Enhanced cardioprotective effects of resveratrol and liraglutide against isoproterenol-induced myocardial injury: role of oxidative stress, inflammation, and mitochondrial function

Enhanced cardioprotective effects of resveratrol and liraglutide against isoproterenol-induced myocardial injury: role of oxidative stress, inflammation, and mitochondrial function | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhanced cardioprotective effects of resveratrol and liraglutide against isoproterenol-induced myocardial injury: role of oxidative stress, inflammation, and mitochondrial function Ahmed El-Sayed Nour El-Deen, Reda Taha, Shaimaa Fawzy Abdellatif Esmail, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9302185/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background: Myocardial infarction (MI) remains a leading cause of global mortality and is strongly associated with oxidative stress, mitochondrial dysfunction, and inflammatory responses that contribute to irreversible cardiac injury. Glucagon-like peptide-1 receptor agonists (GLP-1 RAs), such as liraglutide, have demonstrated cardioprotective effects beyond glycemic control, while resveratrol, a natural polyphenol, exhibits potent antioxidant and mitochondrial-protective properties. However, the combined effects of these agents on mitochondrial function in myocardial injury have not been fully explored. Objective: This study aimed to evaluate the combined cardioprotective effects of resveratrol and a GLP-1 receptor agonist (liraglutide) against isoproterenol (ISO)-induced myocardial injury in rats, with particular emphasis on oxidative stress and mitochondrial dysfunction. Methods: Seventy adult male albino rats were randomly allocated into five groups: Control, ISO (100 mg/kg, s.c., for two consecutive days), Resveratrol + ISO (20 mg/kg/day, orally for 28 days), GLP-1 RA + ISO (liraglutide 0.2 mg/kg/day, s.c., for 10 days), and Resveratrol + GLP-1 RA + ISO. ISO was administered on days 27 and 28. Cardiac injury was assessed using serum biomarkers (cTnI, CK-MB, LDH), oxidative stress markers (MDA, SOD, GSH), inflammatory cytokines (TNF-α, IL-6), and mitochondrial function parameters, including mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) generation. Histopathological evaluation of cardiac tissue was also performed. Results: ISO administration significantly increased cardiac injury markers, lipid peroxidation, and pro-inflammatory cytokines, while reducing antioxidant defenses and impairing mitochondrial function (p < 0.001 vs. control). Pretreatment with either resveratrol or liraglutide partially attenuated these changes. The combined treatment resulted in significantly greater improvement compared to either monotherapy (p < 0.01), including reductions in MDA (~ 45%) and inflammatory cytokines, along with restoration of antioxidant capacity and mitochondrial function. Two-way ANOVA revealed significant interaction effects between treatments (p < 0.05). Histopathological findings supported the biochemical results, showing marked preservation of myocardial architecture in the combination group. Conclusion: The combined administration of resveratrol and liraglutide provides enhanced cardioprotection against ISO-induced myocardial injury, likely through complementary effects on oxidative stress, inflammation, and mitochondrial function. While interaction analysis suggests a greater-than-additive effect, further studies using dedicated synergy models and molecular validation are required. This combination may represent a promising therapeutic strategy for myocardial injury. Resveratrol GLP-1 receptor agonist liraglutide myocardial injury oxidative stress cardio protection and Mitochondrial dysfunction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Myocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide, driven by an imbalance between myocardial oxygen supply and demand. The ensuing hypoxia triggers oxidative stress, mitochondrial dysfunction, and inflammatory cascades that culminate in irreversible cardiomyocyte death and cardiac remodeling [ 1 , 2 ]. Despite advances in reperfusion therapies, the global burden of MI continues to rise, highlighting the urgent need for cardioprotective strategies that target cellular oxidative and mitochondrial pathways. Oxidative stress and mitochondrial dysfunction are central to myocardial injury. Excessive reactive oxygen species (ROS) generation disrupts mitochondrial membrane potential, damages lipids and proteins, and activates NF-κB-driven inflammation (TNF-α, IL-6) [ 3 , 4 ]. In the isoproterenol (ISO)-induced injury model, mitochondrial calcium mishandling and impaired oxidative metabolism directly underlie cardiac dysfunction [ 5 , 6 ]. Therefore, preserving mitochondrial integrity while enhancing antioxidant defenses represents a promising therapeutic approach. Glucagon-like peptide-1 receptor agonists (GLP-1 RAs)—developed for type 2 diabetes—exhibit potent cardioprotective effects independent of glycemia [ 7 ]. Agents such as liraglutide and semaglutide activate PI3K/Akt/eNOS and Nrf2 pathways, reduce apoptosis, and improve endothelial function [ 8 , 9 ]. Large cardiovascular outcome trials (e.g., LEADER, SUSTAIN-6, REWIND) have consistently shown that GLP-1 RAs reduce major adverse cardiovascular events, including nonfatal MI [ 10 , 11 ]. Preclinical studies show that liraglutide enhances antioxidant enzymes (SOD, GPx) and limits oxidative stress [ 12 ]. However, combining GLP-1 receptor agonists with complementary mitochondrial-targeted agents could further potentiate their efficacy. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural polyphenol with well-established antioxidant, anti-inflammatory, and mitochondrial-protective properties. It activates SIRT1/PGC-1α, upregulates SOD, catalase, and GPx, and preserves mitochondrial membrane potential [ 13 , 14 ]. Resveratrol pretreatment reduces infarct size, lowers lipid peroxidation, and attenuates ISO-induced myocardial injury [ 15 , 16 ]. Importantly, resveratrol enhances AMPK activation and mitochondrial respiratory function—mechanisms that may complement GLP-1 RA-mediated signaling [ 17 ]. Given these complementary mechanisms, combining GLP-1 receptor agonists with mitochondrial-targeted antioxidants such as resveratrol represents a rational and potentially synergistic therapeutic strategy. To our knowledge, no previous study has investigated the combined effects of resveratrol and a GLP-1 receptor agonist on mitochondrial function and oxidative stress in ISO-induced myocardial injury. Accordingly, this study evaluated cardiac injury biomarkers, oxidative stress parameters, inflammatory mediators, mitochondrial function, and histopathological alterations to test the hypothesis that the combination of resveratrol and liraglutide confers synergistic cardioprotection—rather than merely additive effects—against ISO-induced myocardial injury in albino rats. 2. Materials and Methods 2.1. Experimental Animals and Ethical Approval Seventy adult male albino rats (200 ± 20 g) were obtained from the animal facility of the Faculty of Medicine, Al-Azhar University. Animals were housed under standard laboratory conditions (22 ± 2°C; 12 h light/dark cycle) with free access to a standard pellet diet and water ad libitum. All efforts were made to minimize animal suffering and to reduce the number of animals used. All experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 2011) [ 19 ]. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Al-Azhar University, Egypt (Approval No. RP/NA/PHY/05/12/2024). All efforts were made to minimize animal suffering and to reduce the number of animals used. The study was conducted in compliance with ARRIVE guidelines for reporting animal research. Clinical trial number: not applicable 2.2. Experimental Design After a one-week acclimatization period, animals were randomly assigned into five groups (n = 14 per group) using a computer-generated randomization schedule. Sample size was determined based on a priori power analysis to detect a 30% difference in primary outcomes with 80% power at a significance level of α = 0.05, consistent with similar preclinical studies [ 15 , 16 ]. The experimental groups were as follows: Control group : Received oral vehicle (0.5% carboxymethyl cellulose, CMC) for 28 days and subcutaneous saline during the last 10 days. ISO group : Received oral vehicle for 28 days, followed by isoproterenol (ISO; 100 mg/kg, s.c.) on days 27 and 28 to induce myocardial injury [ 20 ]. Resveratrol + ISO group : Received resveratrol (20 mg/kg/day, orally, suspended in 0.5% CMC) for 28 days, followed by ISO on days 27 and 28 [ 15 ]. GLP-1 RA + ISO group : Received liraglutide (0.2 mg/kg/day, s.c.) during days 18–27 and oral vehicle for 28 days, followed by ISO on days 27 and 28 [ 12 ]. Combination group : Received resveratrol (20 mg/kg/day, orally) for 28 days and liraglutide (0.2 mg/kg/day, s.c.) during days 18–27, followed by ISO on days 27 and 28. Timeline summary : Event Days Resveratrol/vehicle administration 1–28 Liraglutide/saline administration 18–27 ISO administration 27–28 Sample collection 29 On day 29, animals were deeply anesthetized using ketamine (50 mg/kg) and xylazine (5 mg/kg), administered intraperitoneally. Adequate depth of anesthesia was confirmed by loss of pedal and corneal reflexes. Euthanasia was performed under deep anesthesia to ensure complete loss of consciousness and to minimize suffering, in accordance with established guidelines for humane animal sacrifice. All procedures were performed by trained personnel to ensure minimal distress to the animals. Blood samples were collected via retro-orbital puncture, and hearts were rapidly excised, washed with ice-cold saline, and processed for subsequent biochemical, mitochondrial, and histopathological analyses. All analyses were performed by investigators blinded to group allocation. 2.3. Chemicals and Reagents Isoproterenol hydrochloride (≥ 98% purity) and resveratrol (≥ 99% purity) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Liraglutide was obtained from Novo Nordisk A/S (Bagsværd, Denmark). Resveratrol was freshly suspended in 0.5% CMC prior to administration. All other reagents were of analytical grade. 2.4. Biochemical Analysis 2.4.1. Cardiac Biomarkers Serum was separated by centrifugation (3000 rpm, 15 min, 4°C). Cardiac troponin I (cTnI), lactate dehydrogenase (LDH), and creatine kinase-MB (CK-MB) were measured using commercially available kits (Bio-Diagnostic, Egypt) according to the manufacturer’s instructions [ 21 ]. 2.4.2. Oxidative Stress Markers Cardiac tissue homogenates (10% w/v in phosphate buffer, pH 7.4) were prepared. Protein concentration was determined using the Bradford method [ 22 ]. Lipid peroxidation was assessed by measuring malondialdehyde (MDA) using the TBARS assay [ 23 ]. Antioxidant parameters including superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) were measured using standard methods [ 24 – 26 ]. 2.4.3. Inflammatory Cytokines Serum TNF-α and IL-6 levels were quantified using ELISA kits (MyBioSource, USA) according to the manufacturer’s instructions. 2.4.4. Mitochondrial Function Mitochondria were isolated from fresh cardiac tissue using differential centrifugation at 4°C [ 27 ]. Mitochondrial membrane potential (ΔΨm) was assessed using rhodamine 123 fluorescence [ 28 ], while mitochondrial ROS production was measured using DCFH-DA [ 29 ]. All measurements were performed in triplicate. 2.5. Histopathological Examination Cardiac tissues were fixed in 10% neutral buffered formalin, processed routinely, and embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin (H&E) [ 30 ]. Histological evaluation was performed by a blinded pathologist using a light microscope (Olympus BX51, Japan). 2.6. Statistical Analysis Statistical analysis was performed using SPSS software (version XX, IBM Corp., Armonk, NY, USA). Normality of data distribution was assessed using the Shapiro–Wilk test, and homogeneity of variance was verified using Levene’s test. Data are expressed as mean ± standard error of the mean (SEM). Intergroup comparisons were performed using two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple comparisons. A p-value < 0.05 was considered statistically significant. 3. Results Data are presented as mean ± SEM (n = 14 per group). 3.1. Resveratrol and GLP-1 Receptor Agonist Attenuated ISO-Induced Elevation of Serum Cardiac Biomarkers ISO administration caused a significant elevation in serum cardiac injury markers compared to the control group. As shown in Fig. 1 , serum levels of cardiac troponin I (cTnI), lactate dehydrogenase (LDH), and creatine kinase-MB (CK-MB) were significantly increased in the ISO group (p < 0.001 versus control). Pretreatment with either resveratrol or liraglutide alone significantly reduced these biomarkers compared to the ISO group (p < 0.01). Notably, the combination of resveratrol and liraglutide outperformed both monotherapies (p < 0.01), reducing cTnI levels by approximately 52%, LDH by 48%, and CK-MB by 45% compared to the ISO group. Two-way ANOVA revealed a significant interaction effect between resveratrol and liraglutide (p < 0.05), supporting a true synergistic interaction. 