Levistolide A Alleviates Myocardial Ischemia–Reperfusion Injury Partly by Improving Calcium Homeostasis via the ADORA2B/cAMP/PKA/PLB/SERCA2α Signaling Axis

Abstract

This study aims to investigate the protective effect of the natural phthalide compound Levistolide A (LA) against myocardial ischemia–reperfusion injury (MIRI) and to elucidate its underlying mechanisms. Utilizing network pharmacology, potential targets of LA in the treatment of MIRI were predicted. Subsequently, a hypoxia/reoxygenation (H/R) model was established using rat H9C2 cardiomyocytes to simulate MIRI, and the mechanisms of action were validated through cellular experiments. Network pharmacology analysis indicated that the potential targets of LA in treating MIRI were significantly enriched in calcium signaling pathways, with the adenosine A2B receptor (ADORA2B), a G protein-coupled receptor (GPCR), identified as a key protein. Cellular experiments demonstrated that 24 μM LA significantly alleviated H/R-induced damage in H9C2 cells, enhanced cell viability, and reduced the release of lactate dehydrogenase (LDH), creatine kinase isoenzyme MB (CK-MB), and cardiac troponin I (cTnI). Pre-treatment with LA significantly activated the ADORA2B/Cyclic adenosine monophosphate (cAMP)/Protein kinase A (PKA) signaling axis, promoting the phosphorylation of phospholamban (PLB), enhancing the activity and protein expression of sarco/endoplasmic reticulum Ca²⁺-ATPase 2 alpha (SERCA2α), and effectively mitigating intracellular calcium overload induced by H/R. However, the ADORA2B antagonist MRS 1754 partially reverses the aforementioned protective effects of LA. The findings of this study reveal a novel mechanism by which LA exerts cardioprotective effects through the ADORA2B/cAMP/PKA/PLB/SERCA2α signaling axis, preventing calcium overload and improving calcium homeostasis, and identify potential candidate compounds and precise targets for the treatment of MIRI.

1. Introduction

Myocardial ischemia–reperfusion injury (MIRI) is a prevalent and serious complication in patients with acute myocardial infarction undergoing percutaneous coronary intervention (PCI). It is a significant contributor to heart failure, malignant arrhythmias, and even mortality, with its global health burden escalating annually [1]. Although current emergency PCI techniques effectively restore blood flow to the epicardial layer of the heart by clearing occluded arteries, clinical treatment continues to encounter numerous challenges. Some patients experience microvascular reperfusion failure post-surgery, where blood flow to the epicardial layer is restored, yet myocardial microcirculation remains inadequately perfused, which is closely associated with adverse clinical outcomes [2]. For instance, studies on patients with ST-segment elevation myocardial infarction indicate that approximately 40% of those treated with PCI exhibit a myocardial blush grade (MBG) of only 0–1, signifying low microvascular perfusion. The in-hospital mortality rate for these patients exceeds five times that of patients with MBGs 2–3, and they are more susceptible to severe complications such as recurrent ischemia and acute heart failure [3]. Current clinical treatment strategies primarily focus on preventing ischemic periods (e.g., antiplatelet agents and statins) and enhancing reperfusion techniques (e.g., thrombus aspiration and delayed PCI) [4]. However, there remains a significant lack of effective medications targeting the fundamental mechanisms of myocardial cell death. Consequently, the development of cardioprotective drugs aimed at key pathological pathways of MIRI has become essential to improve patient prognosis and alleviate healthcare burdens.

MIRI is a complex network characterized by the interplay of calcium overload, oxidative stress, mitochondrial dysfunction, inflammatory responses, and various pathways leading to cell death. Among these factors, intracellular calcium overload plays a critical role, serving as both a consequence of multiple upstream mechanisms (including ion channel abnormalities and energy metabolism disorders) and a key driving factor that triggers and amplifies oxidative damage, mitochondrial failure, inflammatory storms, and ultimately cell death [5]. Calcium overload not only induces excessive contraction and structural damage in cardiomyocytes but also exacerbates apoptotic and necrotic injury by activating calcium-dependent proteases (such as calmodulin-dependent protein kinases), inducing the opening of the mitochondrial permeability transition pore, and generating reactive oxygen species (ROS) [6,7,8,9]. This cascade of events creates a vicious cycle that severely impairs cardiac function and disease prognosis. Consequently, regulating calcium homeostasis has emerged as a crucial strategy for intervening in MIRI.

