Norharmane Mitigates Doxorubicin-Induced Heart Failure via PPARγ- Mediated Suppression of Oxidative Stress and Apoptosis

Norharmane Mitigates Doxorubicin-Induced Heart Failure via PPARγ- Mediated Suppression of Oxidative Stress and Apoptosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Norharmane Mitigates Doxorubicin-Induced Heart Failure via PPARγ- Mediated Suppression of Oxidative Stress and Apoptosis HINA SAQIB, YU HOU, RUI WANG, HAOMING YIN, RONGHUI HOU, JINA ZHAO, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8619773/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 We studied the cardioprotective effects of norharman (NH), a natural alkaloid, against doxorubicin (DOX)-induced myocardial damage and its mechanism of action through PPARγ signaling. Clinical efficacy is limited by dose-dependent cardiotoxicity (which can lead to cardiomyopathy and heart failure), highlighting the need for safe cardioprotective agents. This study combined in vivo and in vitro experimental designs. C57BL/6 mice were treated with DOX alone or in combination with NH. Cardiac function was analyzed by echocardiography. Serum creatine kinase and lactate dehydrogenase levels were also measured as indicators of myocardial damage. Myocardial fibrosis, inflammation, and oxidative stress were assessed by histological and biochemical analyses. Cultured cardiomyocytes were exposed to DOX ± NH to evaluate cell viability, reactive oxygen species formation, and apoptosis-associated protein expression. The selective PPARγ inhibitor GW9662 was used to determine pathway specificity. DOX significantly reduced myocardial ejection fraction and fractional shortening, increased oxidative stress and damage biomarkers, and induced myocardial fibrosis. NH co-treatment protected cardiac function, reduced oxidative damage, and attenuated fibrosis and inflammation. In cardiomyocytes, NH increased cell viability, inhibited reactive oxygen species production, upregulated Bcl-2, and downregulated Bax, cleaved caspase-3, and TNF-α. GW9662 reversed NH’s protective effects, confirming PPARγ involvement. These results indicate that NH, as a novel PPARγ activator, effectively attenuates DOX-induced oxidative stress and apoptosis, highlighting its potential as a natural therapeutic agent for preventing anthracycline-associated cardiotoxicity. Doxorubicin Cardiotoxicity Norharmane Apoptosis Oxidative stress PPARγ Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. INTRODUCTION Heart failure is a complex and multifactorial disease categorized by the heart’s inability to adequately perfuse meet the metabolic requirements of the body, emerge in reduced cardiac output including organ perfusion (Khan, Shahid et al. 2022 ). A wide range of pathologies can interrupt this balance, lead to heart failure. Important risk factors include myocardial infarction, chronic hypertension, valvular heart disease, diabetes, obesity, and cardiotoxicity caused by chemotherapeutic agents such as doxorubicin (DOX)(Azer, AlSwaidan et al. 2015 ) . Regardless of advances in medical therapy, heart failure remains a significant contributor to global morbidity and mortality, drastically reducing patients quality of life and placing a substantial burden on health care system (Arnold 2023 , Sapna, Raveena et al. 2023 ). Recent pharmacological approaches for the treatment of heart failure contain Angiotensin-1 receptor antagonists (like losartan and candesartan), angiotensin-converting enzyme (ACE) inhibitors (like enalapril and captopril), beta-blockers, diuretics, and angiotensin receptor blockers (like losartan and candesartan LCZ696) (Beghini, Sammartino et al. 2025 ). These agents mainly focus neurohormonal pathways to moderate the heart's workload, regulate blood pressure, and expand cardiac function. Nevertheless, their long-term effectiveness is restricted, and many patients endure from side effects such as cough, angioedema, renal impairment, and hypotension, leading to poor adherence and suboptimal outcomes (Flather, Yusuf et al. 2000 , Edwards, Price et al. 2023 ). Doxorubicin (DOX) is a legitimate chemotherapeutic agent mainly used to treat various cancers, comprise breast, lung, and ovarian cancer(Varghese, Ambrose et al. 2013 , Hassan, Zulkifli et al. 2015 ) However, its clinical implementation is severely bounded by its dose-dependent cardiotoxic effects, which can trigger heart failure and other cardiovascular complications (Lipshultz, Colan et al. 1991 , Szabó, Volk et al. 2021 , Andreev, Balakin et al. 2024 ). This cardiotoxicity not only reduces