Deciphering the Protective Effects of Carvacrol Against Doxorubicin-Induced Cardiotoxicity In Vitro and In Vivo

Deciphering the Protective Effects of Carvacrol Against Doxorubicin-Induced Cardiotoxicity In Vitro and In Vivo | 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 Article Deciphering the Protective Effects of Carvacrol Against Doxorubicin-Induced Cardiotoxicity In Vitro and In Vivo Rini Retnosari, Muhamad Adib Abdul Ghani, Munirah Majed Alkharji, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4381440/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Doxorubicin (DOX), a widely used chemotherapy, extends its impact beyond cancer cells, notably affecting the heart, leading to substantial concerns about DOX-induced cardiotoxicity (DIC). However, subclinical DIC remains unresolved, necessitating advanced cardio-protection strategies in cancer therapy. Recent research explores carvacrol (CAR), a natural substance with antioxidant and anti-inflammatory properties, as a potential shield against DIC. However, further exploration is warranted, particularly concerning hypertrophy and cardiac fibrosis. This study investigated CAR’s potential cardioprotective properties against DIC in H9c2 cardiomyocytes and rats. Induction with DOX reduced cardiomyocyte viability, while pretreatment with 0.01 µg/mL CAR enhanced the viability of DOX-induced cardiomyocytes. Meanwhile, administration of DOX induced adverse effects in rats, causing decreased total heart weight and left ventricular mass, and lowered blood pressure. DOX also caused cardiac dysfunction, lipid peroxidation, hypertrophy, and fibrosis. In rat models, CAR pretreatment effectively mitigated DOX-induced reductions in blood pressure, hypertrophy, and cardiac fibrosis. However, the pretreatment kept the heart function, oxidative stress, and antioxidant enzymes unaltered. In conclusion, the results show that CAR could be an adjuvant to reduce DIC by ameliorating cardiac fibrosis and hypertrophy. Health sciences/Cardiology Physical sciences/Chemistry Cardioprotection cardiomyocyte carvacrol doxorubicin-induced cardiotoxicity H9c2 cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Cancer stands as a formidable global public health challenge, with an estimated 10 million fatalities in 2020. Prognostic models predict a worrisome trajectory, with an estimated 28.4 million additional cancer cases by 2040. This represents a stunning 47% increase over the 19.3 million cases observed in 2020 1 . Furthermore, from 2007 to 2011, Malaysia registered 103,507 new cancer cases, while the ensuing decade saw a considerable and alarming increase 2 . In 2020 alone, 48,639 new cancer cases were reported 3 . This alarming trend positions cancer as a persistent and formidable contributor to premature mortality in Malaysia, solidifying its status as a secondary leading cause of untimely death in the nation. Malaysia's changing lifestyle landscape, driven by urbanization and globalization, has been a significant component in this health problem. These lifestyle changes have corresponded with a significant increase in the prevalence of non-communicable diseases (NCDs) among Malaysians, including cancer, diabetes, stroke, heart disease, and hypertension 4 . Chemotherapy and radiotherapy stand as indispensable cornerstones in the comprehensive treatment of diverse cancer types, playing a pivotal role in substantially enhancing survival rates across a burgeoning patient population 5 . However, despite its importance, chemotherapy faces significant hurdles in patient care. Beyond the formidable barrier of chemoresistance, an accumulating body of evidence highlights the inherent toxicity linked to drug concentrations, adding a layer of complexity to the treatment landscape 6 . Doxorubicin (DOX) is a powerful and widely used chemotherapeutic medication that can be given alone or in combination with other cancer treatments. However, the impact of DOX extends beyond its intended target, affecting various organs and consequently diminishing the overall quality of life for cancer patients due to the aftermath of chemotherapy side effects. Notably, the heart, a vital organ, bears a considerable brunt during cancer chemotherapy with DOX, potentially inducing heart failure and irreversible impairment of cardiac function, potentially. As of now, the problem of DIC remains a prominent concern, and research on this subject has significantly risen over the years. The prevalent mechanisms associated with DIC included oxidative stress, apoptosis, inflammation, autophagy, mitophagy, endoplasmic reticulum stress, pyroptosis, and ferroptosis. However, oxidative stress remained the primary molecular mechanism of DIC 6 , 7 . Following the entry of DOX into the body, redox metabolism generates ROS, which acts as a blasting fuse, and oxidative stress provides the foundation for further DIC molecular pathways. On the one hand, excessive ROS generation and accumulation harm DNA and mitochondrial protein, leading to mitochondrial dysfunction and tissue damage. ROS, on the other hand, can function as a signal to activate the body's defense mechanisms that result in cell death, such as apoptosis, autophagy, necrosis, pyroptosis, iron death, and so on 8 . Dexrazoxane is currently the only medicine approved by the FDA to treat cardiotoxicity. Dexrazoxane works by binding to iron in the body before it enters cardiac cells, limiting the production of iron-DOX complexes and minimizing the damaging effects of free radicals on the heart. This is accomplished by reducing lipid membrane peroxidation, which results in decreased cardiotoxicity associated with DOX 9 . Despite pre-treatment with dexrazoxane, subclinical DIC is not entirely reduced 10 . To avoid this worrisome complication of cancer therapy, there is a need for the creation of a more effective cardio-protection approach. There is a rising scientific interest in researching the possible cardioprotective properties of natural substances against DIC. CAR, a notable natural ingredient, is prevalent in essential oils generated from aromatic plants. It is generally recognized for its wide range of biological activities, including antioxidant, anti-inflammatory, anticancer, and cardioprotective qualities. CAR's radical scavenging capacity is primarily due to the hydroxyl group (OH) it carries. CAR's weak acid nature allows the donation of hydrogen atoms to unpaired electrons, stabilizing another radical via electron scattering in the molecule's resonance structure 11 . Recently, researchers have demonstrated a keen interest in exploring the potential effectiveness of CAR in alleviating DIC. A study by El-sayed et al. 12 revealed notable improvements in cardiac function and oxidative stress parameters when rats were pretreated with CAR before co-administration of a single dose of DOX. This positive outcome is attributed to the multifaceted properties of CAR, encompassing its antioxidant, anti-inflammatory, and antiapoptotic activities. Importantly, corroborating these findings, Khajavi Rad & Mohebbati 13 and Jafarinezhad et al. 14 demonstrated CAR's efficacy in alleviating oxidative stress damage, improving heart function, and mitigating structural alterations induced by DOX in rats. Despite these encouraging results, it is noteworthy that the effectiveness of CAR in addressing DOX-induced cell death in H9c2 cells and its impact on hypertrophy and cardiac fibrosis in rat models remain unexplored in current research studies. This study represents a pioneering effort to investigate the cytotoxic effects of CAR on H9c2 cells and explore the potential cardioprotective properties of CAR in the context of DOX-induced cell death in H9c2 cells. Additionally, our research delves into the cardioprotective role of CAR in mitigating DOX-induced acute cardiotoxicity in rat models, focusing on examining its impact on hypertrophy and cardiac fibrosis. MATERIALS AND METHODS Drugs and Chemicals CAR, DOX, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (USA). Dulbecco's modified Eagle's medium (DMEM), Fetal Bovine Serum (FBS), Phosphate Buffered Saline (PBS), and penicillin were obtained from Thermo Fisher Scientific. KTX (Ketamine-Xylazine/Tiletamine-Zolazepam) was employed for anesthesia during animal handling, and corn oil served as the solvent for CAR. Krebs-Henseleit Buffer (KHB) solution, an artificial blood substitute during the Langendorff isolated heart procedure, comprised sodium chloride (NaCl), sodium hydrogen carbonate (NaHCO 3 ), hydrated magnesium sulfate (MgSO 4 ∙7H 2 O), glucose (C 6 H 12 O 6 ), and hydrated calcium chloride (CaCl 2 ∙2H 2 O), diluted in distilled water and supplied with 95% gaseous oxygen and 5% gaseous carbon dioxide. H9c2 Cell Culture The H9c2 cardiomyocyte cell line, procured from Thermo Fisher Scientific, was cultured in DMEM supplemented with 10% FBS and 1% penicillin. Cells were maintained in monolayer culture conditions at 37°C and 5% CO 2 , with media refreshed every two days. Subculturing was performed when cells reached 80% confluence. Cardiomyocyte Viability The viability of H9c2 cardiomyocytes was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. H9c2 cells were seeded in 96-well plates at a density of 5000 cells/well and allowed to attach for 48 h. To assess CAR's cytotoxicity, cardiomyocytes were treated with CAR at concentrations ranging from 0.01 to 100 µg/mL for 48 h. Following the treatment, MTT reagent (5 mg/mL) was added to the culture medium for 4 h at 37°C. Formazan formed in each well was dissolved in 100 µL of dimethyl sulfoxide (DMSO), and absorbance at 570 nm was measured using a microplate reader. Subsequently, to determine the protective effects of CAR on the viability of DOX-induced cardiomyocytes, H9c2 cells were pre-treated with or without non-toxic CAR concentrations for 24 h prior to induction with 10 µM DOX for another 24 h. Animals and Experimental Procedure A total of 48 male Sprague-Dawley rats (200–250 g) were provided by the Laboratory Animal Resource Unit, Faculty of Medicine, Universiti Kebangsaan Malaysia (UKM). The Animal Ethics Committee of Universiti Kebangsaan Malaysia (UKMAEC) approved animal study and protocols (Approval No: FSK/2020/SATIRAH/23-SEPT./1131-NOV.