Adenosine is an endogenous protective signaling molecule that is released in response to ischemic stress and mediates its effects through activation of specific receptors, namely A₁, A₂A, A₂B, and A₃. Among these, the adenosine A₂B receptor (ADORA2B), a member of the G protein-coupled receptor family, has garnered increasing interest for its significant cardioprotective properties. Research indicates that ADORA2B expression is selectively upregulated in ischemic human cardiac tissue [10], highlighting its potential role in adaptive responses to oxygen deprivation. Activation of ADORA2B has been shown to confer protection through multiple interrelated mechanisms. For instance, it can mitigate endoplasmic reticulum stress and restore impaired autophagic flux via the cAMP/PKA signaling pathway [11]. Additionally, ADORA2B activation helps adapt myocardial metabolism, enhancing the heart’s ability to utilize carbohydrates more efficiently, which in turn reduces the extent of myocardial infarction [10]. Further investigations suggest that ADORA2B signaling may also alleviate intracellular calcium overload by modulating key calcium-handling proteins, including SERCA2α and ryanodine receptor 2 [12,13]. Collectively, these insights strengthen the rationale for targeting ADORA2B as a promising therapeutic intervention point in the setting of cardiac ischemic injury.

Levistolide A (LA) is a distinctive phthalide compound found in medicinal plants of the Apiaceae family, such as Ligusticum chuanxiong and Angelica sinensis [14,15]. It exhibits multi-target and multi-pathway pharmacological activities, indicating significant clinical potential. In terms of neuroprotection, LA enhances cognitive function in Alzheimer’s disease animal models by regulating the metabolism of β-amyloid and inhibiting Glycogen synthase kinase-3 beta-mediated hyperphosphorylation of tau protein [16]. For renal protection, LA demonstrates anti-fibrotic, antioxidant, and anti-inflammatory effects by inhibiting the renin-angiotensin system, transforming growth factor-β 1 (TGF-β1)/sma- and mad-related proteins (Smad), and toll-like receptor 4 (TLR-4)/nuclear factor kappa-B (NF-κB) signaling pathways [17]. Its anti-tumor activity is characterized by the induction of apoptosis through the ROS/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) axis and the reversal of drug resistance via the downregulation of multidrug resistance proteins [18,19]. Molecular docking and cell membrane chromatography techniques confirm that LA can specifically bind to and inhibit fibroblast growth factor receptor 4 [20]. Furthermore, LA exhibits inhibitory effects on Porcine Epidemic Diarrhea Virus, with mechanisms linked to the induction of ROS and endoplasmic reticulum stress [21]. Pharmacokinetic studies indicate that while the oral bioavailability of LA is low, traditional compound formulations can significantly enhance its absorption [22]. In summary, LA is a candidate compound with various pharmacological effects, including neuroprotection, renal protection, anti-tumor, and antiviral properties. However, its role in cardiac protection remains underexplored. This study first investigates the potential mechanisms of Levistolide A in MIRI using network pharmacology. Subsequently, a rat myocardial cell hypoxia/reoxygenation (H/R) injury model is employed to simulate MIRI, further elucidating the cardiac protective effects of LA and identifying potential molecular targets and novel candidate drugs for the pharmacological treatment of MIRI.

2. Materials and Methods

2.1. Reagents

The main reagents used in the experiment are as follows: Levistolide A (HY-N1472), ADORA2B receptor antagonist MRS 1754 (HY-14121), and thapsigargin (HY-13433) were purchased from MedChemExpress (Monmouth Junction, NJ, USA); Rat creatine kinase isoenzyme MB (CK-MB) ELISA kit (CB10461) and rat cardiac troponin I (cTnI) ELISA kit (CB11507) were provided by Shanghai Keaibo Biotechnology Co., Ltd. (Shanghai, China); Lactate dehydrogenase (LDH) activity assay kit (A020-2-1) and ultra-micro Ca²⁺-ATPase activity assay kit (A070-4-2) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); cAMP ELISA research kit (MM-926395O1) was purchased from Jiangsu Enzyme Immunoengineering Co., Ltd. (Yancheng, China); PKA activity assay kit (EIAPKA) was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA); CCK8 cell proliferation and cytotoxicity assay kit (HYCCK8-500T) was provided by Wuhan Huiyucheng Biotechnology Co., Ltd. (Wuhan, China); Fluo-3 AM cell-permeable calcium fluorescent probe (40703ES50) was purchased from Yeasen Biotechnology (Shanghai) Co., Ltd. (Shanghai, China); DAPI nuclear staining reagent (C1002) was purchased from Beyotime Biotechnology (Shanghai) Co., Ltd. (Shanghai, China). The antibodies used in this study are as follows: adenosine A2B receptor (ADORA2B) (37KDa) antibody (bs-5900R) was purchased from Bioss Biotechnology Co., Ltd. (Beijing, China); phospholamban (PLB) (36KDa) antibody (A01395-1) was purchased from Boster Biological Engineering Co., Ltd. (Wuhan, China); phosphorylated phospholamban-Ser16 (p-PLB-Ser16) (36KDa) antibody (AP0907) was purchased from ABclonal Biotechnology Co., Ltd. (Wuhan, China); SERCA2α (140KDa) antibody (9580T) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); and α-Tubulin (55 KDa) antibody (66031-1-Ig) was purchased from Proteintech Group, Inc. (Wuhan, China).