3.2. Resveratrol and GLP-1 Receptor Agonist Ameliorated ISO-Induced Oxidative Stress in Cardiac Tissue The effects of resveratrol and liraglutide on oxidative stress markers are presented in Fig. 2 . ISO injection resulted in a marked increase in malondialdehyde (MDA) levels (p < 0.001 versus control), indicating enhanced lipid peroxidation. Concomitantly, ISO significantly reduced the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), as well as reduced glutathione (GSH) levels (p < 0.001 versus control). Pretreatment with resveratrol or liraglutide alone partially restored these parameters. However, the combination exhibited superior efficacy (p < 0.01 versus either monotherapy), reducing MDA levels by approximately 45% and increasing SOD and GSH activities by nearly 2-fold compared to the ISO group. Two-way ANOVA also demonstrated a significant interaction effect (p < 0.05), supporting a synergistic effect. These findings suggest that the combination effectively restores redox balance in cardiac tissue. 3.3. Resveratrol and GLP-1 Receptor Agonist Suppressed ISO-Induced Inflammatory Response As shown in Fig. 3 , ISO administration significantly elevated serum levels of pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) compared to the control group (p < 0.001). Treatment with resveratrol or liraglutide alone significantly reduced TNF-α and IL-6 levels (p < 0.01 versus ISO). The combination therapy resulted in a markedly enhanced effect, reducing TNF-α by approximately 50% and IL-6 by 47% compared to the ISO group (p < 0.01 versus either monotherapy). Two-way ANOVA confirmed a significant interaction (p < 0.05), consistent with a synergistic cardioprotective interaction. 3.4. Resveratrol and GLP-1 Receptor Agonist Preserved Mitochondrial Function Mitochondrial dysfunction parameters are presented in Fig. 4 . ISO administration significantly reduced mitochondrial membrane potential (ΔΨm) and increased mitochondrial reactive oxygen species (ROS) production compared to the control group (p < 0.001). Pretreatment with resveratrol or liraglutide alone partially attenuated these changes. The combination outperformed both monotherapies (p < 0.01), restoring ΔΨm by approximately 55% and reducing mitochondrial ROS production by approximately 50% compared to the ISO group. Two-way ANOVA further demonstrated a significant interaction effect (p < 0.05), supporting a synergistic mechanism. These results suggest that the combination preserves mitochondrial integrity, likely through complementary mechanisms. 3.5. Resveratrol and GLP-1 Receptor Agonist Improved ISO-Induced Histopathological Alterations Histopathological examination of H&E-stained cardiac sections is shown in Fig. 5 . The control group exhibited normal myocardial architecture with intact cardiac muscle fibers, no inflammatory infiltration, and preserved nuclear integrity (Fig. 5 A). In contrast, the ISO group showed severe myocardial damage characterized by extensive inflammatory infiltration, disruption of cardiac muscle fibers, interstitial edema, and areas of necrosis (Fig. 5 B). Pretreatment with resveratrol alone (Fig. 5 C) or liraglutide alone (Fig. 5 D) partially improved the histopathological picture, showing reduced inflammatory infiltration and better fiber alignment. Notably, the combination of resveratrol and liraglutide (Fig. 5 E) produced marked histological improvement, with restoration of near-normal myocardial architecture, minimal inflammatory infiltration, and preserved nuclear integrity. Semi-quantitative histopathological scoring (0–3 scale) further confirmed these observations. The ISO group exhibited the highest injury score (2.8 ± 0.1), whereas the combination group showed a significantly lower score (0.6 ± 0.1, p < 0.001 versus ISO), confirming the superior protective effect of the combination. 3.6. Summary of Synergistic Effects Table 1 summarizes the percentage improvements in all measured parameters for the combination group compared to either monotherapy. The combination consistently demonstrated greater efficacy, confirming a synergistic rather than additive interaction between resveratrol and GLP-1 receptor agonism. Table 1 Percentage change relative to the ISO group Parameter Resveratrol alone GLP-1 RA alone Combination cTnI 32% 35% 52% LDH 28% 31% 48% CK-MB 26% 30% 45% MDA reduction 25% 28% 45% SOD activity 1.4-fold 1.5-fold 2.0-fold GSH activity 1.3-fold 1.4-fold 1.9-fold TNF-α reduction 30% 33% 50% IL-6 reduction 28% 32% 47% ΔΨm restoration 32% 35% 55% Mitochondrial ROS reduction 28% 30% 50% *Note: All combination values were significantly different from both monotherapies (p < 0.01, two-way ANOVA).* 4. Discussion 4.1. The Isoproterenol-Induced Myocardial Injury Model Isoproterenol (ISO) is a well-established experimental tool for inducing myocardial injury that closely mimics human ischemic heart disease [ 5 , 20 ]. ISO administration triggers excessive β-adrenoceptor stimulation, leading to increased myocardial oxygen demand, calcium overload, and the generation of reactive oxygen species (ROS) [ 6 ]. These events disrupt mitochondrial membrane potential, deplete endogenous antioxidant reserves, and initiate lipid peroxidation, ultimately resulting in cardiomyocyte necrosis and the release of cardiac-specific biomarkers [ 5 ]. In the present study, ISO injection produced significant elevations in serum cTnI, LDH, and CK-MB, along with increased MDA levels and reduced SOD and GSH activities, confirming successful induction of oxidative stress-mediated myocardial injury. Histopathological examination further revealed extensive inflammatory infiltration, myofiber disruption, and interstitial edema, validating the reliability of the ISO model for evaluating cardioprotective interventions. 4.2. Resveratrol and Liraglutide Attenuated Cardiac Biomarkers and Improved Myocardial Architecture The significant reduction in serum cardiac biomarkers (cTnI, LDH, and CK-MB) observed following pretreatment with resveratrol or liraglutide alone indicates stabilization of cardiomyocyte membrane integrity and reduced cellular leakage. These findings align with previous reports demonstrating that GLP-1 receptor agonists limit infarct size and preserve cardiac function in both diabetic and non-diabetic animal models [ 9 , 12 ]. Similarly, resveratrol has been shown to reduce ISO-induced cardiac enzyme release by enhancing endogenous antioxidant defenses and inhibiting apoptotic pathways [ 15 , 16 ]. Notably, the combination of resveratrol and liraglutide demonstrated superior efficacy compared to both monotherapies, reducing cTnI by 52%, LDH by 48%, and CK-MB by 45% relative to the ISO group. These findings were corroborated by histopathological examination, where the combination group exhibited near-normal myocardial architecture with minimal inflammatory infiltration and preserved nuclear integrity. Semi-quantitative scoring further confirmed the superior protective effect of the combination (injury score: 0.6 ± 0.1 versus 2.8 ± 0.1 in the ISO group). Two-way ANOVA revealed a significant interaction effect between resveratrol and liraglutide (p < 0.05), providing statistical evidence for a greater-than-additive cardioprotective interaction. 4.3. Resveratrol and Liraglutide Ameliorated ISO-Induced Oxidative Stress Oxidative stress is a central pathogenic mechanism in ISO-induced myocardial injury. ISO metabolism generates ROS that overwhelm endogenous antioxidant defenses, leading to lipid peroxidation and membrane damage [ 3 , 4 ]. In the present study, ISO administration resulted in a marked increase in MDA levels and significant reductions in SOD and GSH activities, consistent with previous reports identifying oxidative stress as a primary mediator of ISO cardiotoxicity [ 5 , 6 ]. Pretreatment with resveratrol alone partially restored redox balance, consistent with its well-characterized free radical scavenging properties and its ability to upregulate Nrf2-mediated antioxidant gene expression [ 13 , 17 ]. Liraglutide alone also improved oxidative parameters, likely through activation of the PI3K/Akt/Nrf2 signaling pathway [ 8 , 12 ]. The combination, however, exhibited markedly enhanced efficacy, reducing MDA levels by approximately 45% and increasing SOD and GSH activities by nearly two-fold compared to the ISO group. Two-way ANOVA demonstrated a significant interaction effect (p < 0.05), supporting an amplified antioxidant response. These findings suggest that resveratrol and liraglutide target complementary pathways—resveratrol acting as a direct radical scavenger and SIRT1 activator, while liraglutide enhances Nrf2-dependent antioxidant transcription—thereby achieving greater oxidative stress attenuation than either agent alone. 4.4. Resveratrol and Liraglutide Suppressed ISO-Induced Inflammation Inflammation is a major driver of secondary myocardial damage following ischemic injury. ISO administration activates NF-κB, promoting the expression of pro-inflammatory cytokines including TNF-α and IL-6 [ 4 , 28 ]. In the present study, ISO induced a marked elevation in these cytokines, confirming activation of the inflammatory cascade. Treatment with resveratrol or liraglutide alone significantly reduced inflammatory markers, consistent with their established anti-inflammatory properties. Resveratrol inhibits NF-κB nuclear translocation [ 14 , 17 ], whereas liraglutide reduces cytokine production through AMPK activation and suppression of the NLRP3 inflammasome [ 9 , 12 ]. The combination produced a more pronounced anti-inflammatory response, reducing TNF-α by 50% and IL-6 by 47% relative to the ISO group. Two-way ANOVA confirmed a significant interaction effect (p < 0.05), indicating a greater-than-additive anti-inflammatory interaction. These biochemical findings were further supported by histopathological evidence of reduced inflammatory infiltration in the combination group. 4.5. Resveratrol and Liraglutide Preserved Mitochondrial Function Mitochondrial dysfunction is both a cause and a consequence of oxidative stress in myocardial injury. ISO-induced calcium overload and ROS generation disrupt mitochondrial membrane potential (ΔΨm), impair ATP synthesis, and trigger apoptotic signaling [ 5 , 6 ]. In this study, ISO administration significantly reduced ΔΨm and increased mitochondrial ROS production, indicating severe mitochondrial impairment. Pretreatment with resveratrol alone partially preserved ΔΨm, consistent with activation of the SIRT1/PGC-1α pathway [ 13 , 17 ]. Liraglutide also improved mitochondrial parameters, likely through PI3K/Akt-mediated survival signaling [ 8 , 12 ]. Importantly, the combination outperformed both monotherapies, restoring ΔΨm by approximately 55% and reducing mitochondrial ROS production by approximately 50% compared to the ISO group. Two-way ANOVA revealed a significant interaction effect (p < 0.05), supporting an amplified mitochondrial protective effect. These findings indicate that the two agents converge on mitochondrial preservation through complementary mechanisms: enhanced biogenesis via SIRT1/PGC-1α and improved survival signaling via PI3K/Akt. 4.6. Proposed Mechanisms of Cardioprotective Interaction Based on the present findings and existing literature, the enhanced combined effect of resveratrol and liraglutide appears to arise from convergence of complementary molecular pathways, as outlined in the integrated framework below: Resveratrol-mediated mechanisms : Direct ROS scavenging and inhibition of lipid peroxidation [ 14 , 15 ] Activation of SIRT1 and subsequent PGC-1α-mediated mitochondrial biogenesis [ 13 , 17 ] Upregulation of Nrf2-dependent antioxidant enzymes (SOD, CAT, GPx) [ 17 ] Inhibition of NF-κB signaling and inflammatory cytokine production [ 14 ] Liraglutide-mediated mechanisms : Activation of GLP-1 receptor signaling (cAMP/PKA and PI3K/Akt pathways) [ 8 , 9 ] Enhancement of Nrf2-mediated antioxidant responses [ 12 ] Suppression of the NLRP3 inflammasome and pro-inflammatory cytokines [ 12 ] Promotion of mitochondrial survival through anti-apoptotic signaling [ 9 ] Convergent mechanisms : Dual activation of Nrf2 via distinct upstream regulators Complementary suppression of NF-κB-mediated inflammation Coordinated preservation of mitochondrial function (biogenesis + survival) This integrated framework provides a mechanistic basis for the consistently superior efficacy of the combination across all measured parameters. 4.7. Comparison with Previous Studies To our knowledge, this is the first study to evaluate the combined effects of resveratrol and a GLP-1 receptor agonist on mitochondrial function and oxidative stress in ISO-induced myocardial injury. Previous studies have examined each agent independently. For example, resveratrol-based formulations have been shown to improve cardiac remodeling, while liraglutide has demonstrated cardioprotective effects through modulation of PI3K/Akt-related signaling pathways [ 15 , 16 ]. The present study extends these findings by demonstrating that combining both agents produces significantly greater cardioprotection than either monotherapy. The consistency of this effect across multiple endpoints—biochemical, inflammatory, mitochondrial, and histological—strengthens the validity of the observed interaction. 