-2020-NOV. -2022), and all experiments were performed in accordance with the guidelines and regulations. Furthermore, animal handling training was provided by the Animal Resource Unit of the Faculty of Medicine Laboratory, Universiti Kebangsaan Malaysia, through the Introductory Workshop on the Care and Handling of Research Animals (IWLACM). We also hereby confirm that the study is reported following ARRIVE guidelines 15 . The rats were provided with conventional rodent food and unrestricted access to tap water, maintained under controlled conditions (25°C to 28°C, 12-hour light-dark cycle, and proper ventilation). A one-week adaptation period was allowed for the rats before they were randomly assigned to three groups: Control Group (n = 8): Animals received corn oil for 14 days, followed by 0.5% DMSO on day 15. Doxorubicin Group (DOX) (n = 8): Animals received corn oil for 14 days, followed by DOX (15 mg/kg, i.p) on day 15. Carvacrol + DOX Group (CAR + DOX) (n = 8): This group was pretreated with CAR (50 mg/kg/daily, p.o) for 14 consecutive days 14 . On day 15, each animal received DOX (15 mg/kg, i.p) to induce cardiotoxicity. A two-day recovery period without intervention was allowed for disease development 16 , and all rats were sacrificed on day-18 of the experiment. Blood Pressure Monitoring Rat blood pressure was non-invasively measured via the tail-cuff method using the CODA IITM system (Kent Scientific Corporation, USA) on days 0, 14, and 18. Systolic blood pressure was recorded, and pulse pressure was calculated. Langendorff Perfusion All experimental rats were evaluated for cardiac contractile function using the Langendorff heart preparation, as our lab has previously established 17 . The rat underwent intraperitoneal administration of anticoagulant heparin (500 IU) and intravenous administration of KTX (1 mg/kg). Following losing consciousness, the heart was isolated, perfused retrogradely with Krebs-Henseleit Buffer, and connected to the Langendorff system. Coronary flow (CF), perfusion pressure (PP), and left ventricle-developed pressure (LVDP) were monitored, and LVDP tracings provided values for dP/dtmax and dP/dtmin. These tracings' values were derived electronically using the PowerLab data acquisition system, and the data was analyzed with LabChart Pro 8.0 (AD Instruments, Australia) software. Biochemical Analysis Bradford's method 18 was used to determine the total protein content of heart tissue. Furthermore, the activity of the enzymes responsible for oxidative stress was measured using a spectrophotometer. The quantification of malondialdehyde (MDA) was based on the reaction of MDA with thiobarbituric acid at 100°C. Thiobarbituric acid reactive substances (TBARS) formation was monitored at 532 nm 19 . On the other hand, reduced glutathione (GSH) was detected at 412 nm after reacting homogenate GSH with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) to create a yellow-colored complex 20 . Furthermore, superoxide dismutase (SOD) activity was assessed at 560 nm based on its ability to prevent ferricytochrome reduction 21 . Histopathological Examination of the Heart The left ventricles of rats in separate groups were sectioned and fixed for 24 hours in 10% neutral buffered formalin. Dehydration was obtained after washing with tap water and gradually diluted alcohol. Before embedding in paraffin, samples were cleaned in xylene. Tissue blocks composed of paraffin wax were sliced into 3 m thick pieces using a microtome. Before being examined under a light microscope, the tissue sections were mounted on glass slides, deparaffinized, stained with hematoxylin and eosin, and picrosirius red. Statistical Analysis of Data Data analysis employed GraphPad Prism 8.0 software, presenting results as mean ± standard deviation (SEM). For normally distributed data, one-way ANOVA with post hoc Tukey was applied, while the Kruskal-Wallis test was used for non-normally distributed data. p < 0.05 was considered as significant. RESULTS CAR Enhanced the Viability of DOX-induced H9c2 Cardiomyocytes The initial step in assessing the cardioprotective potential of CAR involved investigating its cytotoxicity in H9c2 cardiomyocytes. Following 48 h of exposure to 0.01–100 µg/mL CAR, the MTT assay was employed to determine cardiomyocyte viability. CAR exhibited a remarkable non-toxic profile against cardiomyocytes within the concentration range of 0.01–0.1 µg/mL. However, reduced cardiomyocyte viability was observed at CAR concentrations ranging from 1–100 µg/mL (Fig. 1 a). Therefore, CAR concentrations of 0.01–0.1 µg/mL were classified as non-toxic and used for the second stage of the MTT assay. In the second stage of the MTT assay, H9c2 cells were pre-treated with or without non-toxic CAR concentrations prior to DOX induction to determine the cardioprotective effect of non-toxic CAR. The results demonstrated that 10 µM DOX significantly reduced cardiomyocyte viability to 46.61 ± 0.55% compared to the control (P < 0.001), indicating the potent cardiotoxic effect of DOX (Fig. 1 b). Remarkably, pre-treatment with 0.01 µg/mL CAR significantly enhanced the viability of DOX-induced cardiomyocytes compared to the untreated DOX group (P < 0.01). However, pretreatment with a higher CAR concentration (0.1 µg/mL) did not elicit a further increase in cardiomyocyte viability compared to the DOX group. CAR Preserved Cardiac Structure and Function in DOX-treated Rats Impact on body weight and heart mass To assess the potential protective role of CAR against DOX-induced acute cardiotoxicity in vivo, we investigated its influence on body weight and heart mass in male rats. Table 1 illustrates that a single DOX treatment (15 mg/kg) significantly reduced body weight compared to controls. Intriguingly, the DOX + CAR group exhibited growth rates similar to the DOX group, with no significant differences observed in heart and left ventricle masses among the three groups. This comprehensive dataset indicates that CAR treatment did not mitigate DOX-induced heart mass and body weight alterations. Table 1 Effect of CAR on body weight gain and heart mass in DOX-treated rats Treatment CON (n = 8) DOX (n = 8) DOX + CAR (n = 8) Increase in body weight (%) 25.86 ± 2.43 16.63 ± 2.06* 18.34 ± 0.71* Heart (g) 1.08 ± 0.02 1.02 ± 0.02 1.01 ± 0.02 Left ventricle (g) 0.78 ± 0.05 0.66 ± 0.05 0.64 ± 0.01 CON: control rats; DOX: doxorubicin; DOX + CAR: doxorubicin and carvacrol co-administration. Values are expressed as mean ± SEM, *significantly different compared to control (p < 0.05) Blood pressure modulation We scrutinized blood pressure parameters, including systolic and pulse pressures (Fig. 2 ), to unravel the impact of CAR on DOX-induced changes. The acute effects of DOX administration on blood pressure were evaluated utilizing blood pressure parameters at day 0, 14, and 18. Notably, no significant differences were observed among the three groups on days 0 and 14. As soon as three days after receiving DOX (day 18), the DOX group displayed a significant decrease in systolic pressures compared to day 14 (p < 0.05), with a non-significant reduction in pulse pressure. Strikingly, CAR administration significantly attenuated both blood pressure parameters, restoring them to levels comparable to the control group. This intriguing observation suggests that CAR possesses the potential to counteract DOX-induced blood pressure alterations. Ex vivo cardiac function evaluation Ex vivo assessment of cardiac function, employing Langendorf-perfused isolated rat hearts, provided crucial insights into the interplay between DOX, CAR, and heart contractility. LVDP, a key parameter reflecting cardiac contractility, was scrutinized to unravel the protective effects of CAR. According to Tukey’s multiple comparison tests, a single DOX dose (15 mg/kg) marginally improved LVDP and End-Diastolic Pressure (EDP) by 10% and 15%, respectively, compared to the control, though insignificantly (Fig. 3 ). Encouragingly, CAR alleviated LVDP and EDP parameters to levels equivalent to the control. Conversely, the maximum rates of contraction and relaxation exhibited a significant decline by 19% and 23%, respectively, after DOX coadministration compared to control. Strikingly, CAR treatment demonstrated a substantial improvement in these parameters, indicating a potential protective effect against DOX-induced cardiac dysfunction. Oxidative stress Despite the promising cardiovascular effects observed, CAR could not rescue DOX-induced lipid peroxidation in rats, as shown in Fig. 4 . A single DOX injection significantly increased MDA levels by 89% in the heart, accompanied by significant decreases in GSH cardiac contents (27%) and SOD activities (35%) compared to the control group. However, CAR administration for 14 days before DOX coadministration did not significantly mitigate MDA levels or enhance GSH cardiac contents. Intriguingly, CAR even decreased SOD activity by 11.35% compared to the DOX group, suggesting a nuanced interplay between CAR and oxidative stress pathways. CAR reverses cardiomyocyte hypertrophy Histological examination of left ventricle heart sections provided visual insights into the cardioprotective effects of CAR against DOX-induced damage. As shown in Fig. 5 , DOX administration induced severe histological alterations, including obvious wavy degeneration of cardiac muscle fibers as seen by the arrows in (image B), the presence of inflammatory infiltration as seen by the arrows in (image C), coagulative necrosis of cardiac muscle fibers as seen by the arrows in (image D) showing pale segments of cardiac tissue, the presence of blood cells (arrow) between muscle fiber in (image E) indicates hemorrhage in DOX group, arrow in (image F) indicates vascular congestion that was evident in DOX. Impressively, pretreatment with CAR ameliorated these abnormalities, demonstrating its efficacy in minimizing histological damage. Still, wavy degeneration of cardiac muscle fibers and coagulative necrosis of cardiac muscle fibers (Image G and H, respectively) were observed. As supported by quantification of the cardiomyocyte area size (Fig. 5 (II) ), the region of cardiomyocytes was significantly enlarged after DOX administration by 13.4% compared to the control group (p < 0.05). Pretreatment with CAR significantly reduced the cardiomyocyte's cross-sectional area (p < 0.05). Reduction of DOX-induced cardiac fibrosis Figure 6 (I) shows microscopic images at 100x magnification comparing collagen deposition (fibrosis) in the perivascular area of coronary arteries in the left ventricular heart section (A, C, and E), as well as in the interstitial area of left ventricular heart tissue sections (B, D, and F) in each group. Image (A and B) shows normal histology in the control group, but image (C and D) shows severe fibrotic damage after DOX administration compared to the control group. Treatment with CAR reduced the fibrotic damage, which is more visually similar to the control group. The quantification of the fibrotic area supported this evidence. After DOX administration, the fibrosis area was significantly enlarged compared to the control group (p < 0.05), as shown in Fig. 6 (II) . In contrast, CAR treatment significantly reduced the fibrotic area compared to the DOX group (p < 0.05). CAR treatment markedly reduced DOX-induced cardiac fibrosis DISCUSSION This study delves into the potential protective effects of CAR against DIC, examining both cellular and rat models. Initial investigations revealed that CAR did not induce cytotoxicity in cardiomyocytes at 0.01 and 0.1 µg/mL concentrations. Remarkably, 0.01 µg/mL CAR enhanced the viability of DOX-induced cardiomyocytes, presenting a novel insight into the protective properties of CAR against DIC in this cellular context. In extending our exploration to rat models, the influence of CAR on DIC was evaluated by performing functional, biochemical, and histological examinations. The induction of cardiomyopathy in rats was achieved by administering a single cumulative intraperitoneal dose of DOX at 15 mg/kg to create a DOX-induced acute cardiotoxicity model, a well-established method documented in previous studies 22 , 23 . In addition, the risk of cardiotoxicity with DOX becomes significant when cumulative doses exceed 200 