patient survival but also significantly decrease the value of life of cancer survivors (Kappel, Tumlinson et al. 2025 ). The main mechanisms of DOX-induced cardiotoxicity embrace reactive oxygen species (ROS) initiation, mitochondrial dysfunction, apoptosis, and inflammatory responses(Woodward 2003 , Linders, Dias et al. 2024 , Vitale, Marzocco et al. 2024 ). DOX produces an excessive amount of ROS in cardiomyocytes, which overwhelms the heart's antioxidant defenses and leads to lipid peroxidation, protein modification, and mitochondrial dysfunction. DOX activates pro-inflammatory pathways, including the NF-κB and NLRP3 inflammasome pathways(Zhang, Hu et al. 2020 ). This causes the body to release cytokines including TNF-α, IL-1β, and IL-6, which make heart inflammation worse and encourage fibrosis. This inflammatory response causes more oxidative stress and kills heart cells, which is a major cause of heart failure and loss of heart muscle(Riehle and Bauersachs 2019 ). Cardiomyocyte death in DOX-treated hearts is largely determined by the intrinsic apoptotic pathway, which is typified by caspase activation, cytochrome c release, and loss of mitochondrial membrane potential(Kim, Kim et al. 2006 ). Norharman (NH) is a naturally occurring β-carboline alkaloid widely found in medicinal plants such as Eurycoma longifolia, Peganum harmala and Banisteriopsis caapi. Structurally, NH belongs to the indole alkaloid family and is characterized by a tricyclic pyrido[3,4-b] indole scaffold, which contributes to its diverse biological activities (Yu, Shen et al. 2025 ). In recent years, NH has attracted considerable pharmacological interest due to its antioxidant, anti-inflammatory, antimicrobial, anticancer and neuroprotective properties (Xie, Cao et al. 2021 ). Several studies have shown that NH can effectively scavenge reactive oxygen species (ROS), upregulate endogenous antioxidant mechanisms and modulate redox-sensitive signaling pathways, including the Nrf2/HO-1 axis (Liu, Han et al. 2023 ). Moreover, its ability to inhibit proinflammatory mediators such as NF-κB, TNF-α and IL-6 highlights its therapeutic potential in diseases associated with oxidative and inflammatory damage (Roh and Sohn 2018 ). Since oxidative stress, inflammation, and apoptosis are the main mechanisms of DOX-induced cardiac injury (Sheibani, Azizi et al. 2022 ), incorporation of NH into a model of DOX-induced cardiotoxicity will allow a mechanistic assessment of its ability to preserve cardiac structure and function during chemotherapy 2. MATERIAL METHODS 2.2 Reagents Norharmane (NH) (Cat.N0. HY-W008566) and Doxorubicin (DOX) (S17092) was provided by Yuanye Biotechnology (Shanghai, China). Creatine kinase (CK) (A032-1-1), lactate dehydrogenase (LDH) assay kits (Cat.NoA020-1-2) were purchased from Nanjing Jincheng (Nanjing, China). Malondialdehyde (MDA) kit (Cat.NO.S0131S) and Masson Trichrome staining kit were purchased from Solarbio (Beijing, China) (CAT. No. G1340). 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Cat.No.M8180 and Picrosirius Red were obtained from Solarbio(S8060) (Beijing, China). TUNEL detection kit (Cat.No.C1089), Reactive Oxygen Species Assay (ROS) Kit (S0033), GSH Assay Kit (Cat.NO.S0053), Mitochondrial Membrane Potential Assay Kit with TMRE (C2001S) and Total Superoxide Dismutase Assay Kit (S0101) with WST-8 (SOD) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). 2.3 Animal design Eight-weeks-old male C57BL/6J mice(weighing 22-25g) were obtained from the animal center of Dalian Medical University. All experimental procedure were approved by the Institutional Animal Care and Use Committee of the Dalian Medical University and conducted in accordance with established ethical guidelines. The mice were randomly allocated into four groups (n = 6 per group): Control group, received intragastric corn oil and an intraperitoneal injection of saline (100µl/mice). NH group received oral administration of (NH) at 10 mg/kg/day and intraperitoneal saline. DOX model group received intraperitoneal injection of doxorubicin (DOX) at 2 mg/kg/2days for 20 days. DOX plus NH group, Received oral administered NH (10 mg/kg/day) along with DOX injection (2 mg/kg/2day) for 20 days. At the end of the treatment period, mice were anesthetized using tribromoethanol. Electrocardiograms (ECG) recordings were performed and then blood, and cardiac tissue samples were collected for further analysis. 