2.2. Network Pharmacology

Initially, the SMILES structure of Levistolide A was retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and submitted to the SwissTargetPrediction platform (http://swisstargetprediction.ch/) for target prediction. Concurrently, a systematic search was performed using the keyword “Levistolide A” in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, https://www.tcmsp-e.com/tcmsp.php (accessed on 2 December 2024)) as well as the ChEMBL database (https://www.ebi.ac.uk/chembl/ (accessed on 4 December 2024)). The predicted target data obtained from the three databases were then consolidated, with duplicates removed, ultimately leading to the identification of potential action targets for Levistolide A. For MIRI, we utilized the search term “Myocardial ischemic reperfusion injury” to obtain disease-related target information from three authoritative databases: GeneCards (https://www.genecards.org/), OMIM (https://www.omim.org/), and TTD (https://db.idrblab.net/ttd/).

Using the online tool Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html (accessed on 15 December 2024)), we conducted an intersection analysis between the potential action targets of Levistolide A and the disease targets associated with Myocardial Ischemia–Reperfusion Injury (MIRI). This analysis enabled us to identify the potential key targets for treating MIRI with Levistolide A. Using the STRING database (https://cn.string-db.org/), a protein–protein interaction network was constructed by setting the species to “Homo sapiens” with a confidence threshold greater than 0.4 (medium confidence) and excluding isolated nodes. Subsequently, the selected key targets underwent Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, utilizing the DAVID database (https://davidbioinformatics.nih.gov/). Finally, the results of the enrichment analysis were visualized through the SRplot web server (http://www.bioinformatics.com.cn/SRplot (accessed on 22 December 2024)).

2.3. Molecular Docking

The three-dimensional crystal structure of the target protein was retrieved from the Protein Data Bank (PDB) database (https://www.rcsb.org/). The protein structure was preprocessed using PyMOL 3.1.1 software, which involved the removal of water molecules and existing ligands. Subsequently, AutoDock Tools 1.5.7 was utilized to add hydrogen atoms to the protein structure and to calculate the charge parameters. The two-dimensional chemical structure of Levistolide A was acquired from the PubChem database, followed by energy minimization optimization using Chem3D version 22.0.0 (64-bit), which converted the structure into the pdbqt format required for molecular docking. Finally, molecular docking experiments were conducted using AutoDock Vina (version 1.2.0) to determine the binding energy values between the compound and the target protein. The results of the molecular docking were visualized in three dimensions using PyMOL 3.1.1 software.

2.4. Establishment of a Cell Model for MIRI

To simulate MIRI, this study utilized a hypoxia/reoxygenation (H/R) method to treat rat H9C2 myocardial cell lines, thereby constructing an in vitro MIRI cell model. The specific steps are as follows: Rat H9C2 myocardial cells in the logarithmic growth phase were inoculated into DMEM high-glucose medium supplemented with 10% fetal bovine serum for routine culture under conditions of 37 °C and 5% CO₂ in a saturated humidity environment. Once the cell confluence reached approximately 80%, hypoxia/reoxygenation treatment was initiated. Initially, the original culture medium was discarded and replaced with a pre-saturated medium composed of a gas mixture of 95% N₂ and 5% CO₂. Subsequently, the cells were placed in a tri-gas incubator containing a gas mixture of 94% N₂, 1% O₂, and 5% CO₂ for continuous hypoxia treatment lasting 6 h. After the hypoxia treatment, the cells were transferred back to a conventional CO₂ incubator for reoxygenation for 12 h [23].

2.5. Drug Concentration Screening

2.5.1. CCK8 Assay

H9C2 cells in optimal growth conditions were seeded at a density of 5 × 10³ cells per well in a 96-well plate, establishing both a normal control group and a drug treatment group. The cells were incubated overnight in a 37 °C incubator with 5% CO₂. The drug treatment group received varying concentrations of Levistolide A (1.5, 3, 6, 12, 24, 48, and 96 μM) for a duration of 24 h. After treatment, 10 μL of CCK-8 detection reagent was added to each well, and incubation was continued for an additional hour. Finally, the absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) to assess the drug’s effect on the cells.

2.5.2. LDH Release Assay

H9C2 cells were categorized into three groups: a normal control group (Control), a MIRI cell model group (Model), and several pre-treatment groups with varying concentrations of Levistolide A. The drug treatment group was pre-treated with Levistolide A at concentrations of 1.5, 3, 6, 12, and 24 μM for 6 h, followed by hypoxia-reoxygenation treatment. Subsequently, the cell culture supernatants from each group were collected, and LDH activity in the supernatants was measured according to the instructions provided in the LDH assay kit. This assay aims to evaluate the extent of myocardial cell damage to determine the protective effect of Levistolide A.

