyltetrazolium chloride (TTC) phosphate buffer (pH = 7.4) in the dark at 37°C for 30 min. The infarct areas were captured and quantified by using a camera and Image J software. Non-TTC-stained area (white) was identified as the infarct area, and the living tissue was stained red.

Apoptosis was evaluated using a TUNEL assay kit (Solarbio, USA). Myocardial tissues were fixed in 4% paraformaldehyde at 4°C, dehydrated in ethanol, and embedded in paraffin for sectioning. Following the protocol provided with the kit, the TUNEL reaction mixture was added and incubated in the dark at 37°C for 1 h. Continue to stain the nuclei with DAPI at room temperature for 10 min and wash twice with PBS. Myocardial tissues were observed and photographed under a fluorescent microscope. Apoptotic cells exhibited green fluorescence in the TUNEL staining, while cell nuclei displayed blue fluorescence. The apoptotic rate was determined by calculating the ratio of TUNEL-positive cells to the total cell count.

Heart tissues were fixed in 4% paraformaldehyde for 24 h and cut into slices of 4 μm thickness (Cleica, Nussloch, Germany). Hematoxylin and eosin (H&E) staining were performed to examine histopathological changes under a microscope at magnifications of ×100 and ×200 (Olympus, Tokyo, Japan). For immunohistochemical staining, following routine deparaffinization, rehydration, and antigen retrieval of tissue sections, primary antibodies against 3-nitrotyrosine (3-NT) (1:100, Abcam) and 4-hydroxynonenal (4-HNE) (1:50, Abcam) were incubated overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary IgG antibodies (1:50, Abcam) at room temperature for 1 h. Immunostaining was visualized using 3,3'-diaminobenzidine chromogen (Servicebio) and counterstained with hematoxylin, followed by observation under an optical microscope.

H9c2 cardiomyocytes were obtained from Nanjing Kaiji Biotechnology Co., Ltd. (Nanjing, China) and cultivated under conditions involving Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, USA), supplemented with 10% fetal bovine serum. The cultivation process was conducted at 37°C with a controlled CO₂ atmosphere. The cells were randomly divided into control, HR, HR+AM1241, and HR+AM1241+ML385 groups. The control group received no intervention, while the HR group was subjected to oxygen and glucose deprivation (OGD), followed by reperfusion, to simulate in vitro myocardial hypoxia-reoxygenation (HR) injury, as previously described [1]. The HR+AM1241 group’s cells were pre-treated with 10 μm AM1241 for 6 h before undergoing HR injury. Cells in HR+AM1241+ML385 group were soaked in 5 μm of ML385 for 1 h after the same treatment as HR+AM1241 group.

HR-injured cardiomyocytes were seeded in a 96-well plate at a density of 1 × 10⁴ cells per well. 100 μL DMEM and 10 μL CCK-8 reagent were added to each well. The absorbance was measured at 450 nm using a microplate reader (Multiskan Spectrum; Thermo Fisher Scientific, Waltham, MA, USA).

After modeling, cells were washed with PBS buffer and then incubated with 500 μL of DCFH-DA staining solution (Jiangsu biyuntian Biotechnology Co., Ltd., Nantong, China) at 37°C for 20 min. Afterward, the staining solution was removed, and the cells were washed with serum-free medium for three times. Using laser confocal fluorescence microscopy (Leica TCS-SP2-AOBS-MP, Germany), green fluorescence was observed. The fluorescence intensity was detected by Accuri C6 flow cytometry (BD, biosciences, CA, USA). The excitation wavelength was 488 nm, and the emission wavelength was 525 nm.

Myocardial tissues were embedded in Polylysine, fixed with precooled 4% paraformaldehyde at room temperature and then sectioned at a thickness of 4 μm. The slices were washed with PBS and subjected to antigen retrieval. After being blocked with PBS containing 2% BSA at room temperature for 1 h, the sections were incubated with Nrf2 polyclonal antibody (1:200, Santa Cruz, USA) and FITC-labeled anti-rabbit secondary antibody (Jackson ImmunoResearc, USA) at 37°C for 1 h. Fluorescence images were captured under laser confocal fluorescence microscopy (Leica TCS-SP2-AOBS-MP, Germany), and 5 different fields of view were randomly taken from each section.

The concentration of total protein extracted from H9c2 cardiomyocytes was detected with a BCA protein assay kit (Beyotime, Shanghai, China). The protein samples were separated by 12% SDS-PAGE and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked in TBST (Tris HCl and Tween 20) solution containing 5% skimmed milk powder at 25°C for 1.5 h and then incubated overnight at 4°C with primary antibodies against Nrf2 (1:1,000, Abcam, Cambridge, UK) or HO-1 (1:1,000, Abcam, Cambridge, UK). On the next day, the membrane was washed with TBST buffer and incubated with alkaline phosphatase labeled IgG (Promega, USA) for 1 h at room temperature. β-actin was included as an internal reference. Immunoreactive proteins were visualized using a chemiluminescence imaging system (EI600, Beyotime, Shanghai, China), and Image J software was used to determine the gray value of protein bands.