4.8. Limitations Several limitations of the present study should be acknowledged. First, although the sample size (n = 14 per group) was adequately powered for preclinical analysis, caution should be exercised when extrapolating these findings to clinical settings. Second, the duration of treatment was relatively short, and the long-term cardioprotective effects as well as the safety profile of the combination were not evaluated. Third, while the study proposes the involvement of key molecular pathways, including Nrf2, SIRT1, and PI3K/Akt, based on existing literature and the observed biochemical and mitochondrial outcomes, direct molecular validation (e.g., protein or gene expression analysis using Western blotting or quantitative PCR) was not performed. Therefore, the mechanistic interpretations remain inferential and warrant further experimental confirmation. Fourth, functional cardiac assessments, such as echocardiography or measurement of left ventricular ejection fraction (LVEF), were not conducted. Although the observed biochemical, mitochondrial, and histopathological improvements provide strong evidence of cardioprotection, they do not directly establish functional recovery. Accordingly, future studies incorporating in vivo functional evaluation are necessary to strengthen the translational relevance of these findings. Fifth, although two-way ANOVA demonstrated significant interaction effects between resveratrol and liraglutide, suggesting a greater-than-additive pharmacological interaction, formal synergy assessment using approaches such as isobolographic analysis or combination index (CI) calculation was not performed. Therefore, the current findings should be interpreted as indicative of an enhanced combined effect rather than definitive pharmacological synergy. Despite these limitations, the consistency of the observed effects across multiple independent endpoints—including cardiac biomarkers, oxidative stress parameters, inflammatory mediators, mitochondrial function, and histopathological findings—strongly supports the robustness and internal validity of the study outcomes. 4.9. Future Research Directions Future studies should focus on molecular validation of the proposed mechanisms, long-term safety and efficacy, dose optimization, and evaluation in comorbid disease models. Incorporating functional cardiac assessments and conducting translational clinical studies will be essential to confirm clinical applicability. 4.10. Clinical Relevance From a translational perspective, the observed enhanced combined effect may be particularly relevant in patients with cardiometabolic disorders, where GLP-1 receptor agonists are already in clinical use. The addition of a nutraceutical agent such as resveratrol could represent a complementary strategy to improve cardiovascular outcomes. 4.11. Conclusion In conclusion, the present study demonstrates that the combination of resveratrol and the GLP-1 receptor agonist liraglutide confers enhanced cardioprotection against isoproterenol-induced myocardial injury. The combination significantly attenuated cardiac injury biomarkers, reduced oxidative stress, suppressed inflammation, preserved mitochondrial function, and improved myocardial architecture compared to either agent alone. Two-way ANOVA confirmed significant interaction effects across multiple endpoints, supporting a greater-than-additive interaction. These findings support the potential of this combination as a therapeutic strategy targeting oxidative stress and mitochondrial dysfunction in myocardial injury. Further studies are warranted to validate these findings and explore their translational potential in human ischemic heart disease. Abbreviations ALT Alanine aminotransferase AMPK AMP-activated protein kinase ANOVA Analysis of variance AST Aspartate aminotransferase ATP Adenosine triphosphate CAT Catalase CK-MB Creatine kinase–myocardial band cTnI Cardiac troponin I DCFH-DA Dichlorofluorescein diacetate ELISA Enzyme-linked immunosorbent assay GSH Reduced glutathione GLP-1 RA Glucagon-like peptide-1 receptor agonist H&E Hematoxylin and eosin HMGB1 High mobility group box 1 IL-6 Interleukin-6 ISO Isoproterenol LDH Lactate dehydrogenase LVEF Left ventricular ejection fraction MDA Malondialdehyde MI Myocardial infarction NF-κB Nuclear factor kappa B NLRP3 NOD-like receptor family pyrin domain containing 3 Nrf2 Nuclear factor erythroid 2–related factor 2 PI3K Phosphoinositide 3-kinase PGC-1α Peroxisome proliferator-activated receptor gamma coactivator 1-alpha ROS Reactive oxygen species SEM Standard error of the mean SIRT1 Sirtuin 1 SOD Superoxide dismutase TBARS Thiobarbituric acid reactive substances TNF-α Tumor necrosis factor-alpha ΔΨm Mitochondrial membrane potentia Declarations Conflict of interest : The authors declare that they have no competing interests. Financial support: This study was funded by the Department of Basic Medical and Dental Sciences, Faculty of Dentistry, Zarqa University, Zarqa, Jordan, without any particular role in the study design, recruitment of individuals, data analysis, or writing of the report. Author's contributions: Ahmed El-Sayed Nour El-Deen: Conceptualization, study design, methodology, data acquisition, statistical analysis, data interpretation, manuscript writing, and final approval. Reda Taha: Data collection, formal analysis, data interpretation, manuscript revision, and final approval. Shaimaa Fawzy Abdellatif Esmaeil: Conceptualization, methodology, data acquisition, manuscript revision, and final approval. Mohamed Hamdy Sayed: Data collection, formal analysis, figure design, manuscript revision, and final approval. Mohamed Zaeim Hafez Ahmed: Data acquisition, investigation, manuscript revision, and final approval. Muhammad Abdelbaeth Elfiky: Data collection, validation, manuscript revision, and final approval. Ahmed A. Abd El-Rhman: Statistical analysis, figure design, data interpretation, manuscript writing, and final approval. Osama Khalil Farag: Data acquisition, investigation, manuscript revision, and final approval. Mohammed Abdel Aziz Mohammed: Data interpretation, manuscript writing, manuscript revision, and final approval. Ahmed F. Abdel Ghany: Data collection, validation, manuscript revision, and final approval. Ahmed Gad Allah: Histopathological examination, data interpretation, manuscript revision, and final approval. Ahmad Taha: Conceptualization, study design, supervision, data interpretation, manuscript writing, and final approval. 6. Ethics approval and consent to participate All experimental procedures involving animals were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 2011). The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Al-Azhar University, Egypt (Approval No. RP/NA/PHY/05/12/2024). All efforts were made to minimize animal suffering, reduce the number of animals used, and ensure humane endpoints. Animals were anesthetized using ketamine (50 mg/kg) and xylazine (5 mg/kg) administered intraperitoneally prior to sample collection, and euthanasia was performed under deep anesthesia to ensure complete loss of consciousness. Consent to participate is not applicable as this study did not involve human subjects. Clinical trial number: not applicable 7. Consent for publication Not applicable. 8. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 9. Competing interests The authors declare that they have no competing interests. 10. Funding The authors extend their appreciation to the Deanship of Scientific Research at Zarqa University for funding this work. 11. Authors’ contributions Nour El-Deen A., Osman A., and Taha A. conceived and designed the study. Osman A. and Mohamed M. performed data collection. Nour El-Deen A. and Rashed F. conducted statistical analysis and figure preparation. Nour El-Deen A., Rashed F., and Mohamed M. contributed to data interpretation. Abd El-Rahman A., Ehab A., Mohamed M., Nour El-Deen A., and Rashed F. drafted the manuscript. All authors critically revised the manuscript and approved the final version for publication. 12. Acknowledgements The authors extend their appreciation to the Deanship of Scientific Research at Zarqa University, Jordan for funding this work References Virani SS, Alonso A, Aparicio HJ, et al. Heart Disease and Stroke Statistics-2023 Update: A Report From the American Heart Association. Circulation. 2023;147(8):e93–621. Roth GA, Mensah GA, Johnson CO, et al. Global Burden of Cardiovascular Diseases and Risk Factors, 1990–2019: Update From the GBD 2019 Study. J Am Coll Cardiol. 2020;76(25):2982–3021. Dubois-Deruy E, Peugnet V, Turkieh A, Pinet F. Oxidative Stress in Cardiovascular Diseases. Antioxid (Basel). 2020;9(9):864. Morgan MJ, Liu ZG. Crosstalk of reactive oxygen species and NF-κB signaling. Cell Res. 2011;21(1):103–15. Willis BC, Salazar-Cantú A, Silva-Platas C, et al. Impaired oxidative metabolism and calcium mishandling underlie cardiac dysfunction in a rat model of post-acute isoproterenol-induced cardiomyopathy. Am J Physiol Heart Circ Physiol. 2015;308(5):H467–77. Vázquez-Martínez O, Galán-Hernández N, Hernández-Muñoz R, et al. Correlation between oxidative Stress and Alteration of Intracellular Calcium Handling in Isoproterenol-Induced Myocardial Infarction. Cardiovasc Toxicol. 2021;21(6):456–70. Haider A, Zainab R, Rafique M, et al. GLP-1 Receptor Agonists: Beyond Glycemic Control - A Multifaceted Approach to Cardiovascular Risk Reduction. J Med Res Rev. 2025;45(2):112–28. Drucker DJ. The Cardiovascular Biology of Glucagon-like Peptide-1. Cell Metab. 2016;24(1):15–30. Ussher JR, Drucker DJ. Glucagon-like peptide 1 receptor agonists: cardiovascular benefits and mechanisms. Nat Rev Cardiol. 2023;20(7):463–74. Marso SP, Daniels GH, Brown-Frandsen K, et al. Liraglutide and Cardiovascular Outcomes in Type 2 Diabetes. N Engl J Med. 2016;375(4):311–22. (LEADER trial). Marx N, Husain M, Lehrke M, Verma S, Sattar N. GLP-1 receptor agonists for the reduction of atherosclerotic cardiovascular risk in patients with type 2 diabetes. Circulation. 2022;146(24):1882–94. Alobaid WA, Elshal MM, El-Sayed WM, et al. Liraglutide Attenuates Diabetic Cardiomyopathy via the ILK/PI3K/AKT/PTEN Signaling Pathway in Rats with Type 2 Diabetes. Pharmaceuticals. 2024;17(8):1024. Rahmani S, Naraki K, Roohbakhsh A, et al. The Cardiovascular Protective Function of Natural Compounds Through AMPK/SIRT1/PGC-1α Signaling Pathway. Food Sci Nutr. 2024;12(12):9998–10009. Berman AY, Motechin RA, Wiesenfeld MY, Holz MK. The therapeutic potential of resveratrol: a review of clinical trials. NPJ Precis Oncol. 2017;1(1):35. Alhusaini AM, Alghamdi AM, Alghamdi SA, et al. Resveratrol-Based Liposomes Improve Cardiac Remodeling Induced by Isoproterenol Partially by Modulating MEF2, Cytochrome C and S100A1 Expression. Dose Response. 2024;22(2):15593258241252624. Abdel-Reheim MA, El-Sayed WM, El-Naga RN, et al. Unlocking the miRNA-34a-5p/TGF-β and HMGB1/PI3K/Akt/mTOR crosstalk participate in the enhanced cardiac protection of liraglutide against isoproterenol-induced acute myocardial injury rat model. Int Immunopharmacol. 2024;128:111529. Kim EN, Lim JH, Kim MY, et al. Resveratrol, an Nrf2 activator, ameliorates aging-related progressive renal injury. Aging. 2018;10(1):83–99. Zhao L, Li M, Wei S, et al. The crosstalk between SIRT1 and GLP-1: implications for cardiometabolic diseases. Front Endocrinol. 2023;14:1205432. National Research Council. Guide for the Care and Use of Laboratory Animals. 8th ed. Washington, DC: National Academies Press. 2011. (NIH Publication No. 85 – 23, revised 2011). Rona G, Chappel CI, Balazs T, Gaudry R. An infarct-like myocardial lesion and other toxic manifestations produced by isoproterenol in the rat. AMA Arch Pathol. 1959;67(4):443–55. Young DS. Effects of drugs on clinical laboratory tests. Ann Clin Biochem. 1997;34(6):579–81. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95(2):351–8. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974;47(3):469–74. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–6. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959;82(1):70–7. Frezza C, Cipolat S, Scorrano L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc. 2007;2(2):287–95. Emaus RK, Grunwald R, Lemasters JJ. Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties. Biochim Biophys Acta. 1986;850(3):436–48. LeBel CP, Ischiropoulos H, Bondy SC. Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol. 1992;5(2):227–31. Bancroft JD, Layton C. The hematoxylin and eosin. In: Suvarna SK, Layton C, Bancroft JD, editors. Bancroft's Theory and Practice of Histological Techniques. 8th ed. Elsevier; 2019. pp. 126–38. Guo Y, et al. Liraglutide attenuates myocardial ischemia-reperfusion injury through activation of the Nrf2/HO-1 pathway. Eur J Pharmacol. 2024;962:176231. Song J, et al. Resveratrol protects against ISO-induced cardiotoxicity via SIRT1/PGC-1α-mediated mitochondrial biogenesis. Biomed Pharmacother. 2023;165:115247. Wang X, et al. GLP-1 receptor agonists improve endothelial function in diabetic patients: a meta-analysis. Cardiovasc Diabetol. 2023;22(1):45. Chen Y, et al. Resveratrol attenuates vascular endothelial dysfunction through activation of AMPK. Vascul Pharmacol. 2022;146:107098. Liu L, et al. NLRP3 inflammasome mediates ISO-induced cardiac inflammation and fibrosis. J Mol Cell Cardiol. 2023;175:1–12. Zhang H, et al. SIRT1 activation by resveratrol prevents ISO-induced cardiac hypertrophy. Front Pharmacol. 2022;13:892345. Kim S, et al. PI3K/Akt signaling in GLP-1 receptor agonist-mediated cardioprotection. Cell Signal. 2023;101:110498. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 19 May, 2026 Reviews received at journal 13 May, 2026 Reviews received at journal 12 May, 2026 Reviewers agreed at journal 08 May, 2026 Reviewers agreed at journal 04 May, 2026 Reviewers agreed at journal 21 Apr, 2026 Reviewers invited by journal 20 Apr, 2026 Editor assigned by journal 20 Apr, 2026 Editor invited by journal 13 Apr, 2026 Submission checks completed at journal 11 Apr, 2026 First submitted to journal 11 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9302185","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626972724,"identity":"260d617c-9b8d-4b0a-9d45-08a93bb21124","order_by":0,"name":"Ahmed El-Sayed Nour El-Deen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYDCCA2DSxo5fAsyQkCFWS1qy5AwGxgagFh5itRxm3HADrIWBsBa+22cPPvi4J43Z+Hbz8Uc3aix4GNgPH92AT4vkubxkwxnPbPjM7hxLbM45BnQYT1raDXxaDM7wmEnzHEhjNruRY9icwwbUIsFjRoyWw4ybZ4C0/CNFywYJoJbcNiK0SJ7hMTaccSAtWeJGWuLs3D4JHjZCfuE7w2P44MMBYFTOSD7wOedbnRw/++FjeLVgAjbSlI+CUTAKRsEowAYArkBHp3gwZHsAAAAASUVORK5CYII=","orcid":"","institution":"Al Azhar University","correspondingAuthor":true,"prefix":"","firstName":"Ahmed","middleName":"El-Sayed Nour","lastName":"El-Deen","suffix":""},{"id":626972725,"identity":"45beb746-548b-4714-8f43-addc7f56cd73","order_by":1,"name":"Reda Taha","email":"","orcid":"","institution":"Port Said University","correspondingAuthor":false,"prefix":"","firstName":"Reda","middleName":"","lastName":"Taha","suffix":""},{"id":626972726,"identity":"59b958f4-338d-48d5-8c5a-fa5f830c796b","order_by":2,"name":"Shaimaa Fawzy Abdellatif Esmail","email":"","orcid":"","institution":"Al Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Shaimaa","middleName":"Fawzy Abdellatif","lastName":"Esmail","suffix":""},{"id":626972727,"identity":"a6eaa368-78cf-4490-b312-51391adfbda2","order_by":3,"name":"Mohamed Hamdy Sayed","email":"","orcid":"","institution":"Al Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"Hamdy","lastName":"Sayed","suffix":""},{"id":626972728,"identity":"3501c0b7-1691-4199-bb31-e31969c9aba0","order_by":4,"name":"Mohamed Zaeim Hafez Ahmed","email":"","orcid":"","institution":"Al Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"Zaeim Hafez","lastName":"Ahmed","suffix":""},{"id":626972729,"identity":"9bc614a9-b866-4522-9093-2a6943bbbfa7","order_by":5,"name":"Muhammad Abdelbaeth Elfiky","email":"","orcid":"","institution":"Al Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Abdelbaeth","lastName":"Elfiky","suffix":""},{"id":626972730,"identity":"18be6ecf-8b15-44a9-9255-2fda917d5e89","order_by":6,"name":"Ahmed A. Abd El-Rhman","email":"","orcid":"","institution":"Al Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"A. Abd","lastName":"El-Rhman","suffix":""},{"id":626972731,"identity":"84f3c494-9672-4f11-966c-4009bbb12d54","order_by":7,"name":"Osama Khalil Farag","email":"","orcid":"","institution":"Al Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Osama","middleName":"Khalil","lastName":"Farag","suffix":""},{"id":626972732,"identity":"23e5d5f6-0ffe-42eb-ad34-6839401f3567","order_by":8,"name":"Mohammed Abdel Aziz Mohammed","email":"","orcid":"","institution":"Al Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Mohammed","middleName":"Abdel Aziz","lastName":"Mohammed","suffix":""},{"id":626972734,"identity":"047ecf71-9e1c-424a-93bf-472d260fe406","order_by":9,"name":"Ahmed F. Abdel Ghany","email":"","orcid":"","institution":"Al Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"F. Abdel","lastName":"Ghany","suffix":""},{"id":626972738,"identity":"cd261023-f272-41bc-8938-e9b652bd9286","order_by":10,"name":"Ahmed Gad Allah","email":"","orcid":"","institution":"Al Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"Gad","lastName":"Allah","suffix":""},{"id":626972739,"identity":"a7da7a4a-903e-465e-b3ef-5e2dc05a71dc","order_by":11,"name":"Ahmad Taha","email":"","orcid":"","institution":"Al Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Ahmad","middleName":"","lastName":"Taha","suffix":""}],"badges":[],"createdAt":"2026-04-02 10:55:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9302185/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9302185/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108392918,"identity":"0d6a2243-d063-4bd5-bfcb-25ed52b71755","added_by":"auto","created_at":"2026-05-04 07:20:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":564608,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of resveratrol and liraglutide on serum cardiac biomarkers in ISO-induced myocardial injury.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Cardiac troponin I (cTnI), (B) lactate dehydrogenase (LDH), and (C) creatine kinase-MB (CK-MB).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroups:\u003c/strong\u003e Control; ISO (isoproterenol 100 mg/kg, s.c., days 27–28); Resveratrol + ISO (20 mg/kg/day, orally, 28 days); GLP-1 RA + ISO (liraglutide 0.2 mg/kg/day, s.c., days 18–27); and combination group.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM (n = 14).\u003c/p\u003e\n\u003cp\u003e*p \u0026lt; 0.001 vs. Control;\u003cbr\u003e\n#p \u0026lt; 0.01 vs. ISO;\u003cbr\u003e\n$p \u0026lt; 0.01 vs. Resveratrol + ISO and GLP-1 RA + ISO.\u003c/p\u003e\n\u003cp\u003eISO significantly increased all cardiac biomarkers. Resveratrol or liraglutide partially reduced these elevations, whereas the combination produced a significantly greater reduction. Two-way ANOVA revealed a significant interaction effect (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9302185/v1/c46ab4c5e5823759607e3402.png"},{"id":108803797,"identity":"fd26cea3-a5d0-4f7f-919c-007fb84eb974","added_by":"auto","created_at":"2026-05-08 15:07:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":415179,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of resveratrol and liraglutide on oxidative stress markers in cardiac tissue.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Superoxide dismutase (SOD) activity, (B) reduced glutathione (GSH) level, and (C) malondialdehyde (MDA) level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroups:\u003c/strong\u003e Control; ISO; Resveratrol + ISO; GLP-1 RA + ISO; and combination group (as described in Figure 1).\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM (n = 14).\u003c/p\u003e\n\u003cp\u003e*p \u0026lt; 0.001 vs. Control;\u003cbr\u003e\n#p \u0026lt; 0.01 vs. ISO;\u003cbr\u003e\n$p \u0026lt; 0.01 vs. Resveratrol + ISO and GLP-1 RA + ISO.\u003c/p\u003e\n\u003cp\u003eISO significantly reduced SOD and GSH levels and increased MDA levels. Resveratrol or liraglutide alone significantly attenuated these changes, whereas the combination demonstrated greater improvement. Two-way ANOVA demonstrated a significant interaction effect (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9302185/v1/d8469791cab78413ce869523.png"},{"id":108492559,"identity":"4ac20702-df21-47fd-a260-10ba12e39834","added_by":"auto","created_at":"2026-05-05 09:58:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":328561,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of resveratrol and liraglutide on serum inflammatory cytokines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Tumor necrosis factor-alpha (TNF-α) and (B) interleukin-6 (IL-6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroups:\u003c/strong\u003e Control; ISO; Resveratrol + ISO; GLP-1 RA + ISO; and combination group (as described in Figure 1).\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM (n = 14).\u003c/p\u003e\n\u003cp\u003e*p \u0026lt; 0.001 vs. Control;\u003cbr\u003e\n#p \u0026lt; 0.01 vs. ISO;\u003cbr\u003e\n$p \u0026lt; 0.01 vs. Resveratrol + ISO and GLP-1 RA + ISO.\u003c/p\u003e\n\u003cp\u003eISO significantly elevated TNF-α and IL-6 levels. Resveratrol or liraglutide alone significantly reduced these cytokines, whereas the combination produced a greater reduction. Two-way ANOVA confirmed a significant interaction effect (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9302185/v1/5c840090f53a90c61eda586f.png"},{"id":108392922,"identity":"1e398f16-34a9-4ae7-9c37-ffddf1eb9306","added_by":"auto","created_at":"2026-05-04 07:20:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":308812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of resveratrol and liraglutide on mitochondrial function parameters.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Mitochondrial membrane potential (ΔΨm) and (B) mitochondrial reactive oxygen species (ROS) production.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroups:\u003c/strong\u003e Control; ISO; Resveratrol + ISO; GLP-1 RA + ISO; and combination group (as described in Figure 1).\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM (n = 14).\u003c/p\u003e\n\u003cp\u003e*p \u0026lt; 0.001 vs. Control;\u003cbr\u003e\n#p \u0026lt; 0.01 vs. ISO;\u003cbr\u003e\n$p \u0026lt; 0.01 vs. Resveratrol + ISO and GLP-1 RA + ISO.\u003c/p\u003e\n\u003cp\u003eISO significantly reduced ΔΨm and increased mitochondrial ROS production. Resveratrol or liraglutide alone significantly attenuated these changes, whereas the combination demonstrated greater preservation. Two-way ANOVA indicated a significant interaction effect (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9302185/v1/05504565c8abc90518d8d85a.png"},{"id":108392921,"identity":"df7d4cbe-d201-4522-8605-2bda6f2e24af","added_by":"auto","created_at":"2026-05-04 07:20:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":946726,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological examination of cardiac tissue (H\u0026amp;E staining, ×400 magnification).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Control group: normal myocardial architecture with intact cardiac muscle fibers and no inflammatory infiltration.\u003cbr\u003e\n(B) ISO group: extensive inflammatory infiltration (arrow), myofiber disruption, interstitial edema (arrowhead), and necrosis.\u003cbr\u003e\n(C) Resveratrol + ISO group: partial improvement with reduced inflammatory infiltration.\u003cbr\u003e\n(D) GLP-1 RA + ISO group: partial improvement with improved fiber alignment.\u003cbr\u003e\n(E) Combination group: near-normal myocardial architecture with minimal inflammatory infiltration and preserved nuclear integrity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroups:\u003c/strong\u003e Control; ISO; Resveratrol + ISO; GLP-1 RA + ISO; and combination group (as described in Figure 1).\u003c/p\u003e\n\u003cp\u003eSemi-quantitative histopathological scoring (0–3 scale): ISO group (2.8 ± 0.1) and combination group (0.6 ± 0.1, p \u0026lt; 0.001 vs. ISO).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\"Histopathological assessment was performed by a pathologist blinded to group allocation.\"\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9302185/v1/0bfa7d78af919d2242049262.png"},{"id":108809036,"identity":"31d05932-8733-43dd-8663-414963ba65cf","added_by":"auto","created_at":"2026-05-08 15:48:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2902669,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9302185/v1/80b9b8c4-e40b-4950-9cb3-56495656815a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced cardioprotective effects of resveratrol and liraglutide against isoproterenol-induced myocardial injury: role of oxidative stress, inflammation, and mitochondrial function","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMyocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide, driven by an imbalance between myocardial oxygen supply and demand. The ensuing hypoxia triggers oxidative stress, mitochondrial dysfunction, and inflammatory cascades that culminate in irreversible cardiomyocyte death and cardiac remodeling [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite advances in reperfusion therapies, the global burden of MI continues to rise, highlighting the urgent need for cardioprotective strategies that target cellular oxidative and mitochondrial pathways.\u003c/p\u003e \u003cp\u003eOxidative stress and mitochondrial dysfunction are central to myocardial injury. Excessive reactive oxygen species (ROS) generation disrupts mitochondrial membrane potential, damages lipids and proteins, and activates NF-κB-driven inflammation (TNF-α, IL-6) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In the isoproterenol (ISO)-induced injury model, mitochondrial calcium mishandling and impaired oxidative metabolism directly underlie cardiac dysfunction [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, preserving mitochondrial integrity while enhancing antioxidant defenses represents a promising therapeutic approach.\u003c/p\u003e \u003cp\u003eGlucagon-like peptide-1 receptor agonists (GLP-1 RAs)\u0026mdash;developed for type 2 diabetes\u0026mdash;exhibit potent cardioprotective effects independent of glycemia [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Agents such as liraglutide and semaglutide activate PI3K/Akt/eNOS and Nrf2 pathways, reduce apoptosis, and improve endothelial function [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Large cardiovascular outcome trials (e.g., LEADER, SUSTAIN-6, REWIND) have consistently shown that GLP-1 RAs reduce major adverse cardiovascular events, including nonfatal MI [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Preclinical studies show that liraglutide enhances antioxidant enzymes (SOD, GPx) and limits oxidative stress [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, combining GLP-1 receptor agonists with complementary mitochondrial-targeted agents could further potentiate their efficacy.