2.6. Experimental Grouping and Drug Intervention

The cell experiments were categorized into five distinct groups: the Normal Control Group (Control), the Hypoxia-Reoxygenation Treatment Group (H/R), the group treated with 24 μM Levistolide A followed by hypoxia-reoxygenation (LA + H/R), the group receiving 1 μM MRS 1754 prior to the 24 μM Levistolide A treatment (MRS 1754 + LA + H/R), and the group with 1 μM MRS 1754 administered only before hypoxia-reoxygenation (MRS 1754 + H/R). All groups, with the exception of the Normal Control Group, underwent hypoxia-reoxygenation treatment. Regarding drug intervention, 24 μM Levistolide A was administered as a 6 h pre-treatment prior to hypoxia, while 1 μM MRS 1754 was added 45 min before the Levistolide A treatment to evaluate its effect on the drug’s efficacy.

2.7. ELISA Method

To assess the extent of damage to cardiomyocytes, supernatants from cell cultures of each group were collected, and the activities or concentrations of LDH, CK-MB, and cTnI in these supernatants were measured using the Enzyme-Linked Immunosorbent Assay (ELISA) method. The procedure must adhere strictly to the technical specifications outlined in the corresponding reagent kits. To evaluate intracellular cAMP content and PKA activity, cells from each group were harvested and subjected to multiple freeze–thaw cycles and ultrasonic disruption to lyse the cells and release their intracellular components. Following this, the cell suspension was centrifuged at 2500 rpm for 20 min at 4 °C, and the supernatant was collected for subsequent analysis. All subsequent operations were performed in strict accordance with the technical specifications of the relevant reagent kits.

2.8. Determination of Calcium Ion (Ca²⁺) Concentration

The intracellular free Ca²⁺ concentration was measured using the fluorescent probe Fluo-3 AM. Cells from each group were collected and washed with PBS buffer, followed by treatment with a working solution containing 5 μM Fluo-3 AM. The cells were then incubated in the dark at 37 °C for 30 min to facilitate adequate probe entry into the cells. After incubation, the cells were washed again with PBS to thoroughly remove unbound probes, thereby minimizing background interference. Subsequently, DAPI staining was performed for nuclear visualization. The cells were observed, and fluorescent images were captured using a fluorescence microscope (Nikon, Nikon Fi3, Tokyo, Japan). The collected immunofluorescence images were analyzed using Image-Pro Plus 6.0 software, with primary measurement parameters including Integrated Optical Density (IOD) and Area. The Mean Density was calculated using the formula: Mean Density = IOD/Area, to reflect the relative changes in intracellular Ca²⁺ concentration. For each experimental group, more than 50 cells from at least three independent biological replicates were quantified to ensure statistical reliability.

2.9. Measurement of SERCA2α Activity

Cells from each group were collected and lysed using RIPA lysis buffer supplemented with protease inhibitors. Following centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatant was collected. The total protein concentration of each sample was determined using a BCA protein assay kit, and all samples were subsequently diluted to a uniform protein concentration with lysis buffer to ensure consistent detection conditions. SERCA2α activity was measured using a commercial Ca²⁺-ATPase activity assay kit [24], adhering strictly to the kit’s instructions. To specifically assess SERCA2α activity, a control well containing the SERCA-specific inhibitor thapsigargin (1 μM) was established. The SERCA activity value was derived by calculating the difference between the total Ca²⁺-ATPase activity and the activity measured in the inhibitor control well. Thapsigargin, a potent and selective inhibitor of SERCA, exerts minimal effects on other calcium pumps, such as plasma membrane calcium ATPase (PMCA). By implementing this specific inhibitor control, interference from other calcium pumps (e.g., PMCA) can be effectively eliminated, thereby accurately reflecting the activity level of SERCA [25,26]. Given that SERCA2α is the predominant subtype expressed in cardiomyocytes, the final calculation formula for SERCA2α activity is: SERCA2α activity (U/mg prot) = (activity value of the measurement well − activity value of the inhibitor control well)/protein concentration (mg prot).

2.10. Western Blot

Total proteins were extracted from each experimental cell group and quantified using the BCA method. A sample containing 30 μg of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following electrophoresis, the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with TBST buffer containing 5% non-fat dry milk at room temperature for 2 h. Subsequently, the following diluted primary antibodies were added: ADORA2B (1:1000), PLB (1:1000), p-PLB Ser16 (1:1000), SERCA2α (1:1000), and α-Tubulin (1:5000) as a loading control, and incubated overnight at 4 °C. The following day, the membrane was thoroughly washed with TBST buffer to eliminate unbound primary antibodies, after which it was incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. Finally, enhanced chemiluminescence (ECL) reagents were employed for detection, and Image-Pro Plus 6.0 image analysis software was utilized for the quantitative analysis of the gray values of the protein bands to evaluate the expression levels of each protein.

2.11. Statistical Analysis

All experiments were independently replicated a minimum of three times. The data are presented as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism software (Version 10.1.2). Inter-group comparisons were analyzed using one-way analysis of variance (ANOVA), followed by post hoc Tukey’s test for normally distributed data, which was confirmed by the Shapiro–Wilk normality test and the Brown-Forsythe variance homogeneity test. The statistical significance level for this study was established at p < 0.05.