mg/m 2 , equivalent to 5 mg/kg in humans 24 . We initiated this by evaluating the increase in body weight of rats following pretreatment with CAR, both with and without co-administration of DOX. Rats in the DOX group exhibited a significant decrease in body weight, consistent with the findings of Junior et al. 25 Intriguingly, pre-administration of CAR at 50 mg/kg also led to a decrease in body weight, aligning with Lee et al.'s observation 26 that CAR suppresses food intake, consequently reducing the percentage of body weight gain. However, in contrast to the cardiotoxicity group, the CAR-treated group exhibited a slight, though statistically non-significant, percentage increase, contradicting the findings of Cota et al. 27 , who employed a higher CAR dose (100 mg/kg) before DOX treatment. Left ventricular hypertrophy (LVH), identified as an adaptive response to pathological volume or pressure aiming to regulate pump function by circulatory demand, was evident in the cardiotoxicity and treatment groups, resulting in left ventricular remodeling 28 . However, intriguingly, when rats were pre-treated with CAR, there were no discernible differences in the heart and left ventricle weights. This finding contradicts the outcomes of studies by Jamhiri et al. 29 , which demonstrated that treating rats with CAR at 50 and 75 mg/kg/day prevented hypertension induced by abdominal aortic banding, decreasing the heart weight-to-body weight ratio. A thorough examination of the blood pressure findings revealed a general decrease in SBP in the DOX group. These results are consistent with previous research indicating impaired cardiac performance in rat models treated with DOX and align with clinical observations 30 , 31 . It is important to note that DOX-induced acute cardiotoxicity typically manifests during or immediately after treatment and usually involves reversible hypotension 32 . Interestingly, CAR administration effectively mitigated all blood pressure parameters, restoring them to levels comparable to the control group. A previous study showed that treatment with CAR induces hypotension and bradycardia in hypertensive rats, influencing the cardiovascular system and promoting antihypertensive mechanisms 33 . This study also showed cardiac dysfunction in Langendorff-perfused rat hearts isolated from DOX-administered rats. LVDP, EDP, LV + dP/dt, and LV -dP/dt as markers for LV contractile markers were impaired in the DOX group, indicating contractile dysfunction, in line with previous results 22 . The study identified several mechanisms underlying cardiac dysfunction triggered by DOX, including heightened generation of reactive oxygen species (ROS) resulting in oxidative stress damage, disturbed calcium ion (Ca 2+ ) homeostasis, and compromised expression of genes associated with the apoptosis and necrosis of cardiomyocytes 34 . This discrepancy might be attributed to structural remodeling of the heart, suggesting an adaptive response to the oxidative stress induced by DOX administration. Although DOX is commonly associated with dilated cardiomyopathy, it is noteworthy that hypertrophic adaptation of cardiomyocytes can occur in the early stages of congestive heart failure progression 35 . In addition, the DOX group's EDP was higher than the control group, likely due to DOX-induced cardiomyocyte hypertrophy, promotion of fibrosis in the heart 36 , and mitochondrial dysfunction 37 . Both factors can impact the EDP value, with fibrosis potentially causing a loss of heart compliance and mitochondrial dysfunction disrupting average ATP generation in the heart. A recent study showed that CAR mitigated DOX-induced cardiac dysfunction. This observation may be attributed to CAR's beneficial properties as an anti-inflammatory and antioxidative agent, reducing the toxic effects of DOX on the heart 38 . Contrary to expectations, the mean dP/dtmin of the CAR + DOX group was the lowest among the three groups, surpassing the mean of the control group. This steep dP/dtmin slope may be associated with CAR's relaxant effect, which affects not only vascular smooth muscle 39 but also influences viable remaining cardiac myocytes by blocking calcium ion channels on the surface of cardiomyocytes 40 . The central mechanistic underpinning of DIC is well-established and primarily rooted in the induction of oxidative stress 41 . This current investigation delved into the intricate interplay of DOX-induced oxidative stress by examining lipid peroxidation, as evidenced by notable alterations in MDA levels, GSH content, and SOD activity. Regrettably, our results indicate that pretreatment with CAR did not mitigate the lipid peroxidation induced by DOX. Specifically, CAR failed to reduce MDA levels significantly and did not impart a significant increase in GSH content. Unexpectedly, CAR pretreatment led to a reduction in SOD activity in cardiac tissue, diverging from the anticipated antioxidant effects attributed to CAR. This outcome contrasts with the findings reported by El-Sayed et al. 12 , possibly attributed to variations in the experimental design, including divergent doses of DOX and CAR employed in our investigation. El-Sayed et al. 12 demonstrated that CAR administration (25 mg/kg) for 14 days before exposure to DOX (10 mg/kg) ameliorated oxidative stress markers. However, our study, employing higher doses of both DOX (15 mg/kg) and CAR (50 mg/kg), revealed a lack of enhancement in oxidative stress parameters with CAR pretreatment. The discrepancy in outcomes prompts an exploration into the nuanced dose-dependent effects of CAR in the context of DOX-induced oxidative stress. While CAR is recognized for its antioxidant properties, interacting with cell membrane phospholipids to mitigate lipid peroxidation and reduce nitric oxide production 42 , 43 , it is essential to acknowledge that, at elevated concentrations, CAR can exhibit a shift in its role towards that of a prooxidant. In this capacity, CAR may potentially induce oxidative stress, either through the generation of reactive oxygen species (ROS) or by depleting cellular antioxidants 44 . The intricate balance between antioxidant and prooxidant activities of CAR may be influenced by dosage and the specific context of the experimental conditions. Consequently, in our study, the inability of CAR to mitigate DIC through its anticipated antioxidant activity suggests a nuanced interplay between CAR, DOX, and oxidative stress. These findings underscore the importance of considering dose-dependent effects and highlight the need for a comprehensive understanding of the intricate mechanisms that govern CAR's actions in the specific context of DIC. Future investigations may benefit from elucidating the threshold at which CAR transitions from an antioxidant to a prooxidant, paving the way for a more refined understanding of its therapeutic potential in managing oxidative stress-associated cardiotoxicity. The DOX group exhibited various histological alterations in the histological observation of H&E staining, including wavy degeneration of cardiac muscle fibers, inflammatory infiltration, coagulative necrosis of cardiac muscle fibers, hemorrhage, and vascular congestion. Wavy degeneration and coagulative necrosis are indicative of ischemia. These findings parallel studies by El-Agamy et al. 16 and Cota et al. 27 , where the induction of cardiotoxicity with DOX resulted in similar histopathological changes, such as necrosis, myocyte rupture, degeneration, edema, and vascular congestion. Remarkably, the administration of CAR at 50 mg/kg/day demonstrated a significant alleviation of hypertrophy induced by DOX. A notable reduction in the cross-sectional areas of cardiomyocytes evidenced this. However, it is worth noting that despite this positive effect, some degree of wavy degeneration and coagulative necrosis of cardiac muscle fibers persisted. Recent studies have shown that CAR administration prevents aortic banding-induced left ventricular hypertrophy in rats. In hypertrophied left ventricles, an increase in cardiomyocyte apoptosis is evident, as indicated by elevated mRNA levels of the pro-apoptotic protein Bcl-2-associated death promoter (BAD) and the anti-apoptotic protein Bcl-2. CAR demonstrated a notable capacity to decrease the expression of the pro-apoptotic factor BAD while simultaneously enhancing the levels of anti-apoptotic factors, specifically Bcl-2 and Bcl-xL, within the left ventricular tissue. This dual effect suggests a promising role for CAR in modulating apoptotic processes associated with ventricular hypertrophy 29 , 45 . The antiapoptotic of CAR was also evidenced in Yu et al.’s result 46 . In rats, CAR exhibited cardioprotective activity against acute myocardial infarction by downregulating caspase-3 and Bax activity and increased Bcl-2 expression at the protein level. Moreover, on a molecular and cellular scale, cardiac hypertrophy is intricately associated with oxidative stress, arising from the imbalance between the antioxidant defense system within the heart and the excessive generation of reactive oxygen species (ROS). Oxidative stress can activate apoptotic factors such as proapoptotic members of the BCL-2 family and caspases 47 . Consequently, CAR demonstrated potential cardioprotective effects in DOX-induced hypertrophy, likely attributed to its anti-apoptotic and antioxidant characteristics. In-depth histological examination through picrosirius red staining illuminated a profound increase in red color staining within the perivascular and interstitial regions of the left ventricle in the DOX group, suggesting an augmented collagen deposition compared to the control group. This observation resonates with Tanaka et al. 48 , who highlighted that picrosirius red staining, particularly in perivascular areas, serves as a distinctive marker for the initial stages of reactive fibrosis. In the current study, administering 15 mg/kg of DOX to induce cardiotoxicity likely propelled the heart samples beyond the early fibrotic stages into a more advanced phase, as evidenced by the conspicuous collagen deposition in both perivascular and interstitial cardiac regions. This progression is further substantiated by a notable expansion in the fibrotic area within the DOX group compared to the control group, a finding consistent with the work of Chang et al. 49 The collective evidence underscores the profound impact of DOX on cardiac fibrosis, suggesting a trajectory beyond mere initiation toward a more complex and extensive fibrotic response. The probable mechanistic action could involve TGF-β1 activation and α-SMA expression as key contributors to the observed cardiac fibrosis in the pathogenesis of DOX-induced fibrotic changes 50 , 51 . Further complexity arises from the sustained elevation of critical fibrotic markers in animals subjected to the highest dose of DOX (15 mg/kg). Increased expression of type I and II collagen, endothelin-1 (ET1), fibroblast growth factor 4 (FGF4), and tumor necrosis factor-alpha (TNFα) could be involved in the inflammatory processes. This dual manifestation suggests a dynamic interplay between fibrosis and inflammation against the backdrop of DOX's pronounced cardiotoxic effects, as elucidated by Podyacheva et al. 52 Unexpectedly, pretreatment