2.4 Electrocardiography (ECG) detection Twenty-four hours after the final DOX injection, the mice were anesthetized with intraperitoneal injection of tribromoethanol (dosage). ECG recording was conducted. Their limbs were attached to four electrode clamps, and heart function parameters were recorded using Power Lab software (ADI instruments).The following interval heart rate (HR), QRS interval, QT interval (an indirect measure of the duration between ventricular depolarization and repolarization), and RRI (the interval from the peak of one QRS complex to the peak of the next) of each mouse were recorded(Podyacheva, Kushnareva et al. 2021 ) . 2.5 Histological and fibrosis Analysis After being preserved in 4% paraformaldehyde, the heart tissue was dehydrated, embedded in paraffin, and cut into 5 µm thick slices for staining with hematoxylin and eosin (H&E), Masson's trichrome, Picrosirius Red, and TUNEL tests. After that, a microscope (Carl Zeiss, Germany) was used to look at and film the slices(Fischer, Jacobson et al. 2008 ). 2.6 Biochemical analysis The Lactate Dehydrogenase (LDH) assay, using an LDH assay kit from Sigma-Aldrich (USA), measured the release of LDH, which shows that the cell membrane is damaged. We used commercial test kits from Beyotime Biotechnology in China to measure the activity of superoxide dismutase (SOD) and the levels of malondialdehyde (MDA). Reduced Glutathione (GSH): We used a colorimetric test kit (Cayman Chemical, USA) to quantify GSH levels. Following the manufacturer's directions, we used a microplate reader (Enspire2300, USA) to measure the activity of CK. 2.7 Cell culture and determination of cellular proliferation H9C2 cells were purchased from ATCC (Manassas, USA) and cultured in DMEM medium supplemented with 10% fetal bovine serum and 100-U/ml penicillin. Cell viability was assessed using the MTT assay (Sigma-Aldrich, USA). After 24 hours of drug treatment, 20 µL of MTT solution (5 mg/mL in PBS) was added to each well and incubated at 37°C for 4 hours. The resulting formazan crystals were dissolved in 150 µL of dimethyl sulfoxide (DMSO) and the absorbance was measured at 570 nm using a microplate reader (Biotech, USA). Cell viability was expressed as a percentage of the control group(Xu, Li et al. 2025 ). 2.8 Intracellular ROS detection Intracellular ROS levels were measured with the Reactive Oxygen Specific Assay Kit (Shanghai Beyotime Institute of Biotechnology Institute) according to the manufacturer's instructions.). Cells were incubated with 10 µM DCFH-DA at 37°C for 30 minutes in the dark. Fluorescence intensity was measured using a fluorescence microplate reader (excitation: 488 nm, emission: 525 nm) (Biotech, USA)(Amaldoss, Pandzic et al. 2022 ). Fluorescence’s Images were captured using an Olympus BX63 fluorescence and confocal microscopy. 2.9 TMRE assay H9C2 cells were seeded in 12-well plates and culture until they reached the appropriate density mitochondrial membrane potential (MMP) was assessed using the TMRE dye. A total of 500 µL of TMRE working solution, (diluted1:1000 in serum-free DMEM medium), was added to each well. After incubating for 15 minutes, the cells were washed twice with DMEM and observed under a fluorescence microscope. 2.10 Western blotting assay H9C2 cells were lysed with RIPA buffer (Solarbio, Beijing) and the supernatant from the lysate was collected after centrifugation and stored as the protein sample. Western blotting was performed to assess protein expression of apoptosis related proteins (Caspase-3, Bax, and Bcl-2) and inflammatory marker (TNF-α). Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with specific primary antibodies (Abcam, USA). Proteins signals were detected using enhanced chemiluminescence (Bio-Rad, USA). 2.11 Tunnel staining measurement After deparaffinization and rehydration, heart tissue sections were incubated with DNase-free protein K (20 µg/mL) at 37°C for 20 minutes and then washed three times with PBS for 10 minutes each. A TdT-labeled nucleotide mixture was added and incubated in the dark at 37°C for 1 hour. Finally, the sections were counterstained with DAPI. 2.12 Cell death and apoptosis analysis Propidium Iodide (PI) Staining: PI staining was performed to assess cell membrane integrity. Cells were stained with 5 µg/mL PI (Sigma-Aldrich, USA) and analyzed by flow cytometry 2.13 Data analysis All data were expressed as the mean ± standard error of mean (SEM). Statistical analysis was performed using GraphPad Prism 7.0 software (San Diego, USA) and analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test when comparing multiple groups. The significant difference was considered if P < 0.05. 