Statistical analyses were performed using GraphPad Prism 8.0 software. The experimental data were expressed as mean ± standard deviation (SD). Differences between multiple groups were analyzed with one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Statistical significance was defined as p < 0.05.

To investigate the impact of AM1241 on cardiac function in rats, we utilized echocardiography to measure cardiac parameters (Fig. 1a–c). MIRI led to a significant impairment in myocardial function, evidenced by a notable decrease in both left ventricular ejection fraction and left ventricular fractional shortening. Serum biomarkers of myocardial injury, including LDH, CK-MB, cardiac troponin I , and ALT, were significantly elevated in the MIRI group compared to the Sham group (Fig. 1a–g). However, AM1241 treatment markedly ameliorated these indices of myocardial injury (Fig. 1). To explore the role of Nrf2 in this protective effect, we administered the Nrf2 inhibitor ML385 (30 mg/kg) alongside AM1241. As depicted in Figure 1, the ameliorative effects of AM1241 were reversed by concomitant treatment with ML385.

We further investigated AM1241’s impact on myocardial infarct size and histomorphology through TTC and H&E staining. As shown by TTC staining, almost no infarct area (white) was identified from rat heart slices in sham group, heart slices in IR group contained large white areas, and significantly smaller infarct area in the IR+AM1241 group than in the IR group, while the Nrf2 inhibitor ML385 reversed the effect of AM1241(Fig. 2a, b). H&E staining revealed myocardial rupture, necrosis, congestion, and inflammation in the IR group, whereas the IR+AM1241 group exhibited normal myocardial structure (Fig. 2c). Notably, administration of AM1241 (6 mg/kg) significantly reduced the number of inflammatory cells in the IR-injured tissues (p < 0.001), while this inhibitory effect of AM1241 was reversed by Nrf2 inhibitor ML385 (p < 0.01) (Fig. 2d).

Oxidative/nitrosative stress is critical in the pathogenesis of MIRI [2, 15]. We assessed the expression of 4-HNE and 3-NT, markers of oxidative and nitrosative damage [10], through immunohistochemical staining. The IR group showed significant increases in 3-NT and 4-HNE levels, which were significantly attenuated by AM1241 (Fig. 2e). This attenuation was reversed by ML385, underscoring the role of the Nrf2 pathway in mediating AM1241’s protective effects.

As shown in Figure 3a, compared to the sham surgery group, the IR group exhibited an increase in TUNEL-positive apoptotic cardiomyocytes stained in green. Quantitative analysis revealed a significant increase in the apoptosis rate (p < 0.001, Fig. 3b). Meanwhile, treatment of IR rats with AM1241 (6 mg/kg) led to a marked decrease in the apoptosis rate (p < 0.01). Notably, co-treatment with ML385 (30 mg/kg) partially reversed AM1241-induced inhibition of apoptosis in IR rats (p < 0.05). Together, these observations indicated that AM1241 inhibited apoptosis in the cardiomyocytes of IR rats.

To investigate the effect of AM1241 on the survival of H9c2 cardiomyocytes, we performed a cell viability assay. As illustrated in Figure 4a, treatment with 10 μm AM1241 significantly increased cell viability compared to control cells (p < 0.01). Consequently, 10 μm AM1241 was selected for subsequent experiments.

We assessed ROS production in rat cardiomyocytes using the DCFH-DA assay. Flow cytometry revealed elevated DCFH-DA fluorescence in HR cells relative to the control group (Fig. 4b), indicating increased intracellular ROS levels (p < 0.001). However, the HR+AM1241 group exhibited significantly reduced ROS generation compared to the HR group (p < 0.001), an effect reversed by ML385 (5 μm) treatment (p < 0.05). Confocal laser fluorescence microscopy (Fig. 4c) confirmed these findings, showing intense green fluorescence in HR cells, signifying substantial ROS accumulation (p < 0.001). This fluorescence was markedly diminished in the HR+AM1241 group (p < 0.001) and increased with ML385 treatment (p < 0.05). These results suggest that AM1241 inhibits HR-induced ROS production, likely via Nrf2 pathway activation.

We further examined Nrf2 and HO-1 expression in the cardiomyocytes. Immunofluorescence assays showed increased green fluorescence intensity in HR-injured cells treated with AM1241, indicating elevated Nrf2 expression (Fig. 5a). Western blot analysis supported these findings, revealing upregulated Nrf2 and HO-1 levels in HR cells treated with AM1241. This upregulation was partially reversed by ML385 co-administration (Fig. 5b). Taken together, these results suggested that the protective effect of AM1241 on MIRI rats may involve Nrf2/HO-1 signaling pathway.