with CAR exhibited a slight increase in red color staining, indicating a minor degree of collagen deposition. Furthermore, a pivotal and promising outcome emerged as a significant decrease in fibrosis area was discerned in the CAR-pretreated group compared to the DOX-alone group. These findings resonate with the outcomes of prior investigations, providing a consistent narrative wherein CAR has demonstrated a remarkable ability to ameliorate cardiac fibrosis 29 . In elucidating the putative mechanisms behind CAR's anti-fibrotic effects on DIC, existing literature posits that CAR exerts its protective influence by inhibiting inflammatory cytokines, specifically tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) 12 . In a distinct investigation, the promising potential of CAR in ameliorating ethanol-induced hepatic fibrosis was prominently demonstrated. The study illuminated a multifaceted mechanism wherein CAR exerted significant therapeutic effects by alleviating oxidative stress, downregulating the pro-fibrotic growth factor (TGF-β1), augmenting the anti-inflammatory cytokine interleukin-10 (IL-10), inhibiting collagen synthesis as evidenced by reduced 4-hydroxyproline (4-HYP) levels, and attenuating hepatic stellate cell (HSC) activation, as manifested by the diminished expression of alpha-smooth muscle actin (α-SMA) in hepatocytes. These findings underscore CAR's profound antioxidant, anti-inflammatory, and anti-fibrotic properties, providing valuable insights into its potential therapeutic utility 53 . Furthermore, CAR's capacity to impede the progression of liver fibrosis through its interaction with the TGF-β signaling pathway was substantiated by Mohseni et al. 54 Their findings echoed the multifaceted anti-fibrotic and anti-inflammatory attributes of CAR, further solidifying its potential as a therapeutic intervention against hepatic fibrosis. In a parallel investigation by Cai et al. 55 , CAR emerged as a pivotal player in reducing carbon tetrachloride (CCl 4 )-induced hepatic fibrogenesis in rats. The study demonstrated that CAR exerted its effects by downregulating the expression of key fibrotic markers such as α-SMA, collagen type I alpha 1 (Col1α1), platelet-derived growth factor (PDGF) receptor, and TGF-β1 receptor. These comprehensive findings underscore CAR's remarkable potential in countering hepatic fibrosis through intricate molecular mechanisms. CONCLUSION The current study demonstrates that CAR reduces DIC in vivo and in vitro. CAR enhances the viability of DOX-induced cardiomyocytes. Additionally, CAR attenuates DOX-induced reductions in blood pressure, cardiac hypertrophy, and cardiac fibrosis in rats. These effects are likely attributed to CAR’s anti-fibrotic, anti-inflammatory, anti-apoptotic, and antioxidant properties. Nevertheless, further research is necessary to elucidate the precise mechanisms through which CAR mitigates DOX's cardiotoxic effects. Understanding the effect of this compound on cardiac function could pave the way for its potential utilization in managing cardiovascular complications associated with DOX therapy. Declarations AUTHOR CONTRIBUTIONS All authors have read and agreed to the published version of the manuscript. FUNDING The authors gratefully acknowledged the financial assistance provided by Universiti Kebangsaan Malaysia (Grant no: DIP-2020-020). The authors also acknowledge the Universitas Negeri Malang and Gifu University for the PhD Scholarship awarded to Rini Retnosari. CONFLICTS OF INTEREST The authors declare no conflict of interest. DATA AVAILABILITY All data generated or analyzed during this study are included in this published article. Acknowledgement The authors gratefully acknowledged the financial assistance provided by Universiti Kebangsaan Malaysia (Grant no: DIP-2020-020). The authors also acknowledge the Universitas Negeri Malang and Gifu University for the PhD Scholarship awarded to Rini Retnosari. References Sung, H. et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 71 (3), 209-249; 10.3322/caac.21660 (2021). Azizah, Ab. M., Nor Saleha, I. T., Noor Hashimah, A., Asmah, Z.A., & Mastulu, W. Malaysian national cancer registry report 2007 – 2011. Preprint at https://www.crc.gov.my/wp-content/uploads/documents/report/MNCRRrepor2007-2011.pdf (2015). World Health Organization (WHO). Cancer today: Malaysia. 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The effect of carvacrol on transcription levels of Bcl‐2 family proteins in hypertrophied heart of rats. Physiol and Pharmacol . 22 (1), 54–62; https://ppj.phypha.ir/article-1-1305-en.html (2018). Yu, W., Liu, Q. & Zhu, S. Carvacrol protects against acute myocardial infarction of rats via anti-oxidative and anti-apoptotic pathways. Biol Pharm Bull. 36 (4), 579-84; 10.1248/bpb.b12-00948 (2013). Mansi, Vikas, G. & Bidya D.S. Carvacrol and its effect on cardiovascular diseases: from molecular mechanism to pharmacological modulation. Food Bioscience . 57 , 103444; 10.1016/j.fbio.2023.103444 (2024). Tanaka, R. et al . Reactive fibrosis precedes doxorubicin-induced heart failure through sterile inflammation. ESC Heart Fail. 7 (2), 588-603; 10.1002/ehf2.12616 (2020). Chang, D., Li, H., Qian, C. & Wang, Y. DiOHF protects against doxorubicin-induced cardiotoxicity through erk1 signaling pathway. Front Pharmacol. 10 , 1081; 10.3389/fphar.2019.01081 (2019). Qi, W. et al . Cardamonin protects against doxorubicin-induced cardiotoxicity in mice by restraining oxidative stress and inflammation associated with Nrf2 signaling. Biomed Pharmacother. 122 ,109547; 10.1016/j.biopha.2019.109547 (2020). Sun, X. et al . Scutellarin attenuates doxorubicin-induced cardiotoxicity by inhibiting myocardial fibrosis, apoptosis and autophagy in rats. Chem Biodivers. 20 (1), e202200450; 10.1002/cbdv.202200450 (2023). Podyacheva, E.Y. et al. Profile of molecular markers of cardiac fibrosis in rats exposed to different doxorubicin doses. J Evol Biochem Phys. 59 , 359–368; 10.1134/S0022093023020059 (2023). Abu-Risha, S.E., Sokar, S.S., Elbohoty, H.R. & Elsisi, A.E. Combined carvacrol and cilostazol ameliorate ethanol-induced liver fibrosis in rats: possible role of sirt1/nrf2/ho-1 pathway. Int Immunopharmacol. 116 , 109750; 10.1016/j.intimp.2023.109750 (2023). Mohseni, R., Karimi, J., Tavilani, H., Khodadadi, I. & Hashemnia, M. Carvacrol ameliorates the progression of liver fibrosis through targeting of Hippo and TGF-β signaling pathways in carbon tetrachloride (CCl 4 )-induced liver fibrosis in rats. Immunopharmacol Immunotoxicol. 41 (1), 163-171; 10.1080/08923973.2019.1566926 (2019). Cai, S. et al . Carvacrol alleviates liver fibrosis by inhibiting TRPM7 and modulating the MAPK signaling pathway. Eur J Pharmacol. 898 , 173982; 10.1016/j.ejphar.2021.173982 (2021). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-4381440","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":304911621,"identity":"c29786d2-be25-4f98-9a65-608f740b398b","order_by":0,"name":"Rini Retnosari","email":"","orcid":"","institution":"Universiti Kebangsaan Malaysia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rini","middleName":"","lastName":"Retnosari","suffix":""},{"id":304911623,"identity":"1375a0cf-f504-4850-be09-9e87cc334efa","order_by":1,"name":"Muhamad Adib Abdul Ghani","email":"","orcid":"","institution":"Universiti Kebangsaan 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(0.01 – 100 µg/mL) for 48 h. \u003cstrong\u003e(b)\u003c/strong\u003e H9c2 was pretreated with non-toxic CAR concentrations (0.01 – 0.1 µg/mL) followed by induction with 10 µM DOX for an additional 24 h. Data are shown as mean ± SEM, n = 6.\u0026nbsp; ****p \u0026lt; 0.001 vs. control, \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.01 vs. DOX-induced group.\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4381440/v1/d4b35776648fa8953221a93f.jpg"},{"id":56947616,"identity":"6e6fe02b-f61d-4d20-940b-d5c5a0c19c5c","added_by":"auto","created_at":"2024-05-22 13:51:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":217496,"visible":true,"origin":"","legend":"\u003cp\u003eCAR pre-treatment alleviates systolic and pulse pressure in rat models of DOX-induced acute cardiotoxicity. Data are presented as mean ± SEM for n=8 per group. \u003csup\u003ea\u003c/sup\u003ep \u0026lt; 0.05 vs. Control, \u003csup\u003eb\u003c/sup\u003ep \u0026lt; 0.05 vs. DOX, \u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05 vs. the group at day-0, and \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05 vs. the group at day-14.\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4381440/v1/749b70a419d0a7d29413348d.jpg"},{"id":56947620,"identity":"08a8b078-6427-4c93-a116-e4c4b029b636","added_by":"auto","created_at":"2024-05-22 13:51:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":261759,"visible":true,"origin":"","legend":"\u003cp\u003eCAR pre-treatment ameliorates cardiac dysfunction in Langendorff-perfused DOX-induced acute cardiotoxicity in rat models. (A) LVDP, (B) EDP, (C) dP/dt(max), (D) dP/dt(min). Data are presented as mean ± SEM for n=8 per group.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4381440/v1/d3e08ffa03e348051595f9bd.jpg"},{"id":56947615,"identity":"a80228a4-6ba5-4cd8-9f06-aee452323d80","added_by":"auto","created_at":"2024-05-22 13:51:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":353555,"visible":true,"origin":"","legend":"\u003cp\u003eCAR pre-treatment did not reduce oxidative stress in rat models of DOX-induced acute cardiotoxicity. (A) Total protein, (B) MDA, (C) GSH, (D) SOD. Data are presented as mean ± SEM for n=8 per group. *p \u0026lt; 0.05 vs. Control. \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05 vs. DOX.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4381440/v1/3f31ee6a9ca1d6671d253f21.jpg"},{"id":56947906,"identity":"1acafc23-f4d3-4f16-b1d0-35cab981d429","added_by":"auto","created_at":"2024-05-22 13:59:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":435292,"visible":true,"origin":"","legend":"\u003cp\u003eCAR pre-treatment reduces cardiomyocyte hypertrophy in rat models of DOX-induced acute cardiotoxicity. Representative images from microscopic observation of heart tissue for each group with (I) H\u0026amp;E staining; 400x magnification. Measurement was taken from each group for (II) cardiomyocytes cross-sectional area from H\u0026amp;E staining. \u0026nbsp;Data are presented as mean ± SEM for n=8 per group. \u003csup\u003ea\u003c/sup\u003ep \u0026lt; 0.05 vs. Control, \u003csup\u003eb\u003c/sup\u003ep \u0026lt; 0.05 vs. DOX.\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4381440/v1/ddf1686affde7f9857a3bade.jpg"},{"id":56947907,"identity":"a1ac9b59-7d32-4f8e-9e08-415f094249ca","added_by":"auto","created_at":"2024-05-22 13:59:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":322118,"visible":true,"origin":"","legend":"\u003cp\u003eCAR pre-treatment reduces cardiac fibrosis in rat models of DOX-induced acute cardiotoxicity. Representative images from microscopic observation of heart tissue for each group with (I) picrosirius red staining; 100x magnification. Measurement was taken from each group for (II) fibrotic area. \u0026nbsp;Data are presented as mean ± SEM for n=8 per group. \u003csup\u003ea\u003c/sup\u003ep \u0026lt; 0.05 vs. Control, \u003csup\u003eb\u003c/sup\u003ep \u0026lt; 0.05 vs. DOX.