\u003c/p\u003e \u003cp\u003eResveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural polyphenol with well-established antioxidant, anti-inflammatory, and mitochondrial-protective properties. It activates SIRT1/PGC-1α, upregulates SOD, catalase, and GPx, and preserves mitochondrial membrane potential [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Resveratrol pretreatment reduces infarct size, lowers lipid peroxidation, and attenuates ISO-induced myocardial injury [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Importantly, resveratrol enhances AMPK activation and mitochondrial respiratory function\u0026mdash;mechanisms that may complement GLP-1 RA-mediated signaling [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven these complementary mechanisms, combining GLP-1 receptor agonists with mitochondrial-targeted antioxidants such as resveratrol represents a rational and potentially synergistic therapeutic strategy. To our knowledge, no previous study has investigated the combined effects of resveratrol and a GLP-1 receptor agonist on mitochondrial function and oxidative stress in ISO-induced myocardial injury.\u003c/p\u003e \u003cp\u003eAccordingly, this study evaluated cardiac injury biomarkers, oxidative stress parameters, inflammatory mediators, mitochondrial function, and histopathological alterations to test the hypothesis that the combination of resveratrol and liraglutide confers synergistic cardioprotection\u0026mdash;rather than merely additive effects\u0026mdash;against ISO-induced myocardial injury in albino rats.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Experimental Animals and Ethical Approval\u003c/h2\u003e \u003cp\u003eSeventy adult male albino rats (200\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) were obtained from the animal facility of the Faculty of Medicine, Al-Azhar University. Animals were housed under standard laboratory conditions (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C; 12 h light/dark cycle) with free access to a standard pellet diet and water ad libitum. All efforts were made to minimize animal suffering and to reduce the number of animals used.\u003c/p\u003e \u003cp\u003e All experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85\u0026ndash;23, revised 2011) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Al-Azhar University, Egypt (Approval No. RP/NA/PHY/05/12/2024).\u003c/p\u003e \u003cp\u003eAll efforts were made to minimize animal suffering and to reduce the number of animals used.\u003c/p\u003e \u003cp\u003e The study was conducted in compliance with ARRIVE guidelines for reporting animal research.\u003c/p\u003e \u003cp\u003eClinical trial number: not applicable\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental Design\u003c/h2\u003e \u003cp\u003eAfter a one-week acclimatization period, animals were randomly assigned into five groups (n\u0026thinsp;=\u0026thinsp;14 per group) using a computer-generated randomization schedule. Sample size was determined based on a priori power analysis to detect a 30% difference in primary outcomes with 80% power at a significance level of α\u0026thinsp;=\u0026thinsp;0.05, consistent with similar preclinical studies [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe experimental groups were as follows:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eControl group\u003c/b\u003e: Received oral vehicle (0.5% carboxymethyl cellulose, CMC) for 28 days and subcutaneous saline during the last 10 days.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eISO group\u003c/b\u003e: Received oral vehicle for 28 days, followed by isoproterenol (ISO; 100 mg/kg, s.c.) on days 27 and 28 to induce myocardial injury [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eResveratrol\u0026thinsp;+\u0026thinsp;ISO group\u003c/b\u003e: Received resveratrol (20 mg/kg/day, orally, suspended in 0.5% CMC) for 28 days, followed by ISO on days 27 and 28 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGLP-1 RA\u0026thinsp;+\u0026thinsp;ISO group\u003c/b\u003e: Received liraglutide (0.2 mg/kg/day, s.c.) during days 18\u0026ndash;27 and oral vehicle for 28 days, followed by ISO on days 27 and 28 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eCombination group\u003c/b\u003e: Received resveratrol (20 mg/kg/day, orally) for 28 days and liraglutide (0.2 mg/kg/day, s.c.) during days 18\u0026ndash;27, followed by ISO on days 27 and 28.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTimeline summary\u003c/b\u003e:\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEvent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDays\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResveratrol/vehicle administration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiraglutide/saline administration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u0026ndash;27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eISO administration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27\u0026ndash;28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample collection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOn day 29, animals were deeply anesthetized using ketamine (50 mg/kg) and xylazine (5 mg/kg), administered intraperitoneally. Adequate depth of anesthesia was confirmed by loss of pedal and corneal reflexes. Euthanasia was performed under deep anesthesia to ensure complete loss of consciousness and to minimize suffering, in accordance with established guidelines for humane animal sacrifice. All procedures were performed by trained personnel to ensure minimal distress to the animals.\u003c/p\u003e \u003cp\u003eBlood samples were collected via retro-orbital puncture, and hearts were rapidly excised, washed with ice-cold saline, and processed for subsequent biochemical, mitochondrial, and histopathological analyses.\u003c/p\u003e \u003cp\u003eAll analyses were performed by investigators blinded to group allocation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Chemicals and Reagents\u003c/h2\u003e \u003cp\u003eIsoproterenol hydrochloride (\u0026ge;\u0026thinsp;98% purity) and resveratrol (\u0026ge;\u0026thinsp;99% purity) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Liraglutide was obtained from Novo Nordisk A/S (Bagsv\u0026aelig;rd, Denmark). Resveratrol was freshly suspended in 0.5% CMC prior to administration. All other reagents were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Biochemical Analysis\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Cardiac Biomarkers\u003c/h2\u003e \u003cp\u003eSerum was separated by centrifugation (3000 rpm, 15 min, 4\u0026deg;C). Cardiac troponin I (cTnI), lactate dehydrogenase (LDH), and creatine kinase-MB (CK-MB) were measured using commercially available kits (Bio-Diagnostic, Egypt) according to the manufacturer\u0026rsquo;s instructions [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Oxidative Stress Markers\u003c/h2\u003e \u003cp\u003eCardiac tissue homogenates (10% w/v in phosphate buffer, pH 7.4) were prepared. Protein concentration was determined using the Bradford method [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Lipid peroxidation was assessed by measuring malondialdehyde (MDA) using the TBARS assay [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Antioxidant parameters including superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) were measured using standard methods [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3. Inflammatory Cytokines\u003c/h2\u003e \u003cp\u003eSerum TNF-α and IL-6 levels were quantified using ELISA kits (MyBioSource, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4. Mitochondrial Function\u003c/h2\u003e \u003cp\u003eMitochondria were isolated from fresh cardiac tissue using differential centrifugation at 4\u0026deg;C [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Mitochondrial membrane potential (ΔΨm) was assessed using rhodamine 123 fluorescence [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], while mitochondrial ROS production was measured using DCFH-DA [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. All measurements were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Histopathological Examination\u003c/h2\u003e \u003cp\u003eCardiac tissues were fixed in 10% neutral buffered formalin, processed routinely, and embedded in paraffin. Sections (5 \u0026micro;m) were stained with hematoxylin and eosin (H\u0026amp;E) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Histological evaluation was performed by a blinded pathologist using a light microscope (Olympus BX51, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using SPSS software (version XX, IBM Corp., Armonk, NY, USA).\u003c/p\u003e \u003cp\u003eNormality of data distribution was assessed using the Shapiro\u0026ndash;Wilk test, and homogeneity of variance was verified using Levene\u0026rsquo;s test.\u003c/p\u003e \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Intergroup comparisons were performed using two-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s post hoc test for multiple comparisons.\u003c/p\u003e \u003cp\u003eA p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;14 per group).\u003c/b\u003e \u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Resveratrol and GLP-1 Receptor Agonist Attenuated ISO-Induced Elevation of Serum Cardiac Biomarkers\u003c/h2\u003e \u003cp\u003eISO administration caused a significant elevation in serum cardiac injury markers compared to the control group. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, serum levels of cardiac troponin I (cTnI), lactate dehydrogenase (LDH), and creatine kinase-MB (CK-MB) were significantly increased in the ISO group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 versus control).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePretreatment with either resveratrol or liraglutide alone significantly reduced these biomarkers compared to the ISO group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Notably, the combination of resveratrol and liraglutide outperformed both monotherapies (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), reducing cTnI levels by approximately 52%, LDH by 48%, and CK-MB by 45% compared to the ISO group. Two-way ANOVA revealed a significant interaction effect between resveratrol and \u003cb\u003eliraglutide\u003c/b\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), supporting a true synergistic interaction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Resveratrol and GLP-1 Receptor Agonist Ameliorated ISO-Induced Oxidative Stress in Cardiac Tissue\u003c/h2\u003e \u003cp\u003eThe effects of resveratrol and liraglutide on oxidative stress markers are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. ISO injection resulted in a marked increase in malondialdehyde (MDA) levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 versus control), indicating enhanced lipid peroxidation. Concomitantly, ISO significantly reduced the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), as well as reduced glutathione (GSH) levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 versus control).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePretreatment with resveratrol or liraglutide alone partially restored these parameters. However, the combination exhibited superior efficacy (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 versus either monotherapy), reducing MDA levels by approximately 45% and increasing SOD and GSH activities by nearly 2-fold compared to the ISO group. Two-way ANOVA also demonstrated a significant interaction effect (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), supporting a synergistic effect. These findings suggest that the combination effectively restores redox balance in cardiac tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Resveratrol and GLP-1 Receptor Agonist Suppressed ISO-Induced Inflammatory Response\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, ISO administration significantly elevated serum levels of pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Treatment with resveratrol or liraglutide alone significantly reduced TNF-α and IL-6 levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 versus ISO). The combination therapy resulted in a markedly enhanced effect, reducing TNF-α by approximately 50% and IL-6 by 47% compared to the ISO group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 versus either monotherapy). Two-way ANOVA confirmed a significant interaction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), consistent with a synergistic cardioprotective interaction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Resveratrol and GLP-1 Receptor Agonist Preserved Mitochondrial Function\u003c/h2\u003e \u003cp\u003eMitochondrial dysfunction parameters are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. ISO administration significantly reduced mitochondrial membrane potential (ΔΨm) and increased mitochondrial reactive oxygen species (ROS) production compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Pretreatment with resveratrol or liraglutide alone partially attenuated these changes. The combination outperformed both monotherapies (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), restoring ΔΨm by approximately 55% and reducing mitochondrial ROS production by approximately 50% compared to the ISO group. Two-way ANOVA further demonstrated a significant interaction effect (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), supporting a synergistic mechanism. These results suggest that the combination preserves mitochondrial integrity, likely through complementary mechanisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Resveratrol and GLP-1 Receptor Agonist Improved ISO-Induced Histopathological Alterations\u003c/h2\u003e \u003cp\u003eHistopathological examination of H\u0026amp;E-stained cardiac sections is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The control group exhibited normal myocardial architecture with intact cardiac muscle fibers, no inflammatory infiltration, and preserved nuclear integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In contrast, the ISO group showed severe myocardial damage characterized by extensive inflammatory infiltration, disruption of cardiac muscle fibers, interstitial edema, and areas of necrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePretreatment with resveratrol alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) or liraglutide alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) partially improved the histopathological picture, showing reduced inflammatory infiltration and better fiber alignment. Notably, the combination of resveratrol and liraglutide (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) produced marked histological improvement, with restoration of near-normal myocardial architecture, minimal inflammatory infiltration, and preserved nuclear integrity.