3. Results

3.1. Network Pharmacology Analysis Indicates That LA Confers Protection Against MIRI Through Modulation of Calcium Signaling Pathways

Based on predictions retrieved from the TCMSP, SwissTargetPrediction, and ChEMBL databases, 125 potential target genes of LA were identified. Meanwhile, 1441 disease-related targets associated with MIRI were collected from the GeneCards, OMIM, and TTD databases. Venn diagram analysis (Figure 1A) revealed 48 overlapping targets, which were initially proposed as potential key targets for LA intervention in MIRI. Subsequent construction of a protein–protein interaction network demonstrated that 44 of these intersecting targets exhibited mutual interactions (Figure 1B). GO enrichment analysis showed that the targets linked to LA treatment of MIRI were significantly enriched in multiple biological processes. At the biological process (BP) level, these targets were primarily involved in G protein-coupled receptor signaling, inflammatory response, and calcium ion-mediated signal transduction; at the cellular component (CC) level, they were mainly localized to the plasma membrane, extracellular region, and cell surface; at the molecular function (MF) level, they were chiefly associated with protein binding, ATP binding, and identical protein binding (Figure 1C). KEGG pathway enrichment analysis further indicated that these targets were significantly enriched in neuroactive ligand-receptor interaction pathways, NOD-like receptor signaling pathways, and calcium signaling pathways (Figure 1D). Together, these results imply that LA may protect against MIRI by coordinately regulating key pathological processes such as inflammatory response, calcium signal transduction, and ion channel activity.

To validate interactions between LA and the potential targets, molecular docking simulations were conducted between LA and each of the 48 intersecting targets. The docking outcomes were sorted by binding energy from lowest to highest, with the top 10 targets possessing the most favorable (lowest) binding energies presented in

Table 1. Molecular docking binding energies (Top 10) between LA and potential targets.

Ingredients	Targets	Binding Energy (kcal/mol)
Levistolide A	BCHE	−10
Levistolide A	RORA	−9.9
Levistolide A	IDH1	−9.7
Levistolide A	TSPO	−9.3
Levistolide A	PPP1CA	−9.1
Levistolide A	IDO1	−9
Levistolide A	ADORA2B	−8.8
Levistolide A	CACNA1C	−8.7
Levistolide A	PTAFR	−8.5
Levistolide A	KDR	−8.4
Levistolide A	BCHE	−10

. Further inspection indicated that most of these high-affinity targets were enriched in calcium-related signaling pathways, consistent with the GO and KEGG findings, and supporting the notion that calcium signaling may play a pivotal role in LA-mediated alleviation of MIRI. Among these, ADORA2B (binding energy: −8.8 kcal/mol) emerged as a key protein within this pathway (Figure 2A). Figure 2B illustrates the molecular docking pose between LA and the ADORA2B protein. These data offer a foundation for subsequent target verification and mechanistic exploration. Among the top 10 potential targets, we selected ADORA2B for further validation based on the following considerations: (1) ADORA2B is a G protein-coupled receptor (GPCR) with rapid signal transduction, making it a classic target category for pharmacological intervention; (2) Existing literature indicates that ADORA2B expression is abnormal in ischemic cardiac tissue, suggesting its potentially significant role in the myocardial ischemic stress response; (3) Molecular docking studies show that LA has a high binding affinity for ADORA2B, and its known Stimulatory G protein (Gs)/cAMP signaling pathways are closely related to calcium homeostasis regulation.

3.2. Screening the Optimal Concentration of LA for Attenuating H9C2 Cell Hypoxia/Reoxygenation Injury

To evaluate the potential cytotoxicity of LA, cell viability was assessed using the CCK-8 assay following treatment with varying concentrations of LA (Figure 3A). Compared with the Control group, no significant change in viability was observed at LA concentrations of 1.5, 3, 6, 12, or 24 μM, indicating that LA does not exhibit significant cytotoxicity within this range. In contrast, treatment with 48 μM and 96 μM LA resulted in a marked decrease in cell viability, demonstrating pronounced cytotoxic effects at these higher concentrations.

To investigate the protective effect of LA against H/R injury, LDH activity in the culture supernatant was measured (Figure 3B). The Model group displayed a significant increase in LDH release compared with the Control, confirming substantial cellular damage following H/R insult. Treatment with increasing concentrations of LA notably attenuated LDH release in a dose-dependent manner. The most pronounced reduction was observed at 24 μM LA, which effectively suppressed LDH activity without compromising cell viability. Based on these results, 24 μM LA was selected as the optimal concentration for subsequent experiments, as it provided maximal protection against H/R-induced injury while maintaining a favorable safety profile.