\u003c/p\u003e","description":"","filename":"floatimage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4381440/v1/ef6367b55945e98d91b22b57.jpg"},{"id":59090320,"identity":"372143ef-aecb-4f33-a661-19434f1e5782","added_by":"auto","created_at":"2024-06-26 08:47:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2501667,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4381440/v1/c827f4e7-fb84-48a0-b2e5-f4c47ee3de2c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Deciphering the Protective Effects of Carvacrol Against Doxorubicin-Induced Cardiotoxicity In Vitro and In Vivo","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCancer stands as a formidable global public health challenge, with an estimated 10\u0026nbsp;million fatalities in 2020. Prognostic models predict a worrisome trajectory, with an estimated 28.4\u0026nbsp;million additional cancer cases by 2040. This represents a stunning 47% increase over the 19.3\u0026nbsp;million cases observed in 2020\u003csup\u003e1\u003c/sup\u003e. Furthermore, from 2007 to 2011, Malaysia registered 103,507 new cancer cases, while the ensuing decade saw a considerable and alarming increase\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In 2020 alone, 48,639 new cancer cases were reported\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This alarming trend positions cancer as a persistent and formidable contributor to premature mortality in Malaysia, solidifying its status as a secondary leading cause of untimely death in the nation. Malaysia's changing lifestyle landscape, driven by urbanization and globalization, has been a significant component in this health problem. These lifestyle changes have corresponded with a significant increase in the prevalence of non-communicable diseases (NCDs) among Malaysians, including cancer, diabetes, stroke, heart disease, and hypertension\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eChemotherapy and radiotherapy stand as indispensable cornerstones in the comprehensive treatment of diverse cancer types, playing a pivotal role in substantially enhancing survival rates across a burgeoning patient population\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, despite its importance, chemotherapy faces significant hurdles in patient care. Beyond the formidable barrier of chemoresistance, an accumulating body of evidence highlights the inherent toxicity linked to drug concentrations, adding a layer of complexity to the treatment landscape\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDoxorubicin (DOX) is a powerful and widely used chemotherapeutic medication that can be given alone or in combination with other cancer treatments. However, the impact of DOX extends beyond its intended target, affecting various organs and consequently diminishing the overall quality of life for cancer patients due to the aftermath of chemotherapy side effects. Notably, the heart, a vital organ, bears a considerable brunt during cancer chemotherapy with DOX, potentially inducing heart failure and irreversible impairment of cardiac function, potentially. As of now, the problem of DIC remains a prominent concern, and research on this subject has significantly risen over the years. The prevalent mechanisms associated with DIC included oxidative stress, apoptosis, inflammation, autophagy, mitophagy, endoplasmic reticulum stress, pyroptosis, and ferroptosis. However, oxidative stress remained the primary molecular mechanism of DIC\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFollowing the entry of DOX into the body, redox metabolism generates ROS, which acts as a blasting fuse, and oxidative stress provides the foundation for further DIC molecular pathways. On the one hand, excessive ROS generation and accumulation harm DNA and mitochondrial protein, leading to mitochondrial dysfunction and tissue damage. ROS, on the other hand, can function as a signal to activate the body's defense mechanisms that result in cell death, such as apoptosis, autophagy, necrosis, pyroptosis, iron death, and so on\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e Dexrazoxane is currently the only medicine approved by the FDA to treat cardiotoxicity. Dexrazoxane works by binding to iron in the body before it enters cardiac cells, limiting the production of iron-DOX complexes and minimizing the damaging effects of free radicals on the heart. This is accomplished by reducing lipid membrane peroxidation, which results in decreased cardiotoxicity associated with DOX\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Despite pre-treatment with dexrazoxane, subclinical DIC is not entirely reduced\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. To avoid this worrisome complication of cancer therapy, there is a need for the creation of a more effective cardio-protection approach.\u003c/p\u003e \u003cp\u003eThere is a rising scientific interest in researching the possible cardioprotective properties of natural substances against DIC. CAR, a notable natural ingredient, is prevalent in essential oils generated from aromatic plants. It is generally recognized for its wide range of biological activities, including antioxidant, anti-inflammatory, anticancer, and cardioprotective qualities. CAR's radical scavenging capacity is primarily due to the hydroxyl group (OH) it carries. CAR's weak acid nature allows the donation of hydrogen atoms to unpaired electrons, stabilizing another radical via electron scattering in the molecule's resonance structure\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecently, researchers have demonstrated a keen interest in exploring the potential effectiveness of CAR in alleviating DIC. A study by El-sayed et al.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e revealed notable improvements in cardiac function and oxidative stress parameters when rats were pretreated with CAR before co-administration of a single dose of DOX. This positive outcome is attributed to the multifaceted properties of CAR, encompassing its antioxidant, anti-inflammatory, and antiapoptotic activities. Importantly, corroborating these findings, Khajavi Rad \u0026amp; Mohebbati\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and Jafarinezhad et al.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e demonstrated CAR's efficacy in alleviating oxidative stress damage, improving heart function, and mitigating structural alterations induced by DOX in rats. Despite these encouraging results, it is noteworthy that the effectiveness of CAR in addressing DOX-induced cell death in H9c2 cells and its impact on hypertrophy and cardiac fibrosis in rat models remain unexplored in current research studies.\u003c/p\u003e \u003cp\u003eThis study represents a pioneering effort to investigate the cytotoxic effects of CAR on H9c2 cells and explore the potential cardioprotective properties of CAR in the context of DOX-induced cell death in H9c2 cells. Additionally, our research delves into the cardioprotective role of CAR in mitigating DOX-induced acute cardiotoxicity in rat models, focusing on examining its impact on hypertrophy and cardiac fibrosis.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDrugs and Chemicals\u003c/h2\u003e \u003cp\u003eCAR, DOX, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (USA). Dulbecco's modified Eagle's medium (DMEM), Fetal Bovine Serum (FBS), Phosphate Buffered Saline (PBS), and penicillin were obtained from Thermo Fisher Scientific. KTX (Ketamine-Xylazine/Tiletamine-Zolazepam) was employed for anesthesia during animal handling, and corn oil served as the solvent for CAR. Krebs-Henseleit Buffer (KHB) solution, an artificial blood substitute during the Langendorff isolated heart procedure, comprised sodium chloride (NaCl), sodium hydrogen carbonate (NaHCO\u003csub\u003e3\u003c/sub\u003e), hydrated magnesium sulfate (MgSO\u003csub\u003e4\u003c/sub\u003e∙7H\u003csub\u003e2\u003c/sub\u003eO), glucose (C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e), and hydrated calcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e∙2H\u003csub\u003e2\u003c/sub\u003eO), diluted in distilled water and supplied with 95% gaseous oxygen and 5% gaseous carbon dioxide.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eH9c2 Cell Culture\u003c/h3\u003e\n\u003cp\u003eThe H9c2 cardiomyocyte cell line, procured from Thermo Fisher Scientific, was cultured in DMEM supplemented with 10% FBS and 1% penicillin. Cells were maintained in monolayer culture conditions at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e, with media refreshed every two days. Subculturing was performed when cells reached 80% confluence.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCardiomyocyte Viability\u003c/h2\u003e \u003cp\u003eThe viability of H9c2 cardiomyocytes was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. H9c2 cells were seeded in 96-well plates at a density of 5000 cells/well and allowed to attach for 48 h. To assess CAR's cytotoxicity, cardiomyocytes were treated with CAR at concentrations ranging from 0.01 to 100 \u0026micro;g/mL for 48 h. Following the treatment, MTT reagent (5 mg/mL) was added to the culture medium for 4 h at 37\u0026deg;C. Formazan formed in each well was dissolved in 100 \u0026micro;L of dimethyl sulfoxide (DMSO), and absorbance at 570 nm was measured using a microplate reader. Subsequently, to determine the protective effects of CAR on the viability of DOX-induced cardiomyocytes, H9c2 cells were pre-treated with or without non-toxic CAR concentrations for 24 h prior to induction with 10 \u0026micro;M DOX for another 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and Experimental Procedure\u003c/h2\u003e \u003cp\u003eA total of 48 male Sprague-Dawley rats (200\u0026ndash;250 g) were provided by the Laboratory Animal Resource Unit, Faculty of Medicine, Universiti Kebangsaan Malaysia (UKM). The Animal Ethics Committee of Universiti Kebangsaan Malaysia (UKMAEC) approved animal study and protocols (Approval No: FSK/2020/SATIRAH/23-SEPT./1131-NOV.-2020-NOV. -2022), and all experiments were performed in accordance with the guidelines and regulations. Furthermore, animal handling training was provided by the Animal Resource Unit of the Faculty of Medicine Laboratory, Universiti Kebangsaan Malaysia, through the Introductory Workshop on the Care and Handling of Research Animals (IWLACM). We also hereby confirm that the study is reported following ARRIVE guidelines\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe rats were provided with conventional rodent food and unrestricted access to tap water, maintained under controlled conditions (25\u0026deg;C to 28\u0026deg;C, 12-hour light-dark cycle, and proper ventilation). A one-week adaptation period was allowed for the rats before they were randomly assigned to three groups:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eControl Group (n\u0026thinsp;=\u0026thinsp;8): Animals received corn oil for 14 days, followed by 0.5% DMSO on day 15.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eDoxorubicin Group (DOX) (n\u0026thinsp;=\u0026thinsp;8): Animals received corn oil for 14 days, followed by DOX (15 mg/kg, i.p) on day 15.