\u003c/p\u003e \u003cp\u003eSemi-quantitative histopathological scoring (0\u0026ndash;3 scale) further confirmed these observations. The ISO group exhibited the highest injury score (2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1), whereas the combination group showed a significantly lower score (0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 versus ISO), confirming the superior protective effect of the combination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Summary of Synergistic Effects\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the percentage improvements in all measured parameters for the combination group compared to either monotherapy. The combination consistently demonstrated greater efficacy, confirming a synergistic rather than additive interaction between resveratrol and GLP-1 receptor agonism.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePercentage change relative to the ISO group\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResveratrol alone\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGLP-1 RA alone\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCombination\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecTnI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e52%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e48%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCK-MB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e45%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDA reduction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e45%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOD activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.4-fold\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5-fold\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e2.0-fold\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGSH activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.3-fold\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.4-fold\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e1.9-fold\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNF-α reduction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e50%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-6 reduction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e47%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔΨm restoration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e55%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMitochondrial ROS reduction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e50%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e*Note: All combination values were significantly different from both monotherapies (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, two-way ANOVA).*\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.1. The Isoproterenol-Induced Myocardial Injury Model\u003c/h2\u003e \u003cp\u003eIsoproterenol (ISO) is a well-established experimental tool for inducing myocardial injury that closely mimics human ischemic heart disease [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. ISO administration triggers excessive β-adrenoceptor stimulation, leading to increased myocardial oxygen demand, calcium overload, and the generation of reactive oxygen species (ROS) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These events disrupt mitochondrial membrane potential, deplete endogenous antioxidant reserves, and initiate lipid peroxidation, ultimately resulting in cardiomyocyte necrosis and the release of cardiac-specific biomarkers [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, ISO injection produced significant elevations in serum cTnI, LDH, and CK-MB, along with increased MDA levels and reduced SOD and GSH activities, confirming successful induction of oxidative stress-mediated myocardial injury. Histopathological examination further revealed extensive inflammatory infiltration, myofiber disruption, and interstitial edema, validating the reliability of the ISO model for evaluating cardioprotective interventions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Resveratrol and Liraglutide Attenuated Cardiac Biomarkers and Improved Myocardial Architecture\u003c/h2\u003e \u003cp\u003eThe significant reduction in serum cardiac biomarkers (cTnI, LDH, and CK-MB) observed following pretreatment with resveratrol or \u003cb\u003eliraglutide\u003c/b\u003e alone indicates stabilization of cardiomyocyte membrane integrity and reduced cellular leakage. These findings align with previous reports demonstrating that GLP-1 receptor agonists limit infarct size and preserve cardiac function in both diabetic and non-diabetic animal models [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Similarly, resveratrol has been shown to reduce ISO-induced cardiac enzyme release by enhancing endogenous antioxidant defenses and inhibiting apoptotic pathways [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, the combination of resveratrol and liraglutide demonstrated superior efficacy compared to both monotherapies, reducing cTnI by 52%, LDH by 48%, and CK-MB by 45% relative to the ISO group. These findings were corroborated by histopathological examination, where the combination group exhibited near-normal myocardial architecture with minimal inflammatory infiltration and preserved nuclear integrity. Semi-quantitative scoring further confirmed the superior protective effect of the combination (injury score: 0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 versus 2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 in the ISO group). Two-way ANOVA revealed a significant interaction effect between resveratrol and liraglutide (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), providing statistical evidence for a greater-than-additive cardioprotective interaction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Resveratrol and Liraglutide Ameliorated ISO-Induced Oxidative Stress\u003c/h2\u003e \u003cp\u003eOxidative stress is a central pathogenic mechanism in ISO-induced myocardial injury. ISO metabolism generates ROS that overwhelm endogenous antioxidant defenses, leading to lipid peroxidation and membrane damage [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In the present study, ISO administration resulted in a marked increase in MDA levels and significant reductions in SOD and GSH activities, consistent with previous reports identifying oxidative stress as a primary mediator of ISO cardiotoxicity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePretreatment with resveratrol alone partially restored redox balance, consistent with its well-characterized free radical scavenging properties and its ability to upregulate Nrf2-mediated antioxidant gene expression [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Liraglutide alone also improved oxidative parameters, likely through activation of the PI3K/Akt/Nrf2 signaling pathway [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe combination, however, exhibited markedly enhanced efficacy, reducing MDA levels by approximately 45% and increasing SOD and GSH activities by nearly two-fold compared to the ISO group. Two-way ANOVA demonstrated a significant interaction effect (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), supporting an amplified antioxidant response. These findings suggest that resveratrol and liraglutide target complementary pathways\u0026mdash;resveratrol acting as a direct radical scavenger and SIRT1 activator, while liraglutide enhances Nrf2-dependent antioxidant transcription\u0026mdash;thereby achieving greater oxidative stress attenuation than either agent alone.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Resveratrol and Liraglutide Suppressed ISO-Induced Inflammation\u003c/h2\u003e \u003cp\u003eInflammation is a major driver of secondary myocardial damage following ischemic injury. ISO administration activates NF-κB, promoting the expression of pro-inflammatory cytokines including TNF-α and IL-6 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In the present study, ISO induced a marked elevation in these cytokines, confirming activation of the inflammatory cascade.\u003c/p\u003e \u003cp\u003eTreatment with resveratrol or liraglutide alone significantly reduced inflammatory markers, consistent with their established anti-inflammatory properties. Resveratrol inhibits NF-κB nuclear translocation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], whereas liraglutide reduces cytokine production through AMPK activation and suppression of the NLRP3 inflammasome [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe combination produced a more pronounced anti-inflammatory response, reducing TNF-α by 50% and IL-6 by 47% relative to the ISO group. Two-way ANOVA confirmed a significant interaction effect (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating a greater-than-additive anti-inflammatory interaction. These biochemical findings were further supported by histopathological evidence of reduced inflammatory infiltration in the combination group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Resveratrol and Liraglutide Preserved Mitochondrial Function\u003c/h2\u003e \u003cp\u003eMitochondrial dysfunction is both a cause and a consequence of oxidative stress in myocardial injury. ISO-induced calcium overload and ROS generation disrupt mitochondrial membrane potential (ΔΨm), impair ATP synthesis, and trigger apoptotic signaling [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In this study, ISO administration significantly reduced ΔΨm and increased mitochondrial ROS production, indicating severe mitochondrial impairment.\u003c/p\u003e \u003cp\u003ePretreatment with resveratrol alone partially preserved ΔΨm, consistent with activation of the SIRT1/PGC-1α pathway [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Liraglutide also improved mitochondrial parameters, likely through PI3K/Akt-mediated survival signaling [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImportantly, the combination outperformed both monotherapies, restoring ΔΨm by approximately 55% and reducing mitochondrial ROS production by approximately 50% compared to the ISO group. Two-way ANOVA revealed a significant interaction effect (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), supporting an amplified mitochondrial protective effect. These findings indicate that the two agents converge on mitochondrial preservation through complementary mechanisms: enhanced biogenesis via SIRT1/PGC-1α and improved survival signaling via PI3K/Akt.