3.3. LA Enhances the Viability of H9C2 Cells Subjected to Hypoxia/Reoxygenation

To evaluate the influence of LA on the survival of H9C2 cardiomyocytes exposed to H/R injury, cell activity was measured using the CCK-8 assay across the experimental groups. As presented in Figure 4, cell viability in the H/R group was significantly reduced compared with the Control group, confirming successful establishment of the H/R-injury model. Pretreatment with LA prior to H/R (LA + H/R group) markedly improved cell viability relative to the H/R group, demonstrating that LA effectively counteracts the H/R-induced decline in cell survival. To explore the molecular basis of this protection, LA was combined with the adenosine A2B receptor antagonist MRS 1754. The results revealed that cell viability in the MRS 1754 + LA + H/R group was significantly lower than in the LA + H/R group; however, no statistically significant differences were observed among the MRS 1754 + LA + H/R, MRS 1754 + H/R, and H/R groups. In summary, LA significantly enhances cardiomyocyte viability under H/R stress, and the adenosine A2B receptor antagonist MRS 1754 attenuates this protective effect, suggesting that LA-mediated cytoprotection likely depends on activation of the adenosine A2B receptor.

3.4. LA Protects H9C2 Cells from Hypoxia/Reoxygenation Injury

To assess the cardioprotective effect of LA against H/R injury, the release of three established myocardial injury markers (LDH, CK-MB, and cTnI) was measured in cell-culture supernatants. As illustrated in Figure 5, compared with the Control group, the activities/concentrations of LDH, CK-MB, and cTnI were significantly elevated in the H/R group, indicating successful induction of substantial injury in H9C2 cells. Relative to the H/R group, all three marker levels were significantly reduced in the LA + H/R group, confirming that LA exerts a clear protective effect against H/R-induced cardiomyocyte damage. No significant differences were detected between the H/R and MRS 1754 + H/R groups for any of the three markers. In contrast, co-treatment with MRS 1754 (MRS 1754 + LA + H/R group) significantly increased LDH, CK-MB, and cTnI levels compared with the LA + H/R group. These findings indicate that LA effectively attenuates H/R-induced myocardial injury, and that the adenosine A2B receptor antagonist MRS 1754 reverses this protection, implying that LA’s beneficial action is likely mediated through activation of the adenosine A2B receptor.

3.5. LA Attenuates Hypoxia/Reoxygenation-Induced Calcium Overload in H9C2 Cells

To examine the effect of LA on intracellular calcium homeostasis after H/R injury, free calcium ions (Ca²⁺) were labeled with Fluo-3 AM fluorescent probes. As shown in Figure 6A, the Control group exhibited a weak fluorescence signal, whereas H/R treatment provoked a marked increase in calcium-associated fluorescence, indicating severe intracellular calcium overload. Pretreatment with LA substantially reversed this fluorescence enhancement. When MRS 1754 was co-administered (MRS 1754 + LA + H/R group), the ability of LA to suppress fluorescence was partially blocked. Quantitative analysis (Figure 6B) corroborated the imaging results: compared with the Control group, H/R significantly elevated the mean intracellular Ca²⁺ fluorescence intensity. The LA + H/R group showed a significant reduction in fluorescence relative to the H/R group. Co-treatment with MRS 1754 significantly attenuated LA’s effect, raising the fluorescence intensity again compared with the LA + H/R group. These data demonstrate that LA effectively alleviates H/R-induced calcium overload in H9C2 cells, an effect that appears to depend on adenosine A2B receptor activation.

3.6. LA Improves SERCA2α Activity Following Hypoxia/Reoxygenation Injury in H9C2 Cells

To elucidate the mechanism by which LA mitigates myocardial calcium overload, the activity of SERCA2α was measured. SERCA2α plays a critical role in intracellular calcium homeostasis by actively pumping cytoplasmic Ca²⁺ back into the sarcoplasmic/endoplasmic reticulum. As displayed in Figure 7, H/R injury significantly suppressed SERCA2α activity in H9C2 cells compared with the Control group. Pretreatment with LA before H/R markedly reversed this decline, suggesting that LA’s capacity to reduce calcium overload may involve preservation of SERCA2α function. To determine whether this effect is mediated via the adenosine A2B receptor, MRS 1754 was co-applied with LA. SERCA2α activity in the MRS 1754 + LA + H/R group was significantly lower than in the LA + H/R group, whereas MRS 1754 alone did not produce a significant effect. These results imply that H/R injury inhibits SERCA2α activity, and that LA can partially improve SERCA2α function through adenosine A2B receptor activation, which may be crucial for its protection against calcium overload.