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eCarvacrol\u0026thinsp;+\u0026thinsp;DOX Group (CAR\u0026thinsp;+\u0026thinsp;DOX) (n\u0026thinsp;=\u0026thinsp;8): This group was pretreated with CAR (50 mg/kg/daily, p.o) for 14 consecutive days\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. On day 15, each animal received DOX (15 mg/kg, i.p) to induce cardiotoxicity.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eA two-day recovery period without intervention was allowed for disease development\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and all rats were sacrificed on day-18 of the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBlood Pressure Monitoring\u003c/h2\u003e \u003cp\u003eRat blood pressure was non-invasively measured via the tail-cuff method using the CODA IITM system (Kent Scientific Corporation, USA) on days 0, 14, and 18. Systolic blood pressure was recorded, and pulse pressure was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLangendorff Perfusion\u003c/h2\u003e \u003cp\u003eAll experimental rats were evaluated for cardiac contractile function using the Langendorff heart preparation, as our lab has previously established\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The rat underwent intraperitoneal administration of anticoagulant heparin (500 IU) and intravenous administration of KTX (1 mg/kg). Following losing consciousness, the heart was isolated, perfused retrogradely with Krebs-Henseleit Buffer, and connected to the Langendorff system. Coronary flow (CF), perfusion pressure (PP), and left ventricle-developed pressure (LVDP) were monitored, and LVDP tracings provided values for dP/dtmax and dP/dtmin. These tracings' values were derived electronically using the PowerLab data acquisition system, and the data was analyzed with LabChart Pro 8.0 (AD Instruments, Australia) software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical Analysis\u003c/h2\u003e \u003cp\u003eBradford's method\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e was used to determine the total protein content of heart tissue. Furthermore, the activity of the enzymes responsible for oxidative stress was measured using a spectrophotometer. The quantification of malondialdehyde (MDA) was based on the reaction of MDA with thiobarbituric acid at 100\u0026deg;C. Thiobarbituric acid reactive substances (TBARS) formation was monitored at 532 nm\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. On the other hand, reduced glutathione (GSH) was detected at 412 nm after reacting homogenate GSH with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) to create a yellow-colored complex\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Furthermore, superoxide dismutase (SOD) activity was assessed at 560 nm based on its ability to prevent ferricytochrome reduction\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eHistopathological Examination of the Heart\u003c/h2\u003e \u003cp\u003eThe left ventricles of rats in separate groups were sectioned and fixed for 24 hours in 10% neutral buffered formalin. Dehydration was obtained after washing with tap water and gradually diluted alcohol. Before embedding in paraffin, samples were cleaned in xylene. Tissue blocks composed of paraffin wax were sliced into 3 m thick pieces using a microtome. Before being examined under a light microscope, the tissue sections were mounted on glass slides, deparaffinized, stained with hematoxylin and eosin, and picrosirius red.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis of Data\u003c/h2\u003e \u003cp\u003eData analysis employed GraphPad Prism 8.0 software, presenting results as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SEM). For normally distributed data, one-way ANOVA with post hoc Tukey was applied, while the Kruskal-Wallis test was used for non-normally distributed data. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCAR Enhanced the Viability of DOX-induced H9c2 Cardiomyocytes\u003c/h2\u003e \u003cp\u003eThe initial step in assessing the cardioprotective potential of CAR involved investigating its cytotoxicity in H9c2 cardiomyocytes. Following 48 h of exposure to 0.01\u0026ndash;100 \u0026micro;g/mL CAR, the MTT assay was employed to determine cardiomyocyte viability. CAR exhibited a remarkable non-toxic profile against cardiomyocytes within the concentration range of 0.01\u0026ndash;0.1 \u0026micro;g/mL. However, reduced cardiomyocyte viability was observed at CAR concentrations ranging from 1\u0026ndash;100 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Therefore, CAR concentrations of 0.01\u0026ndash;0.1 \u0026micro;g/mL were classified as non-toxic and used for the second stage of the MTT assay. In the second stage of the MTT assay, H9c2 cells were pre-treated with or without non-toxic CAR concentrations prior to DOX induction to determine the cardioprotective effect of non-toxic CAR. The results demonstrated that 10 \u0026micro;M DOX significantly reduced cardiomyocyte viability to 46.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55% compared to the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating the potent cardiotoxic effect of DOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Remarkably, pre-treatment with 0.01 \u0026micro;g/mL CAR significantly enhanced the viability of DOX-induced cardiomyocytes compared to the untreated DOX group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, pretreatment with a higher CAR concentration (0.1 \u0026micro;g/mL) did not elicit a further increase in cardiomyocyte viability compared to the DOX group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCAR Preserved Cardiac Structure and Function in DOX-treated Rats\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003eImpact on body weight and heart mass\u003c/h2\u003e \u003cp\u003eTo assess the potential protective role of CAR against DOX-induced acute cardiotoxicity in vivo, we investigated its influence on body weight and heart mass in male rats. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates that a single DOX treatment (15 mg/kg) significantly reduced body weight compared to controls. Intriguingly, the DOX\u0026thinsp;+\u0026thinsp;CAR group exhibited growth rates similar to the DOX group, with no significant differences observed in heart and left ventricle masses among the three groups. This comprehensive dataset indicates that CAR treatment did not mitigate DOX-induced heart mass and body weight alterations.\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\u003eEffect of CAR on body weight gain and heart mass in DOX-treated rats\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=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCON\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDOX\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDOX\u0026thinsp;+\u0026thinsp;CAR\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIncrease in body weight (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25.86\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e16.63\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e18.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeart (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeft ventricle (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eCON: control rats; DOX: doxorubicin; DOX\u0026thinsp;+\u0026thinsp;CAR: doxorubicin and carvacrol co-administration. Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, *significantly different compared to control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBlood pressure modulation\u003c/h2\u003e \u003cp\u003eWe scrutinized blood pressure parameters, including systolic and pulse pressures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), to unravel the impact of CAR on DOX-induced changes. The acute effects of DOX administration on blood pressure were evaluated utilizing blood pressure parameters at day 0, 14, and 18. Notably, no significant differences were observed among the three groups on days 0 and 14. As soon as three days after receiving DOX (day 18), the DOX group displayed a significant decrease in systolic pressures compared to day 14 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with a non-significant reduction in pulse pressure. Strikingly, CAR administration significantly attenuated both blood pressure parameters, restoring them to levels comparable to the control group. This intriguing observation suggests that CAR possesses the potential to counteract DOX-induced blood pressure alterations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEx vivo cardiac function evaluation\u003c/h2\u003e \u003cp\u003eEx vivo assessment of cardiac function, employing Langendorf-perfused isolated rat hearts, provided crucial insights into the interplay between DOX, CAR, and heart contractility. LVDP, a key parameter reflecting cardiac contractility, was scrutinized to unravel the protective effects of CAR. According to Tukey\u0026rsquo;s multiple comparison tests, a single DOX dose (15 mg/kg) marginally improved LVDP and End-Diastolic Pressure (EDP) by 10% and 15%, respectively, compared to the control, though insignificantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Encouragingly, CAR alleviated LVDP and EDP parameters to levels equivalent to the control. Conversely, the maximum rates of contraction and relaxation exhibited a significant decline by 19% and 23%, respectively, after DOX coadministration compared to control. Strikingly, CAR treatment demonstrated a substantial improvement in these parameters, indicating a potential protective effect against DOX-induced cardiac dysfunction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eOxidative stress\u003c/h2\u003e \u003cp\u003eDespite the promising cardiovascular effects observed, CAR could not rescue DOX-induced lipid peroxidation in rats, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. A single DOX injection significantly increased MDA levels by 89% in the heart, accompanied by significant decreases in GSH cardiac contents (27%) and SOD activities (35%) compared to the control group. However, CAR administration for 14 days before DOX coadministration did not significantly mitigate MDA levels or enhance GSH cardiac contents. Intriguingly, CAR even decreased SOD activity by 11.35% compared to the DOX group, suggesting a nuanced interplay between CAR and oxidative stress pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCAR reverses cardiomyocyte hypertrophy\u003c/h2\u003e \u003cp\u003eHistological examination of left ventricle heart sections provided visual insights into the cardioprotective effects of CAR against DOX-induced damage. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, DOX administration induced severe histological alterations, including obvious wavy degeneration of cardiac muscle fibers as seen by the arrows in (image B), the presence of inflammatory infiltration as seen by the arrows in (image C), coagulative necrosis of cardiac muscle fibers as seen by the arrows in (image D) showing pale segments of cardiac tissue, the presence of blood cells (arrow) between muscle fiber in (image E) indicates hemorrhage in DOX group, arrow in (image F) indicates vascular congestion that was evident in DOX. Impressively, pretreatment with CAR ameliorated these abnormalities, demonstrating its efficacy in minimizing histological damage. Still, wavy degeneration of cardiac muscle fibers and coagulative necrosis of cardiac muscle fibers (Image G and H, respectively) were observed. As supported by quantification of the cardiomyocyte area size (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e(II)\u003c/b\u003e), the region of cardiomyocytes was significantly enlarged after DOX administration by 13.4% compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Pretreatment with CAR significantly reduced the cardiomyocyte's cross-sectional area (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eReduction of DOX-induced cardiac fibrosis\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e(I)\u003c/b\u003e shows microscopic images at 100x magnification comparing collagen deposition (fibrosis) in the perivascular area of coronary arteries in the left ventricular heart section (A, C, and E), as well as in the interstitial area of left ventricular heart tissue sections (B, D, and F) in each group. Image (A and B) shows normal histology in the control group, but image (C and D) shows severe fibrotic damage after DOX administration compared to the control group. Treatment with CAR reduced the fibrotic damage, which is more visually similar to the control group. The quantification of the fibrotic area supported this evidence. After DOX administration, the fibrosis area was significantly enlarged compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e(II)\u003c/b\u003e. In contrast, CAR treatment significantly reduced the fibrotic area compared to the DOX group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). CAR treatment markedly reduced DOX-induced cardiac fibrosis\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study delves into the potential protective effects of CAR against DIC, examining both cellular and rat models. Initial investigations revealed that CAR did not induce cytotoxicity in cardiomyocytes at 0.01 and 0.1 \u0026micro;g/mL concentrations. Remarkably, 0.01 \u0026micro;g/mL CAR enhanced the viability of DOX-induced cardiomyocytes, presenting a novel insight into the protective properties of CAR against DIC in this cellular context.\u003c/p\u003e \u003cp\u003eIn extending our exploration to rat models, the influence of CAR on DIC was evaluated by performing functional, biochemical, and histological examinations. The induction of cardiomyopathy in rats was achieved by administering a single cumulative intraperitoneal dose of DOX at 15 mg/kg to create a DOX-induced acute cardiotoxicity model, a well-established method documented in previous studies\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In addition, the risk of cardiotoxicity with DOX becomes significant when cumulative doses exceed 200 mg/m\u003csup\u003e2\u003c/sup\u003e, equivalent to 5 mg/kg in humans\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe initiated this by evaluating the increase in body weight of rats following pretreatment with CAR, both with and without co-administration of DOX. Rats in the DOX group exhibited a significant decrease in body weight, consistent with the findings of Junior et al.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Intriguingly, pre-administration of CAR at 50 mg/kg also led to a decrease in body weight, aligning with Lee et al.'s observation\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e that CAR suppresses food intake, consequently reducing the percentage of body weight gain. However, in contrast to the cardiotoxicity group, the CAR-treated group exhibited a slight, though statistically non-significant, percentage increase, contradicting the findings of Cota et al.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, who employed a higher CAR dose (100 mg/kg) before DOX treatment. Left ventricular hypertrophy (LVH), identified as an adaptive response to pathological volume or pressure aiming to regulate pump function by circulatory demand, was evident in the cardiotoxicity and treatment groups, resulting in left ventricular remodeling\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. However, intriguingly, when rats were pre-treated with CAR, there were no discernible differences in the heart and left ventricle weights. This finding contradicts the outcomes of studies by Jamhiri et al.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, which demonstrated that treating rats with CAR at 50 and 75 mg/kg/day prevented hypertension induced by abdominal aortic banding, decreasing the heart weight-to-body weight ratio.\u003c/p\u003e \u003cp\u003eA thorough examination of the blood pressure findings revealed a general decrease in SBP in the DOX group. These results are consistent with previous research indicating impaired cardiac performance in rat models treated with DOX and align with clinical observations\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. It is important to note that DOX-induced acute cardiotoxicity typically manifests during or immediately after treatment and usually involves reversible hypotension\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Interestingly, CAR administration effectively mitigated all blood pressure parameters, restoring them to levels comparable to the control group. A previous study showed that treatment with CAR induces hypotension and bradycardia in hypertensive rats, influencing the cardiovascular system and promoting antihypertensive mechanisms\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study also showed cardiac dysfunction in Langendorff-perfused rat hearts isolated from DOX-administered rats. LVDP, EDP, LV\u0026thinsp;+\u0026thinsp;dP/dt, and LV -dP/dt as markers for LV contractile markers were impaired in the DOX group, indicating contractile dysfunction, in line with previous results\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The study identified several mechanisms underlying cardiac dysfunction triggered by DOX, including heightened generation of reactive oxygen species (ROS) resulting in oxidative stress damage, disturbed calcium ion (Ca\u003csup\u003e2+\u003c/sup\u003e) homeostasis, and compromised expression of genes associated with the apoptosis and necrosis of cardiomyocytes\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis discrepancy might be attributed to structural remodeling of the heart, suggesting an adaptive response to the oxidative stress induced by DOX administration. Although DOX is commonly associated with dilated cardiomyopathy, it is noteworthy that hypertrophic adaptation of cardiomyocytes can occur in the early stages of congestive heart failure progression\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In addition, the DOX group's EDP was higher than the control group, likely due to DOX-induced cardiomyocyte hypertrophy, promotion of fibrosis in the heart\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, and mitochondrial dysfunction\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Both factors can impact the EDP value, with fibrosis potentially causing a loss of heart compliance and mitochondrial dysfunction disrupting average ATP generation in the heart. A recent study showed that CAR mitigated DOX-induced cardiac dysfunction. This observation may be attributed to CAR's beneficial properties as an anti-inflammatory and antioxidative agent, reducing the toxic effects of DOX on the heart\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Contrary to expectations, the mean dP/dtmin of the CAR\u0026thinsp;+\u0026thinsp;DOX group was the lowest among the three groups, surpassing the mean of the control group. This steep dP/dtmin slope may be associated with CAR's relaxant effect, which affects not only vascular smooth muscle\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e but also influences viable remaining cardiac myocytes by blocking calcium ion channels on the surface of cardiomyocytes\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe central mechanistic underpinning of DIC is well-established and primarily rooted in the induction of oxidative stress\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. This current investigation delved into the intricate interplay of DOX-induced oxidative stress by examining lipid peroxidation, as evidenced by notable alterations in MDA levels, GSH content, and SOD activity. Regrettably, our results indicate that pretreatment with CAR did not mitigate the lipid peroxidation induced by DOX. Specifically, CAR failed to reduce MDA levels significantly and did not impart a significant increase in GSH content. Unexpectedly, CAR pretreatment led to a reduction in SOD activity in cardiac tissue, diverging from the anticipated antioxidant effects attributed to CAR. This outcome contrasts with the findings reported by El-Sayed et al.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, possibly attributed to variations in the experimental design, including divergent doses of DOX and CAR employed in our investigation. El-Sayed et al.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e demonstrated that CAR administration (25 mg/kg) for 14 days before exposure to DOX (10 mg/kg) ameliorated oxidative stress markers. However, our study, employing higher doses of both DOX (15 mg/kg) and CAR (50 mg/kg), revealed a lack of enhancement in oxidative stress parameters with CAR pretreatment.\u003c/p\u003e \u003cp\u003eThe discrepancy in outcomes prompts an exploration into the nuanced dose-dependent effects of CAR in the context of DOX-induced oxidative stress. While CAR is recognized for its antioxidant properties, interacting with cell membrane phospholipids to mitigate lipid peroxidation and reduce nitric oxide production\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, it is essential to acknowledge that, at elevated concentrations, CAR can exhibit a shift in its role towards that of a prooxidant. In this capacity, CAR may potentially induce oxidative stress, either through the generation of reactive oxygen species (ROS) or by depleting cellular antioxidants\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The intricate balance between antioxidant and prooxidant activities of CAR may be influenced by dosage and the specific context of the experimental conditions.\u003c/p\u003e \u003cp\u003eConsequently, in our study, the inability of CAR to mitigate DIC through its anticipated antioxidant activity suggests a nuanced interplay between CAR, DOX, and oxidative stress. These findings underscore the importance of considering dose-dependent effects and highlight the need for a comprehensive understanding of the intricate mechanisms that govern CAR's actions in the specific context of DIC. Future investigations may benefit from elucidating the threshold at which CAR transitions from an antioxidant to a prooxidant, paving the way for a more refined understanding of its therapeutic potential in managing oxidative stress-associated cardiotoxicity.\u003c/p\u003e \u003cp\u003eThe DOX group exhibited various histological alterations in the histological observation of H\u0026amp;E staining, including wavy degeneration of cardiac muscle fibers, inflammatory infiltration, coagulative necrosis of cardiac muscle fibers, hemorrhage, and vascular congestion. Wavy degeneration and coagulative necrosis are indicative of ischemia. These findings parallel studies by El-Agamy et al.