IR injury in the heart could lead to changes in myocardial morphology, metabolism and function. Previous studies have suggested that IR injury may be linked to calcium overload, inflammatory responses, dysfunction of energy metabolism, and increased ROS production [16, 17]. In this study, we showed that rats with MIRI display inflammatory cell infiltration and necrosis followed by a marked increase in myocardial apoptosis, providing consistent data with the previous reports [18, 19]. AM1241, a selective agonist of cannabinoid CB2 receptor, has been shown to improve cardiac function and alleviate myocardial fibrosis through Nrf2-mediated TGF-β1/Smad3 signaling pathway, while preventing ethanol-induced cardiotoxicity [20, 21]. However, the effects of AM1241 on ischemic heart disease and its cellular mechanisms remain underexplored. This study aimed to investigate the role of AM1241 in MIRI and its underlying molecular mechanisms. Our findings reveal that AM1241 treatment significantly reduced infarction size and decreased serum levels of cardiac injury markers (cTnl, AST, LDH, CK-MB), indicating a protective effect of AM1241 against ischemia-induced myocardial damage. Activation of the CB2 receptor has been linked to reduced oxidative/nitrosative stress and inflammatory responses, contributing to its cardioprotective effects [2, 10, 22]. For instance, HU308, a CB2 receptor agonist, has been shown to mitigate ROS and inflammatory responses in IR models [22], while JWH-133, another CB2 agonist, demonstrated similar protective effects in ovarian IR [23]. Additionally, both natural and synthetic CB2 agonists have exhibited antioxidative properties in various models of atherosclerosis and MIRI [24]. Specifically, JWH-133 reduced oxidative/nitrosative stress markers such as 4-HNE and 3-NT in diabetic-induced myocardial injury [10]. We observed that AM1241 suppressed the elevated levels of 3-NT and 4-HNE and reduced inflammatory responses in IR-injured cardiac tissues, suggesting its role in mitigating oxidative/nitrosative damage and inflammation. To further elucidate the relationship between oxidative stress and IR injury, we conducted in vitro experiments using H9c2 cardiomyocytes to model HR injury. HR injury, akin to MIRI, leads to ROS overproduction and oxidative stress [25, 26]. Using the DCFH-DA assay, we found that AM1241 significantly inhibited ROS production in HR-injured cardiomyocytes, confirming its antioxidant effects. This aligns with previous observations of AM1241’s antioxidative properties in hepatic stellate cells [27].

In early studies, activation of CB2 receptors may stimulate the PI3K/Akt pathway, which is involved in cellular survival and antioxidative responses [28]. AM1241’s cardioprotective effects in ischemic hearts may be mediated through the activation of the PI3K/Akt/Nrf2 pathway, supporting endogenous myocardial regeneration [29]. Nrf2, a critical transcription factor, orchestrates the cellular response to oxidative stress. For instance, Nrf2 activation has been shown to mitigate oxidative damage in renal IR injury [4]. HO-1, a downstream target of Nrf2, plays a multifaceted role in antioxidant defense, anti-inflammatory activities, and signal transduction. Studies have demonstrated that HO-1 can protect against IR injury in various organs, including the heart and intestines [3]. In this study, AM1241 upregulated the expression of HO-1 and Nrf2, providing consistent data with the previous research [13]. Moreover, the use of the Nrf2 inhibitor ML385 partially reversed these effects, suggesting that AM1241 exerts its protective effects against MIRI through the Nrf2/HO-1 pathway.

However, our study has certain limitations. Based on prior RNA sequencing studies, there is minimal expression of CB2 receptors in human and murine cardiomyocytes [10, 30]. Additionally, previous investigations relied on the use of nonspecific CB2 antibodies, and to date, there is no available specific CB2 antibody. Consequently, some effects of AM1241, particularly observed in in vitro cell lines, may not be mediated by CB2 receptors [31]. Further research is warranted to delve into the underlying molecular mechanisms, which will aid in the development of its pharmacotherapeutics.

This study provides evidence demonstrating that the cannabinoid receptor-2 agonist AM1241 can ameliorate cardiac function in MIRI rats by attenuating cardiac oxidative stress, inflammation, and cell apoptosis to alleviate myocardial infarction. Further investigations suggest that it may suppress HR-induced oxidative stress by activating the Nrf2/HO-1 signaling pathway. Therefore, AM1241 holds promise as a therapeutic agent for MIRI, although whether its effects are mediated by CB2 receptors remains to be further elucidated.

All experimental protocols were approved by the Animal Experimentation Ethics Committee of Wenzhou Medical University (wydw2023-0125).

The authors report there are no competing interests to declare.

This study was supported by the Wenzhou Science and Technology Bureau (No. Y20210790).

Mingxiao Zhang proposed the concept, designed the methodology. Formal analysis, data investigations and management were completed by Qingxin Tian. Jianlong Liu made critical revisions to the manuscript. All authors wrote, read and approved the final manuscript.

The datasets used and/or analyzed in the study are available from the corresponding author on reasonable request.