3.7. LA Protects H9C2 Cells from Hypoxia/Reoxygenation Injury by Upregulating cAMP and PKA Levels

To preliminarily investigate the signaling pathways involved in LA’s protection against H/R injury, intracellular cAMP levels and PKA activity were measured using ELISA. The results are presented in Figure 8. Regarding cAMP levels (Figure 8A), the H/R model group showed a significant decrease compared with the Control group. LA pretreatment significantly elevated cAMP levels relative to H/R alone. Co-administration of MRS 1754 with LA blocked this increase, with cAMP levels returning to values not significantly different from those in the H/R group. A similar trend was observed for PKA activity (Figure 8B): H/R significantly reduced PKA activity, LA pretreatment partly reversed this reduction, and co-treatment with MRS 1754 inhibited the LA-induced increase. Neither MRS 1754 alone nor LA alone significantly altered cAMP or PKA levels compared with the Control. These findings suggest that H/R injury suppresses the cAMP/PKA signaling pathway in H9C2 cells and that LA likely activates this pathway via the adenosine A2B receptor, contributing to its cytoprotective effects.

3.8. LA Promotes Calcium Homeostasis by Upregulating Key Proteins in the Calcium Signaling Pathway

To further delineate the molecular mechanisms underlying LA’s alleviation of H/R-induced calcium overload, the expression levels of ADORA2B, p-PLB, total PLB, and SERCA2α proteins were examined by Western blotting. As shown in Figure 9, H/R significantly downregulated ADORA2B protein expression compared with the Control group (Figure 9B). LA pretreatment (LA + H/R) significantly increased ADORA2B expression relative to H/R alone, an effect that was prevented by co-treatment with the antagonist MRS 1754 (MRS 1754 + LA + H/R). This suggests that the protective effect of LA may be related to its antagonism of H/R-induced downregulation of ADORA2B expression. Regarding PLB phosphorylation (Figure 9C), the ratio of p-PLB to total PLB was significantly reduced after H/R, indicating decreased phosphorylation and consequently enhanced inhibitory action on SERCA2α. LA pretreatment restored this phosphorylation ratio significantly, and the addition of MRS 1754 blocked LA’s effect. For SERCA2α expression (Figure 9D), H/R injury caused a significant decrease in its protein level. LA preconditioning prevented this decrease, whereas co-treatment with MRS 1754 diminished the protective effect. Collectively, these results indicate that H/R suppresses ADORA2B expression, reduces PLB phosphorylation, and downregulates SERCA2α protein. LA activates the adenosine A2B receptor, upregulates its expression, promotes PLB phosphorylation to relieve its inhibition of SERCA2α, and maintains SERCA2α expression—core mechanisms that contribute to improving calcium transport and protecting cardiomyocytes from H/R injury.

4. Discussion

This study demonstrates for the first time in an in vitro H/R model that the natural product Levistolide A regulates the downstream cAMP/PKA/PLB signaling cascade by activating the adenosine A2B receptor (ADORA2B), thereby improving calcium ion homeostasis in cardiomyocytes and protecting cardiomyocytes from hypoxia/reoxygenation injury. Based on network pharmacology analysis, it is suggested that LA may treat MIRI by regulating calcium signaling pathways. Molecular docking simulations further reveal that LA exhibits low binding energy with the key protein ADORA2B in the calcium signaling pathway, preliminarily indicating the potential of ADORA2B as a target for LA’s action. In an in vitro H/R injury model constructed using H9C2 rat cardiomyocytes, LA pretreatment significantly enhanced cell survival following H/R and reduced the release of myocardial injury markers. Mechanistic investigations demonstrated that LA treatment upregulated the protein expression level of ADORA2B and reversed the abnormal increase in intracellular calcium ion concentration ([Ca²⁺]i) induced by H/R. However, the protective effects of LA were significantly diminished when the ADORA2B receptor antagonist MRS 1754 was administered. The results of this antagonistic replenishment experiment are highly consistent with the previous molecular docking predictions, which suggest a high-affinity interaction between LA and ADORA2B, thereby strongly confirming ADORA2B as a core molecular target for LA’s cardioprotective effects.

This study elucidates the specific signaling pathways through which LA regulates downstream calcium homeostasis, building upon a clear identification of upstream targets. ELISA results demonstrate that LA treatment increases intracellular cAMP levels and PKA activity in hypoxic/reoxygenated cardiomyocytes. Furthermore, Western Blot analysis reveals that LA significantly enhances the protein expression levels of phosphorylated PLB (p-PLB Ser16) and SERCA2α. Additionally, LA treatment markedly increases the enzymatic activity of SERCA2α. The precise regulation of calcium homeostasis in cardiomyocytes relies on the synergistic action of several key proteins. SERCA2α serves as the core pump protein responsible for reuptaking cytosolic Ca²⁺ back into the sarcoplasmic reticulum (SR), and its activity directly influences cardiac diastolic function and the calcium storage capacity of the SR [27,28]. PLB acts as an endogenous negative regulator of SERCA2α, binding to SERCA2α in its non-phosphorylated state and inhibiting its activity [29]. Protein kinase A (PKA) catalyzes the phosphorylation of serine 16 on PLB (p-PLB Ser16), thereby alleviating the inhibition of PLB on SERCA2α and enhancing the efficiency of calcium ion reuptake, which is a crucial mechanism for improving cardiac function [30]. The activity of PKA itself is modulated by the levels of its upstream second messenger cAMP [31]. ADORA2B, a member of the G protein-coupled receptor family, promotes the generation of cAMP in cardiomyocytes by activating the Gs protein-adenylate cyclase pathway [32,33]. H/R injury leads to a decrease in PLB phosphorylation levels and inhibits SERCA2α activity, resulting in impaired calcium clearance. This study demonstrates that LA restores the inhibitory phosphorylation modification of PLB through the ADORA2B/cAMP/PKA signaling axis, effectively “removing the brakes” and significantly enhancing the pump activity of SERCA2α. This represents a key molecular mechanism by which LA improves calcium homeostasis in hypoxic/reoxygenated myocardial cells and alleviates myocardial injury (Figure 10).