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and Cota et al.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, where the induction of cardiotoxicity with DOX resulted in similar histopathological changes, such as necrosis, myocyte rupture, degeneration, edema, and vascular congestion. Remarkably, the administration of CAR at 50 mg/kg/day demonstrated a significant alleviation of hypertrophy induced by DOX. A notable reduction in the cross-sectional areas of cardiomyocytes evidenced this. However, it is worth noting that despite this positive effect, some degree of wavy degeneration and coagulative necrosis of cardiac muscle fibers persisted. Recent studies have shown that CAR administration prevents aortic banding-induced left ventricular hypertrophy in rats. In hypertrophied left ventricles, an increase in cardiomyocyte apoptosis is evident, as indicated by elevated mRNA levels of the pro-apoptotic protein Bcl-2-associated death promoter (BAD) and the anti-apoptotic protein Bcl-2. CAR demonstrated a notable capacity to decrease the expression of the pro-apoptotic factor BAD while simultaneously enhancing the levels of anti-apoptotic factors, specifically Bcl-2 and Bcl-xL, within the left ventricular tissue. This dual effect suggests a promising role for CAR in modulating apoptotic processes associated with ventricular hypertrophy\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe antiapoptotic of CAR was also evidenced in Yu et al.\u0026rsquo;s result\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In rats, CAR exhibited cardioprotective activity against acute myocardial infarction by downregulating caspase-3 and Bax activity and increased Bcl-2 expression at the protein level. Moreover, on a molecular and cellular scale, cardiac hypertrophy is intricately associated with oxidative stress, arising from the imbalance between the antioxidant defense system within the heart and the excessive generation of reactive oxygen species (ROS). Oxidative stress can activate apoptotic factors such as proapoptotic members of the BCL-2 family and caspases\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Consequently, CAR demonstrated potential cardioprotective effects in DOX-induced hypertrophy, likely attributed to its anti-apoptotic and antioxidant characteristics.\u003c/p\u003e \u003cp\u003eIn-depth histological examination through picrosirius red staining illuminated a profound increase in red color staining within the perivascular and interstitial regions of the left ventricle in the DOX group, suggesting an augmented collagen deposition compared to the control group. This observation resonates with Tanaka et al.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, who highlighted that picrosirius red staining, particularly in perivascular areas, serves as a distinctive marker for the initial stages of reactive fibrosis. In the current study, administering 15 mg/kg of DOX to induce cardiotoxicity likely propelled the heart samples beyond the early fibrotic stages into a more advanced phase, as evidenced by the conspicuous collagen deposition in both perivascular and interstitial cardiac regions.\u003c/p\u003e \u003cp\u003eThis progression is further substantiated by a notable expansion in the fibrotic area within the DOX group compared to the control group, a finding consistent with the work of Chang et al.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e The collective evidence underscores the profound impact of DOX on cardiac fibrosis, suggesting a trajectory beyond mere initiation toward a more complex and extensive fibrotic response. The probable mechanistic action could involve TGF-β1 activation and α-SMA expression as key contributors to the observed cardiac fibrosis in the pathogenesis of DOX-induced fibrotic changes\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurther complexity arises from the sustained elevation of critical fibrotic markers in animals subjected to the highest dose of DOX (15 mg/kg). Increased expression of type I and II collagen, endothelin-1 (ET1), fibroblast growth factor 4 (FGF4), and tumor necrosis factor-alpha (TNFα) could be involved in the inflammatory processes. This dual manifestation suggests a dynamic interplay between fibrosis and inflammation against the backdrop of DOX's pronounced cardiotoxic effects, as elucidated by Podyacheva et al.\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eUnexpectedly, pretreatment with CAR exhibited a slight increase in red color staining, indicating a minor degree of collagen deposition. Furthermore, a pivotal and promising outcome emerged as a significant decrease in fibrosis area was discerned in the CAR-pretreated group compared to the DOX-alone group. These findings resonate with the outcomes of prior investigations, providing a consistent narrative wherein CAR has demonstrated a remarkable ability to ameliorate cardiac fibrosis\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn elucidating the putative mechanisms behind CAR's anti-fibrotic effects on DIC, existing literature posits that CAR exerts its protective influence by inhibiting inflammatory cytokines, specifically tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In a distinct investigation, the promising potential of CAR in ameliorating ethanol-induced hepatic fibrosis was prominently demonstrated. The study illuminated a multifaceted mechanism wherein CAR exerted significant therapeutic effects by alleviating oxidative stress, downregulating the pro-fibrotic growth factor (TGF-β1), augmenting the anti-inflammatory cytokine interleukin-10 (IL-10), inhibiting collagen synthesis as evidenced by reduced 4-hydroxyproline (4-HYP) levels, and attenuating hepatic stellate cell (HSC) activation, as manifested by the diminished expression of alpha-smooth muscle actin (α-SMA) in hepatocytes. These findings underscore CAR's profound antioxidant, anti-inflammatory, and anti-fibrotic properties, providing valuable insights into its potential therapeutic utility\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, CAR's capacity to impede the progression of liver fibrosis through its interaction with the TGF-β signaling pathway was substantiated by Mohseni et al.\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e Their findings echoed the multifaceted anti-fibrotic and anti-inflammatory attributes of CAR, further solidifying its potential as a therapeutic intervention against hepatic fibrosis. In a parallel investigation by Cai et al.\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, CAR emerged as a pivotal player in reducing carbon tetrachloride (CCl\u003csub\u003e4\u003c/sub\u003e)-induced hepatic fibrogenesis in rats. The study demonstrated that CAR exerted its effects by downregulating the expression of key fibrotic markers such as α-SMA, collagen type I alpha 1 (Col1α1), platelet-derived growth factor (PDGF) receptor, and TGF-β1 receptor. These comprehensive findings underscore CAR's remarkable potential in countering hepatic fibrosis through intricate molecular mechanisms.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe current study demonstrates that CAR reduces DIC in vivo and in vitro. CAR enhances the viability of DOX-induced cardiomyocytes. Additionally, CAR attenuates DOX-induced reductions in blood pressure, cardiac hypertrophy, and cardiac fibrosis in rats. These effects are likely attributed to CAR\u0026rsquo;s anti-fibrotic, anti-inflammatory, anti-apoptotic, and antioxidant properties. Nevertheless, further research is necessary to elucidate the precise mechanisms through which CAR mitigates DOX's cardiotoxic effects. Understanding the effect of this compound on cardiac function could pave the way for its potential utilization in managing cardiovascular complications associated with DOX therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledged the financial assistance provided by\u0026nbsp;Universiti Kebangsaan Malaysia (Grant no: DIP-2020-020).\u0026nbsp;The authors also acknowledge the Universitas Negeri Malang and Gifu University for the PhD Scholarship awarded to Rini Retnosari.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledged the financial assistance provided by Universiti Kebangsaan Malaysia (Grant no: DIP-2020-020). 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Carvacrol alleviates liver fibrosis by inhibiting TRPM7 and modulating the MAPK signaling pathway. \u003cem\u003eEur J Pharmacol. \u003c/em\u003e\u003cstrong\u003e898\u003c/strong\u003e, 173982; 10.1016/j.ejphar.2021.173982 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cardioprotection, cardiomyocyte, carvacrol, doxorubicin-induced cardiotoxicity, H9c2 cells","lastPublishedDoi":"10.21203/rs.3.rs-4381440/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4381440/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDoxorubicin (DOX), a widely used chemotherapy, extends its impact beyond cancer cells, notably affecting the heart, leading to substantial concerns about DOX-induced cardiotoxicity (DIC). However, subclinical DIC remains unresolved, necessitating advanced cardio-protection strategies in cancer therapy. Recent research explores carvacrol (CAR), a natural substance with antioxidant and anti-inflammatory properties, as a potential shield against DIC. However, further exploration is warranted, particularly concerning hypertrophy and cardiac fibrosis. This study investigated CAR\u0026rsquo;s potential cardioprotective properties against DIC in H9c2 cardiomyocytes and rats. Induction with DOX reduced cardiomyocyte viability, while pretreatment with 0.01 \u0026micro;g/mL CAR enhanced the viability of DOX-induced cardiomyocytes. Meanwhile, administration of DOX induced adverse effects in rats, causing decreased total heart weight and left ventricular mass, and lowered blood pressure. DOX also caused cardiac dysfunction, lipid peroxidation, hypertrophy, and fibrosis. In rat models, CAR pretreatment effectively mitigated DOX-induced reductions in blood pressure, hypertrophy, and cardiac fibrosis. However, the pretreatment kept the heart function, oxidative stress, and antioxidant enzymes unaltered. In conclusion, the results show that CAR could be an adjuvant to reduce DIC by ameliorating cardiac fibrosis and hypertrophy.\u003c/p\u003e","manuscriptTitle":"Deciphering the Protective Effects of Carvacrol Against Doxorubicin-Induced Cardiotoxicity In Vitro and In Vivo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-22 13:51:47","doi":"10.21203/rs.3.rs-4381440/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ef458d5f-e4ce-4a9a-b0b2-6dfa9184ce71","owner":[],"postedDate":"May 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":32191366,"name":"Health sciences/Cardiology"},{"id":32191367,"name":"Physical sciences/Chemistry"}],"tags":[],"updatedAt":"2024-06-26T08:38:57+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-22 13:51:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4381440","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4381440","identity":"rs-4381440","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}