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e4.6. Proposed Mechanisms of Cardioprotective Interaction\u003c/h2\u003e \u003cp\u003eBased on the present findings and existing literature, the enhanced combined effect of resveratrol and liraglutide appears to arise from convergence of complementary molecular pathways, as outlined in the integrated framework below:\u003c/p\u003e \u003cp\u003e \u003cb\u003eResveratrol-mediated mechanisms\u003c/b\u003e:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eDirect ROS scavenging and inhibition of lipid peroxidation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eActivation of SIRT1 and subsequent PGC-1α-mediated mitochondrial biogenesis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eUpregulation of Nrf2-dependent antioxidant enzymes (SOD, CAT, GPx) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eInhibition of NF-κB signaling and inflammatory cytokine production [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLiraglutide-mediated mechanisms\u003c/b\u003e:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eActivation of GLP-1 receptor signaling (cAMP/PKA and PI3K/Akt pathways) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eEnhancement of Nrf2-mediated antioxidant responses [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eSuppression of the NLRP3 inflammasome and pro-inflammatory cytokines [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003ePromotion of mitochondrial survival through anti-apoptotic signaling [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eConvergent mechanisms\u003c/b\u003e:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eDual activation of Nrf2 via distinct upstream regulators\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eComplementary suppression of NF-κB-mediated inflammation\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eCoordinated preservation of mitochondrial function (biogenesis\u0026thinsp;+\u0026thinsp;survival)\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThis integrated framework provides a mechanistic basis for the consistently superior efficacy of the combination across all measured parameters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e4.7. Comparison with Previous Studies\u003c/h2\u003e \u003cp\u003eTo our knowledge, this is the first study to evaluate the combined effects of resveratrol and a GLP-1 receptor agonist on mitochondrial function and oxidative stress in ISO-induced myocardial injury. Previous studies have examined each agent independently. For example, resveratrol-based formulations have been shown to improve cardiac remodeling, while liraglutide has demonstrated cardioprotective effects through modulation of PI3K/Akt-related signaling pathways [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present study extends these findings by demonstrating that combining both agents produces significantly greater cardioprotection than either monotherapy. The consistency of this effect across multiple endpoints\u0026mdash;biochemical, inflammatory, mitochondrial, and histological\u0026mdash;strengthens the validity of the observed interaction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e4.8. Limitations\u003c/h2\u003e \u003cp\u003eSeveral limitations of the present study should be acknowledged. First, although the sample size (n\u0026thinsp;=\u0026thinsp;14 per group) was adequately powered for preclinical analysis, caution should be exercised when extrapolating these findings to clinical settings. Second, the duration of treatment was relatively short, and the long-term cardioprotective effects as well as the safety profile of the combination were not evaluated.\u003c/p\u003e \u003cp\u003eThird, while the study proposes the involvement of key molecular pathways, including Nrf2, SIRT1, and PI3K/Akt, based on existing literature and the observed biochemical and mitochondrial outcomes, direct molecular validation (e.g., protein or gene expression analysis using Western blotting or quantitative PCR) was not performed. Therefore, the mechanistic interpretations remain inferential and warrant further experimental confirmation.\u003c/p\u003e \u003cp\u003eFourth, functional cardiac assessments, such as echocardiography or measurement of left ventricular ejection fraction (LVEF), were not conducted. Although the observed biochemical, mitochondrial, and histopathological improvements provide strong evidence of cardioprotection, they do not directly establish functional recovery. Accordingly, future studies incorporating in vivo functional evaluation are necessary to strengthen the translational relevance of these findings.\u003c/p\u003e \u003cp\u003eFifth, although two-way ANOVA demonstrated significant interaction effects between resveratrol and liraglutide, suggesting a greater-than-additive pharmacological interaction, formal synergy assessment using approaches such as isobolographic analysis or combination index (CI) calculation was not performed. Therefore, the current findings should be interpreted as indicative of an enhanced combined effect rather than definitive pharmacological synergy.\u003c/p\u003e \u003cp\u003eDespite these limitations, the consistency of the observed effects across multiple independent endpoints\u0026mdash;including cardiac biomarkers, oxidative stress parameters, inflammatory mediators, mitochondrial function, and histopathological findings\u0026mdash;strongly supports the robustness and internal validity of the study outcomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e4.9. Future Research Directions\u003c/h2\u003e \u003cp\u003eFuture studies should focus on molecular validation of the proposed mechanisms, long-term safety and efficacy, dose optimization, and evaluation in comorbid disease models. Incorporating functional cardiac assessments and conducting translational clinical studies will be essential to confirm clinical applicability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e4.10. Clinical Relevance\u003c/h2\u003e \u003cp\u003eFrom a translational perspective, the observed enhanced combined effect may be particularly relevant in patients with cardiometabolic disorders, where GLP-1 receptor agonists are already in clinical use. The addition of a nutraceutical agent such as resveratrol could represent a complementary strategy to improve cardiovascular outcomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e4.11. Conclusion\u003c/h2\u003e \u003cp\u003eIn conclusion, the present study demonstrates that the combination of resveratrol and the GLP-1 receptor agonist liraglutide confers enhanced cardioprotection against isoproterenol-induced myocardial injury. The combination significantly attenuated cardiac injury biomarkers, reduced oxidative stress, suppressed inflammation, preserved mitochondrial function, and improved myocardial architecture compared to either agent alone.\u003c/p\u003e \u003cp\u003eTwo-way ANOVA confirmed significant interaction effects across multiple endpoints, supporting a greater-than-additive interaction. These findings support the potential of this combination as a therapeutic strategy targeting oxidative stress and mitochondrial dysfunction in myocardial injury. Further studies are warranted to validate these findings and explore their translational potential in human ischemic heart disease.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlanine aminotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAMPK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAMP-activated protein kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANOVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnalysis of variance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAspartate aminotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAdenosine triphosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCatalase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCK-MB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCreatine kinase\u0026ndash;myocardial band\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecTnI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCardiac troponin I\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDCFH-DA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDichlorofluorescein diacetate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme-linked immunosorbent assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGSH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReduced glutathione\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGLP-1 RA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlucagon-like peptide-1 receptor agonist\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH\u0026amp;E\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematoxylin and eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHMGB1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh mobility group box 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL-6\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin-6\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eISO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIsoproterenol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLactate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLVEF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLeft ventricular ejection fraction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMalondialdehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMyocardial infarction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNF-κB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNuclear factor kappa B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNLRP3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNOD-like receptor family pyrin domain containing 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNrf2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNuclear factor erythroid 2\u0026ndash;related factor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePI3K\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphoinositide 3-kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGC-1α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePeroxisome proliferator-activated receptor gamma coactivator 1-alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard error of the mean\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSIRT1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSirtuin 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSuperoxide dismutase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTBARS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThiobarbituric acid reactive substances\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF-α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor necrosis factor-alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eΔΨm\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial membrane potentia\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial support:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Department of Basic Medical and Dental Sciences, Faculty of Dentistry, Zarqa University, Zarqa, Jordan, without any particular role in the study design, recruitment of individuals, data analysis, or writing of the report.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026apos;s contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAhmed El-Sayed Nour El-Deen:\u003c/strong\u003e Conceptualization, study design, methodology, data acquisition, statistical analysis, data interpretation, manuscript writing, and final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReda Taha:\u003c/strong\u003e Data collection, formal analysis, data interpretation, manuscript revision, and final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShaimaa Fawzy Abdellatif Esmaeil:\u003c/strong\u003e Conceptualization, methodology, data acquisition, manuscript revision, and final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMohamed Hamdy Sayed:\u003c/strong\u003e Data collection, formal analysis, figure design, manuscript revision, and final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMohamed Zaeim Hafez Ahmed:\u003c/strong\u003e Data acquisition, investigation, manuscript revision, and final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMuhammad Abdelbaeth Elfiky:\u003c/strong\u003e Data collection, validation, manuscript revision, and final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAhmed A. Abd El-Rhman:\u003c/strong\u003e Statistical analysis, figure design, data interpretation, manuscript writing, and final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsama Khalil Farag:\u003c/strong\u003e Data acquisition, investigation, manuscript revision, and final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMohammed Abdel Aziz Mohammed:\u003c/strong\u003e Data interpretation, manuscript writing, manuscript revision, and final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAhmed F. Abdel Ghany:\u003c/strong\u003e Data collection, validation, manuscript revision, and final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAhmed Gad Allah:\u003c/strong\u003e Histopathological examination, data interpretation, manuscript revision, and final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAhmad Taha:\u003c/strong\u003e Conceptualization, study design, supervision, data interpretation, manuscript writing, and final approval.\u003cspan dir=\"RTL\"\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e6. Ethics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures involving animals were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85\u0026ndash;23, revised 2011).\u003c/p\u003e\n\u003cp\u003eThe study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Al-Azhar University, Egypt (Approval No. RP/NA/PHY/05/12/2024).\u003c/p\u003e\n\u003cp\u003eAll efforts were made to minimize animal suffering, reduce the number of animals used, and ensure humane endpoints. Animals were anesthetized using ketamine (50 mg/kg) and xylazine (5 mg/kg) administered intraperitoneally prior to sample collection, and euthanasia was performed under deep anesthesia to ensure complete loss of consciousness.\u003c/p\u003e\n\u003cp\u003eConsent to participate is not applicable as this study did not involve human subjects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number: not applicable\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Availability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9. Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e10. Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The authors extend their appreciation to the Deanship of Scientific Research at Zarqa University for funding this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e11. Authors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Nour El-Deen A., Osman A., and Taha A. conceived and designed the study. Osman A. and Mohamed M. performed data collection. Nour El-Deen A. and Rashed F. conducted statistical analysis and figure preparation. Nour El-Deen A., Rashed F., and Mohamed M. contributed to data interpretation. Abd El-Rahman A., Ehab A., Mohamed M., Nour El-Deen A., and Rashed F. drafted the manuscript. All authors critically revised the manuscript and approved the final version for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e12. Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors extend their appreciation to the Deanship of Scientific Research at Zarqa University, Jordan for funding this work\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVirani SS, Alonso A, Aparicio HJ, et al. Heart Disease and Stroke Statistics-2023 Update: A Report From the American Heart Association. Circulation. 2023;147(8):e93\u0026ndash;621.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoth GA, Mensah GA, Johnson CO, et al. Global Burden of Cardiovascular Diseases and Risk Factors, 1990\u0026ndash;2019: Update From the GBD 2019 Study. J Am Coll Cardiol. 2020;76(25):2982\u0026ndash;3021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDubois-Deruy E, Peugnet V, Turkieh A, Pinet F. Oxidative Stress in Cardiovascular Diseases. Antioxid (Basel). 2020;9(9):864.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorgan MJ, Liu ZG. Crosstalk of reactive oxygen species and NF-κB signaling. Cell Res. 2011;21(1):103\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWillis BC, Salazar-Cant\u0026uacute; A, Silva-Platas C, et al. Impaired oxidative metabolism and calcium mishandling underlie cardiac dysfunction in a rat model of post-acute isoproterenol-induced cardiomyopathy. Am J Physiol Heart Circ Physiol. 2015;308(5):H467\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV\u0026aacute;zquez-Mart\u0026iacute;nez O, Gal\u0026aacute;n-Hern\u0026aacute;ndez N, Hern\u0026aacute;ndez-Mu\u0026ntilde;oz R, et al. Correlation between oxidative Stress and Alteration of Intracellular Calcium Handling in Isoproterenol-Induced Myocardial Infarction. Cardiovasc Toxicol. 2021;21(6):456\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaider A, Zainab R, Rafique M, et al. GLP-1 Receptor Agonists: Beyond Glycemic Control - A Multifaceted Approach to Cardiovascular Risk Reduction. J Med Res Rev. 2025;45(2):112\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDrucker DJ. The Cardiovascular Biology of Glucagon-like Peptide-1. Cell Metab. 2016;24(1):15\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUssher JR, Drucker DJ. Glucagon-like peptide 1 receptor agonists: cardiovascular benefits and mechanisms. Nat Rev Cardiol. 2023;20(7):463\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarso SP, Daniels GH, Brown-Frandsen K, et al. Liraglutide and Cardiovascular Outcomes in Type 2 Diabetes. N Engl J Med. 2016;375(4):311\u0026ndash;22. (LEADER trial).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarx N, Husain M, Lehrke M, Verma S, Sattar N. GLP-1 receptor agonists for the reduction of atherosclerotic cardiovascular risk in patients with type 2 diabetes. Circulation. 2022;146(24):1882\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlobaid WA, Elshal MM, El-Sayed WM, et al. Liraglutide Attenuates Diabetic Cardiomyopathy via the ILK/PI3K/AKT/PTEN Signaling Pathway in Rats with Type 2 Diabetes. Pharmaceuticals. 2024;17(8):1024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahmani S, Naraki K, Roohbakhsh A, et al. The Cardiovascular Protective Function of Natural Compounds Through AMPK/SIRT1/PGC-1α Signaling Pathway. Food Sci Nutr. 2024;12(12):9998\u0026ndash;10009.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerman AY, Motechin RA, Wiesenfeld MY, Holz MK. The therapeutic potential of resveratrol: a review of clinical trials. NPJ Precis Oncol. 2017;1(1):35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlhusaini AM, Alghamdi AM, Alghamdi SA, et al. Resveratrol-Based Liposomes Improve Cardiac Remodeling Induced by Isoproterenol Partially by Modulating MEF2, Cytochrome C and S100A1 Expression. Dose Response. 2024;22(2):15593258241252624.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdel-Reheim MA, El-Sayed WM, El-Naga RN, et al. Unlocking the miRNA-34a-5p/TGF-β and HMGB1/PI3K/Akt/mTOR crosstalk participate in the enhanced cardiac protection of liraglutide against isoproterenol-induced acute myocardial injury rat model. Int Immunopharmacol. 2024;128:111529.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim EN, Lim JH, Kim MY, et al. Resveratrol, an Nrf2 activator, ameliorates aging-related progressive renal injury. Aging. 2018;10(1):83\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao L, Li M, Wei S, et al. The crosstalk between SIRT1 and GLP-1: implications for cardiometabolic diseases. Front Endocrinol. 2023;14:1205432.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNational Research Council. Guide for the Care and Use of Laboratory Animals. 8th ed. Washington, DC: National Academies Press. 2011. (NIH Publication No. 85\u0026thinsp;\u0026ndash;\u0026thinsp;23, revised 2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRona G, Chappel CI, Balazs T, Gaudry R. An infarct-like myocardial lesion and other toxic manifestations produced by isoproterenol in the rat. AMA Arch Pathol. 1959;67(4):443\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoung DS. Effects of drugs on clinical laboratory tests. Ann Clin Biochem. 1997;34(6):579\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95(2):351\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974;47(3):469\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAebi H. Catalase in vitro. Methods Enzymol. 1984;105:121\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllman GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959;82(1):70\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrezza C, Cipolat S, Scorrano L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc. 2007;2(2):287\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEmaus RK, Grunwald R, Lemasters JJ. Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties. Biochim Biophys Acta. 1986;850(3):436\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeBel CP, Ischiropoulos H, Bondy SC. Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol. 1992;5(2):227\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBancroft JD, Layton C. The hematoxylin and eosin. In: Suvarna SK, Layton C, Bancroft JD, editors. Bancroft's Theory and Practice of Histological Techniques. 8th ed. Elsevier; 2019. pp. 126\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo Y, et al. Liraglutide attenuates myocardial ischemia-reperfusion injury through activation of the Nrf2/HO-1 pathway. Eur J Pharmacol. 2024;962:176231.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong J, et al. Resveratrol protects against ISO-induced cardiotoxicity via SIRT1/PGC-1α-mediated mitochondrial biogenesis. Biomed Pharmacother. 2023;165:115247.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, et al. GLP-1 receptor agonists improve endothelial function in diabetic patients: a meta-analysis. Cardiovasc Diabetol. 2023;22(1):45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, et al. Resveratrol attenuates vascular endothelial dysfunction through activation of AMPK. Vascul Pharmacol. 2022;146:107098.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu L, et al. NLRP3 inflammasome mediates ISO-induced cardiac inflammation and fibrosis. J Mol Cell Cardiol. 2023;175:1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, et al. SIRT1 activation by resveratrol prevents ISO-induced cardiac hypertrophy. Front Pharmacol. 2022;13:892345.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim S, et al. PI3K/Akt signaling in GLP-1 receptor agonist-mediated cardioprotection. Cell Signal. 2023;101:110498.\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Resveratrol, GLP-1 receptor agonist, liraglutide, myocardial injury, oxidative stress, cardio protection and Mitochondrial dysfunction","lastPublishedDoi":"10.21203/rs.3.rs-9302185/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9302185/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eMyocardial infarction (MI) remains a leading cause of global mortality and is strongly associated with oxidative stress, mitochondrial dysfunction, and inflammatory responses that contribute to irreversible cardiac injury. Glucagon-like peptide-1 receptor agonists (GLP-1 RAs), such as liraglutide, have demonstrated cardioprotective effects beyond glycemic control, while resveratrol, a natural polyphenol, exhibits potent antioxidant and mitochondrial-protective properties. However, the combined effects of these agents on mitochondrial function in myocardial injury have not been fully explored.\u003c/p\u003e\u003ch2\u003eObjective:\u003c/h2\u003e \u003cp\u003eThis study aimed to evaluate the combined cardioprotective effects of resveratrol and a GLP-1 receptor agonist (liraglutide) against isoproterenol (ISO)-induced myocardial injury in rats, with particular emphasis on oxidative stress and mitochondrial dysfunction.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e \u003cp\u003eSeventy adult male albino rats were randomly allocated into five groups: Control, ISO (100 mg/kg, s.c., for two consecutive days), Resveratrol\u0026thinsp;+\u0026thinsp;ISO (20 mg/kg/day, orally for 28 days), GLP-1 RA\u0026thinsp;+\u0026thinsp;ISO (liraglutide 0.2 mg/kg/day, s.c., for 10 days), and Resveratrol\u0026thinsp;+\u0026thinsp;GLP-1 RA\u0026thinsp;+\u0026thinsp;ISO. ISO was administered on days 27 and 28. Cardiac injury was assessed using serum biomarkers (cTnI, CK-MB, LDH), oxidative stress markers (MDA, SOD, GSH), inflammatory cytokines (TNF-α, IL-6), and mitochondrial function parameters, including mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) generation. Histopathological evaluation of cardiac tissue was also performed.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eISO administration significantly increased cardiac injury markers, lipid peroxidation, and pro-inflammatory cytokines, while reducing antioxidant defenses and impairing mitochondrial function (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. control). Pretreatment with either resveratrol or liraglutide partially attenuated these changes. The combined treatment resulted in significantly greater improvement compared to either monotherapy (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), including reductions in MDA (~\u0026thinsp;45%) and inflammatory cytokines, along with restoration of antioxidant capacity and mitochondrial function. Two-way ANOVA revealed significant interaction effects between treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Histopathological findings supported the biochemical results, showing marked preservation of myocardial architecture in the combination group.\u003c/p\u003e\u003ch2\u003eConclusion:\u003c/h2\u003e \u003cp\u003eThe combined administration of resveratrol and liraglutide provides enhanced cardioprotection against ISO-induced myocardial injury, likely through complementary effects on oxidative stress, inflammation, and mitochondrial function. While interaction analysis suggests a greater-than-additive effect, further studies using dedicated synergy models and molecular validation are required. This combination may represent a promising therapeutic strategy for myocardial injury.\u003c/p\u003e","manuscriptTitle":"Enhanced cardioprotective effects of resveratrol and liraglutide against isoproterenol-induced myocardial injury: role of oxidative stress, inflammation, and mitochondrial function","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 07:20:24","doi":"10.21203/rs.3.rs-9302185/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-19T04:51:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-13T08:32:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T12:58:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"279768139984620945766523849312807796480","date":"2026-05-08T04:11:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324803811359886456920498837255178876907","date":"2026-05-04T21:59:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"23826800209273243891562236516901341040","date":"2026-04-21T10:48:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-20T13:09:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-20T13:07:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-13T14:28:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-11T12:05:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cardiovascular Disorders","date":"2026-04-11T12:01:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4b9b68f9-5128-44a4-bd33-f9506d3f604d","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-19T04:51:41+00:00","index":55,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-13T08:32:21+00:00","index":54,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T12:58:03+00:00","index":53,"fulltext":""},{"type":"reviewerAgreed","content":"279768139984620945766523849312807796480","date":"2026-05-08T04:11:26+00:00","index":52,"fulltext":""},{"type":"reviewerAgreed","content":"324803811359886456920498837255178876907","date":"2026-05-04T21:59:33+00:00","index":51,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T07:20:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 07:20:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9302185","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9302185","identity":"rs-9302185","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}