Another noteworthy finding is that LA treatment can upregulate the protein expression level of SERCA2α. This effect may occur through regulation at either the transcriptional or translational level, thereby enhancing the cellular calcium handling capacity by increasing the quantity of “pump proteins”. This could represent an additional mechanism through which LA exerts its cardioprotective effects.

Currently, research on the role of LA in the prevention and treatment of cardiovascular diseases is relatively limited. This study explicitly anchors the protective effect of LA to the myocardial ADORA2B receptor, marking an important advancement. The protective role of the adenosine system, particularly the A1 and A3 receptors, has been widely recognized in cardiac ischemic preconditioning and postconditioning [34,35]. In contrast, the role of ADORA2B exhibits a context-dependent complexity. On one hand, some studies suggest that ADORA2B may promote inflammatory responses; for example, its activation is associated with pro-inflammatory and pro-fibrotic processes in pulmonary diseases [36]. Under hypoxic conditions, ADORA2B can modulate macrophage function via hypoxia-inducible factor 1-alpha (HIF-1α), promoting the production of fibrotic mediators such as interleukin-6 [37]. Adenosine promotes autoimmune phenotypes in mice through the ADORA2B receptor, emphasizing the role of the ATPase Inhibitory Factor 1/ATP synthase axis in tissue immune responses [38]. On the other hand, there is also evidence supporting the protective role of ADORA2B under hypoxic conditions: for instance, in models of renal or hepatic ischemia–reperfusion injury, activation of ADORA2B can alleviate tissue damage and inflammatory responses [39,40]. The APAF1-interacting protein has been shown to stabilize ADORA2B and is crucial for the prevention of myocardial infarction [41]. The seemingly paradoxical dual role of ADORA2B can be primarily attributed to its context-dependent functionality. First, differences in pathological backgrounds play a significant role; its transient activation under acute stress, such as ischemia, often mediates adaptive protection, whereas persistent activation in chronic inflammation may drive pathological processes. Second, the specificity of cell types contributes to this duality, as its expression in cardiomyocytes, immune cells, and other cell types varies, along with the downstream signaling networks, leading to distinctly different biological effects. The results of this study support the notion that, under pharmacological intervention, ADORA2B can serve as an active cardioprotective target, thereby providing a new theoretical basis for the application of LA.

This study presents several limitations that warrant attention in future research. Firstly, the current conclusions are primarily derived from network pharmacology analysis and the H/R model utilizing rat H9C2 cells. Although this model is widely used for preliminary investigations into the mechanisms of MIRI and the protective effects of drugs, there are certain differences between H9C2 cells and mature cardiac myocytes in vivo. Subsequent validations in primary cardiomyocytes, more complex three-dimensional culture systems, and in vivo ischemia/reperfusion (I/R) models in animals are necessary to confirm the physiological and pathophysiological relevance of these findings. Secondly, previous pharmacokinetic studies indicate that the oral bioavailability of LA is relatively low; however, traditional compound formulations, such as Danggui Shaoyao San, can significantly enhance its absorption rate. This suggests that future efforts should prioritize the structural optimization of this compound or the design of novel drug delivery strategies to improve its oral bioavailability. Finally, while this study primarily focuses on validating the ADORA2B-PLB signaling axis, it is important to note that protein kinase A (PKA) may also phosphorylate other downstream targets, such as cardiac troponin I and potassium channels. At the same time, ADORA2B may also couple with other second messenger pathways, such as the ERK/MAPK signaling pathway. The involvement of these targets in the protective effects of LA remains unclear and warrants further investigation.

In summary, this study confirms that LA promotes the phosphorylation of PLB by activating the ADORA2B/cAMP/PKA signaling pathway, thereby enhancing SERCA2α activity and improving calcium homeostasis in cardiomyocytes following H/R, thus protecting cardiomyocytes. This work not only provides new mechanistic insights into LA as a potential cardioprotective agent but also enriches therapeutic strategies targeting the ADORA2B/cAMP/PKA/PLB axis for the intervention of MIRI.