3. Results 3.1 NH Alleviates Doxorubicin-Induced Cardiac Dysfunction in Mice To clarify the cardioprotective effect of NH, a heart failure model in doxorubicin (DOX)-induced cardiac injury was established by intravenous injection of DOX with concomitant treatment with NH (Fig. 1 A, B). Morphological examination of whole animals and excised hearts shows that DOX-treated mice are emaciated, with visibly atrophied hearts, whereas mice in the DOX + NH group exhibits significant preservation of cardiac structure (Fig. 1 C, D). Furthermore, DOX administration significantly reduced the heart weight to body weight ratio, indicating cardiac atrophy (Fig. 1 E). This reduction was substantially restored with NH treatment. To validate the model and evaluate the functional continuation of NH, ECG recordings were performed in each experimental group (Fig. 1 F). DOX treated mice exhibits a marked QT interval(Fig. 1 G) and prolongation of QRS (Fig. 1 H) and, reflecting reduced heart rate(Fig. 1 I), disturbed electrical activity, and impaired cardiac functions. Markers of cardiac injury are also assessed. Serum creatine kinase (CK) levels are significantly elevated in DOX-treated mice (Fig. 1 J), indicating myocardial damage. Significantly, Norharmane substantially reduces this increase. Similarly, lactate dehydrogenase (LDH) levels increases in the DOX group and decreases in the DOX + NH group (Fig. 1 K). These data suggest that NH alleviates doxorubicin-induced cardiac dysfunction in mice. 3.2 Mitigated Doxorubicin-Induced Histopathological Cardiac Injury in Mice Histopathological analyses are performed on cardiac tissue specimens derived from experimental mice to evaluate the influence of NH on DOX-induced myocardial injury. H&E staining shows that the cardiomyocytes in the control (CON) and NH-only groups have typical shapes, with a well-organized cellular structure and regular alignment (Fig. 2 A). However, the group that got DOX shows clear pathological changes, such as disorganized cardiac fibers, more gaps between cells, and less clear cellular architecture. Interestingly, these pathological changes were significantly reversed in the DOX + NH group, showing that NH protects the heart tissue from damage. To further evaluate the cardioprotective effect of NH we used Masson staining which reveals equally pronounced fibrotic remodeling in the DOX group as in the CON group. NH administration significantly attenuates this fibrosis, resulting in collagen levels like controls ( Fig. 2 B, C). These findings are further confirmed by Sirius Red staining to evaluate the deposition of collagen fibers. Quantitative analysis of red-stained fibrotic areas relative to total tissue area shows minimal collagen accumulation in both the CON and NH groups, with occasional perivascular deposition observed (Fig. 2 D, E). In contrast, significant collagen accumulation was observed in the interstitial spaces and around blood vessels in the DOX group. NH treatment significantly reduces collagen accumulation in these regions, supporting the antifibrotic potential of collagen. These data revealed that NH mitigates DOX-induced histopathological cardiac injury in mice. 3.3 NH Attenuates Doxorubicin-Induced Cardiomyocyte Toxicity In Vitro To assess the protective effects of norharmane (NH) against DOX-induced cardiomyocyte toxicity, cell viability studies are performed under diverse treatment conditions. The protective dose of cardiomyocytes of NH (0.01–0.5µM) and cardiomyocyte toxic dose of DOX (0.5µM) was confirmed by MTT assay (Fig. 3 A, B). As shown in Fig. 3 C, NH dose-dependently increases H9C2 cell viability under DOX exposure. Consistent with the MTT results, morphological results shows that NH reverses the cell death induced by DOX (Fig. 3 D, E). Cells exhibit a significant structural damage in DOX alone group, reflecting by cell shrinkage, membrane blebs, and overall morphological deterioration. In contrast, cells treated with NH showed preserved morphology. Those findings reveal that NH attenuates DOX-induced cardiomyocyte toxicity in vitro. 3.4 NH Reduces Doxorubicin-Induced Oxidative Stress in Vitro and in Vivo. Next, we evaluate the role of Norharmane (NH) in DOX-induced oxidative stress in vitro. As shown in Fig. 4 A and B. DOX treatment resulted in a significant increase in intracellular ROS levels, as indicated by strong green fluorescence. In contrast, co-treatment with NH significantly reduced ROS accumulation in DOX-treated cells, suggesting strong antioxidant activity. As we known, high levels ROS accumulation impairs the mitochondrial member potential (MMP)(Gorospe, Carvalho et al. 2023 ). Then, TMRE staining assay was used (Fig. 4 C, D). As expected, DOX caused mitochondrial depolarization, which was shown by the drop in TMRE fluorescence. NH restored the MMP in DOX-treated cells, indicating the preservation of mitochondrial function. In vivo, DOX caused a significant decrease of GSH/GSSG (Fig. 4 E, F) in heart tissues and serum. Nevertheless, NH reversed these changes. Meanwhile, NH decreased the enhanced MDA concentration induced by DOX (Fig. 4 G, H). In accordance with the GSH/GSSG results, NH increases the activity of SOD after DOX exposure in heart tissues and serum (Fig. 4 I, J). All these results implicated that NH reduces oxidative stress induced by DOX in vitro and in vivo. 3.5 NH Inhibits Doxorubicin-Induced Apoptosis in Cardiomyocytes To investigate the effect of NH against DOX-induced cardiotoxicity, transcriptome analysis was first performed. Gene ontology (GO) enrichment revealed that DOX treatment significantly activated apoptosis-related biological processes, including the regulation of apoptotic signaling and post-translational protein modification (Fig. 5 A). Western blot analysis also confirmed that DOX treatment significantly increased the expression of the pro-apoptotic proteins Bax, cleaved caspase-3, and TNF-α, along with a decrease of the anti-apoptotic protein Bcl-2 in H9C2 cells (Fig. 5 B-F). In contrast, co-treatment with NH significantly reversed these changes, suggesting that NH inhibits DOX-induced cell apoptosis. In confirmation of these results, TUNEL staining in heart tissue sections showed a significant increase in the number of TUNEL-positive nuclei upon DOX exposure, suggesting increased apoptosis, whereas cotreatment with NH significantly reduced the number of apoptotic cells (Fig. 5 G, H). Consistent with these results, PI staining of H9C2 cells showed a significant increase in cell death upon DOX treatment, which was significantly attenuated by NH (Fig. 5 I, J). Taken together, these results suggest that NH effectively attenuates DOX-induced cardiomyocyte apoptosis both in vitro and in vivo. 3.6 NH Attenuates Doxorubicin-Induced Cardiocytes Oxidative Stress and Apoptosis via Upregulating PPARγ To investigate the underlying molecular mechanism of NH-mediated cardio protection, a Venn diagram was constructed to identify the common target genes between NH and DOX-induced heart injury (DIH). A total of 37 overlapping genes were identified (Fig. 6 A). Protein–protein interaction (PPI) network analysis revealed that several of these genes were functionally associated with PPARγ signaling (Fig. 6 B). Western blotting results show that DOX markedly downregulates PPARγ expression, whereas NH treatment restores its levels (Fig. 6 C, D). MTT analysis confirmed that NH significantly improved cell viability compared with DOX alone, and this effect was diminished by GW9662 (Fig. 7 E). Apoptotic cell death, as assessed by PI staining, was significantly increased in DOX-treated cells but was attenuated by NH; however, inhibition of PPARγ reversed the anti-apoptotic effect of NH (Fig. 6 E, F). Similarly, intracellular ROS accumulation, as assessed by DCFH-DA staining, increased after DOX exposure but was significantly decreased by NH treatment, an effect that was reversed by GW9662 (Fig. 6 G, H). Further analysis of apoptosis- and inflammation-related proteins showed that DOX increased the expression of pro-apoptotic Bax and cleave-caspase3 and pro-inflammatory TNF-α, while decreasing the expression of anti-apoptotic Bcl-2. Treatment with NH reversed these changes, whereas co-treatment with GW9662 abolished the protective effects (Fig. 6 I-N). Taken together, these results indicate that NH potentiates DOX-induced apoptosis and oxidative stress in H9C2 cells through a PPARγ-dependent mechanism. 3.7 NH can Enhance the Sensitivity to DOX in A549 Lung Cancer Cell NH itself has some antitumor effects, and to understand the effect of NH on the antitumor effect of DOX, we performed an MTT experiment to explore it. The results showed that when DOX and NH were used alone to treat A549 lung cancer cells, they could inhibit the viability of A549 cells, and the higher the drug concentration, the stronger the inhibitory effect; When DOX and NH were used in combination, NH could enhance the inhibitory effect of DOX on the viability of A549 cells. The results indicate that NH can enhance the sensitivity of A549 lung cancer cells to DOX (Fig. 7 A–C). 4. DISCUSSION DOX, originally extracted from Streptomyces paucities, has been wildly used as an anti-cancer chemotherapeutic drug to treat solid tumors and acute leukemias(Starobin, Danford et al. 2009 ). Although DOX has been identified as one of the most effective and safe antitumor medicines, the spectrum of its clinical application is somewhat restricted due to its high toxicity to liver, kidney and heart (Arcamone, Cassinelli et al. 1969 , Thorn, Oshiro et al. 2011 ). DOX can induce production of a large amount of ROS and subsequent mitochondrial dysfunctions and cell damages, particularly to cardiomyocytes(Yang, Teves et al. 2014 ). Therefore, DOX can be wildly used to induce heart failure in animal models. In current study, we used DOX-induced heart failure in mice to determine if NH treatment can protect against cardiac dysfunctions. Consistent with previous studies, we also found that DOX caused abnormal ECG, increased serum CK and LDH levels, and induced cardiac fibrosis (Fig. 2 , Fig. 3 ). More importantly, we observed that the damages to heart caused by DOX were substantially attenuated by NH treatment. The cardio protection of NH should be attributed to its inhibition of ROS production, inflammation and apoptosis. Primary electrocardiographic studies provided preliminary evidence of cardiac stress in DOX-treated mice, showing prolonged QRS and QTc intervals and reduced heart rate, which are typically associated with electrical remodeling and early cardiomyopathy(Christidi and Brunham 2021 ). Interestingly, ECG parameters and the ability to maintain electrophysiological stability were preserved with co-administration of NH This finding is consistent with previous studies demonstrating the role of natural antioxidants in stabilizing cardiac conduction during toxic stress(Van Ommen, Kessler et al. 2021 ) . Additional support was provided by biochemical markers. CK and LDH levels in cardiac lesions were significantly increased in the DOX group, while NH administration significantly decreased these markers, mimicking myocardial protection. Histological evaluations confirmed that the HE-stained myocardial tissue in the DOX group showed significant cell disorganization and vacuolization, while the NH maintained a normal structure. These data suggest that NH may suppress fibrotic remodeling, most likely by interfering with pro-fibrotic mediators such as TGF-β1 or matrix metalloproteinases(Saadat, Noureddini et al. 2021 ). The main contributor of oxidative stress to DOX-induced cardiotoxicity, which induces reactive oxygen species (ROS) responses and endogenous antioxidant activity, is myocyte membrane degradation (Tadokoro, Ikeda et al. 2020 ). Excessive ROS in mitochondria induces apoptotic degradation(Liu, Han et al. 2019 ). Cardiomyocyte apoptosis has been demonstrated to be the direct cause of DOX-induced cardiotoxicity(Songbo, Lang et al. 2019 ). NH models significantly improved the GSH/GSSG ratio, increased total SOD activity, and decreased MDA synthesis in serum and tissues. High levels of antioxidants are essential for lipid peroxidation. Microscopic studies confirmed that ROS are not sequestered and reflect mitochondrial membrane potential (TMRE assay) and mitochondria are hemostatically and oxidatively induced. The anti-apoptotic potential of NH could not be evaluated by TUNEL and PI staining, as there were significantly lower numbers of apoptotic nuclei in the NH + DOX group than in the DOX group. Western blot analysis was based on the NH module and the critical regulation of apoptosis, which upregulates Bcl-2 and downregulates Bax. Caspase-3 cleavage restores the balance between pro- and anti-apoptotic signals. These results are consistent with previous studies on natural cardioprotective compounds, as it is resistant to curcumin, which further induces the effects of mitochondrial apoptosis (Koss-Mikołajczyk, Todorovic et al. 2021 ). Our mechanistic studies showed that NH-induced cardio protection is strongly mediated by peroxisome proliferator-activated receptor gamma (PPARγ) activation. In vitro, NH restored cardiomyocyte viability, reduced ROS accumulation, and inhibited apoptosis, as evidenced by increased Bcl-2 and decreased levels of Bax, cleaved caspase-3, and TNF-α. Importantly, these effects were reversed in the presence of GW9662, a selective PPARγ antagonist. This confirms that PPARγ activation is essential for the cardioprotective effects of NH (Kim and Yang 2013 ). Its activation in cardiomyocytes is increasingly recognized as a therapeutic target to ameliorate oxidative damage and apoptosis. Therefore, our findings place NH among the natural small molecules that act as PPARγ agonists, thus providing a novel mechanistic rationale for its protective efficacy. The anti-apoptotic effects of NH, demonstrated by TMRE and PI staining, are consistent with its ability to activate PPARγ, a transcription factor known to modulate the apoptotic gene network. (Yuan, Yi et al. 2024 ). By targeting both apoptotic and inflammatory signaling pathways in a PPARγ-dependent manner, NH disrupted the self-reinforcing cycle of oxidative stress and cardiomyocyte death that underlies anthracycline-induced cardiotoxicity(Xiang, Xin et al. 2024 ). Our findings identify NH as a potent cardioprotective agent that exerts beneficial effects primarily through activation of PPARγ signaling. The abrogation of NH effects by GW9662 provides compelling evidence for this mechanism. Since synthetic PPARγ agonists are already used clinically in metabolic diseases, our data suggest a high potential translation of NH as a natural PPARγ modulator in cancer patients at risk of cardiotoxicity. Future studies should investigate whether NH alters the antitumor efficacy of DOX and explores its long-term safety and pharmacokinetic profile, thus allowing for clinical translation. 5. CONCLUSION In summary, our results demonstrate that (NH), a natural beta-carboline alkaloid, provides effective protection against doxorubicin-induced cardiotoxicity through activation of PPARγ signaling pathways. NH preserves cardiac structure and function by reducing oxidative stress, suppressing inflammatory responses, and inhibiting mitochondrial apoptosis. Interestingly, pharmacological suppression of PPARγ with GW9662 abrogated these benefits, confirming PPARγ activation as the primary mechanism of the cardioprotective effect of NH. Our findings suggest that NH may serve as a useful adjunct to alleviate anthracycline-induced cardiac injury. Further research is needed to elucidate the pharmacokinetics, long-term safety, and potential interactions with the antitumor activity of doxorubicin to facilitate clinical implementation of NH. Abbreviations Bax B-cell lymphoma-2 associated X protein Bcl-2 B-cell lymphoma-2 BF Bright Field CK-MB Creatine kinase-MB DOX Doxorubicin EF Ejection fraction FS Fractional shortening GSH Glutathione (Reduced Glutathione) H E Hematoxylin Eosin LDH Lactate dehydrogenase MDA Malondialdehyde NH Norharmane PI. Propidium Iodide PPARγ Peroxisome proliferator-activated receptor gamma ROS Reactive oxygen species SOD Superoxide Dismutase TNF-α Tumor necrosis factor-alpha TMRE. Tetramethyl rhodamine Ethyl Ester Declarations Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Author Contribution Credit author statementSaqib Hina, Yu Hou, and Rui Wang designed and conducted the experiments. Saqib Hina wrote the manuscript. 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"Doxorubicin-induced apoptosis is exacerbated by MG53 and associated with altered Akt signaling in H9c2 cells." Molecular Pharmacology 107 (5): 100032. Yang, F., S. S. Teves, C. J. Kemp and S. Henikoff (2014). "Doxorubicin, DNA torsion, and chromatin dynamics." Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 1845 (1): 84-89. Yu, L., N. Shen, J. Ren, H. Xin and Y. Cui (2025). "Resource distribution, pharmacological activity, toxicology and clinical drugs of β-Carboline alkaloids: An updated and systematic review." Fitoterapia 180 : 106326. Yuan, H., N. Yi, D. Li, C. Xu, G.-R. Yin, C. Zhuang, Y.-J. Wang and S. Ni (2024). "PPARγ regulates osteoarthritis chondrocytes apoptosis through caspase-3 dependent mitochondrial pathway." Scientific Reports 14 (1): 11237. Zhang, X., C. Hu, C.-Y. Kong, P. Song, H.-M. Wu, S.-C. Xu, Y.-P. Yuan, W. Deng, Z.-G. Ma and Q.-Z. Tang (2020). "FNDC5 alleviates oxidative stress and cardiomyocyte apoptosis in doxorubicin-induced cardiotoxicity via activating AKT." Cell Death & Differentiation 27 (2): 540-555. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8619773","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":592804131,"identity":"4de5d1ae-c49e-491f-952a-9b5c48d70165","order_by":0,"name":"HINA SAQIB","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYJCCwwwGMMYPGyDF2HiAeC2MPWkgLQ0EtTAjGGyHwQy8WviOH394uKDgjrx8++GNhwt4ztutbT8MtKXGJhqXFskzCQmHZxg8M9xwJq3g8AyL28nbziQCtRxLy23AocXgQMKBwzwGhxk3MOQYHObhuZ1sdgCohbHhMG4t5x82gLTYz+9/A9TCdi7ZDCiCX8uNZAaQlsSGGyBb2A7Ymd0gYIvkjWdgLckbbjwrODyzJznB7AbQlgQ8fuE7n/74M8+fw7bz+5M3fy74YWdvdj794YMPNTY4tSBHAThCE8EqE3Apx6bFHp/iUTAKRsEoGJkAANnObuHOcr1YAAAAAElFTkSuQmCC","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"HINA","middleName":"","lastName":"SAQIB","suffix":""},{"id":592804132,"identity":"125551e8-bf6f-4da3-a3d4-d65c2bfe0b63","order_by":1,"name":"YU HOU","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"YU","middleName":"","lastName":"HOU","suffix":""},{"id":592804133,"identity":"4cc28c3a-7f31-4443-8e0d-5bb30541b6af","order_by":2,"name":"RUI WANG","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"RUI","middleName":"","lastName":"WANG","suffix":""},{"id":592804134,"identity":"f88bda12-96b7-48c5-b6e9-511956e64831","order_by":3,"name":"HAOMING YIN","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"HAOMING","middleName":"","lastName":"YIN","suffix":""},{"id":592804135,"identity":"7f04c1c7-d4bd-417d-ab2b-2f93936ad130","order_by":4,"name":"RONGHUI HOU","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"RONGHUI","middleName":"","lastName":"HOU","suffix":""},{"id":592804136,"identity":"1acffa8f-61c3-4942-bf76-8c409c42b114","order_by":5,"name":"JINA ZHAO","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"JINA","middleName":"","lastName":"ZHAO","suffix":""},{"id":592804137,"identity":"a8bbcb60-60f0-4660-9767-50c9dacbbe9a","order_by":6,"name":"HUAIDONG 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WANG","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"MAI","middleName":"","lastName":"WANG","suffix":""},{"id":592804142,"identity":"33edd6bb-b412-4ddf-809e-697e47df36ea","order_by":11,"name":"HASNAIN BALTTE","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"HASNAIN","middleName":"","lastName":"BALTTE","suffix":""},{"id":592804143,"identity":"e8e68878-b4b6-4e5f-8e55-63f31214557a","order_by":12,"name":"NEHA BAQI","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"NEHA","middleName":"","lastName":"BAQI","suffix":""},{"id":592804144,"identity":"e580f2ab-801b-4ea6-b2b9-f1e84e0028b9","order_by":13,"name":"JINJIN PAN","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"JINJIN","middleName":"","lastName":"PAN","suffix":""},{"id":592804146,"identity":"360497f9-3d67-4de4-be28-fb0a89caac97","order_by":14,"name":"YUHUI YUAN","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"YUHUI","middleName":"","lastName":"YUAN","suffix":""}],"badges":[],"createdAt":"2026-01-16 14:13:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8619773/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8619773/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102910597,"identity":"9a6405a6-4c05-4a32-8dea-e6c250a2dbee","added_by":"auto","created_at":"2026-02-18 10:06:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":662470,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of NH treatment on cardiac function and histopathology in mice. \u003c/strong\u003eThe chemical structure of NH (A) and its administration regimen in a DOX-induced mouse cardiotoxicity model(B). (C) Images of mouse in each group. (D) Heart images of mouse in each group. (E) Quantitative data pertaining to heart weight is presented in panel. (F) ECG assessments were performed on all mice, with representative graphs displayed in panel. (G-I) Quantitative data pertaining to QTc interval, and QRS interval, heart rate is presented in panel. At the conclusion of the experiment, blood and heart tissue samples were collected from the mice. The blood was processed to obtain serum, which was then analyzed for serum LDH (J,) and CK (K) activity levels. n=6. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. ns: no significance.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8619773/v1/3c7601ad20ead54bd0894b1b.png"},{"id":102964436,"identity":"2692269c-b125-4ddf-a383-57dcf6d3c676","added_by":"auto","created_at":"2026-02-19 04:22:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2386196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNH alleviates DOX-induced pathological damage\u003c/strong\u003e. (A) H\u0026amp;E staining images of heart tissue sections from mice in each group. (B, C) Masson staining and corresponding quantitative analysis of tissue sections from each group. (D, E) Sirius Red staining and corresponding quantitative analysis of tissue sections from each group. Scale bar = 100 μm. n=6. **p \u0026lt; 0.01, ***p \u0026lt; 0.001. ns: no significance.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8619773/v1/57b704eed65eec237c52fb78.png"},{"id":102963568,"identity":"089a01d2-47e7-4d02-a0e1-28bd511f6e8c","added_by":"auto","created_at":"2026-02-19 04:19:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":751056,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNH alleviates DOX-induced cardiomyocyte toxicity\u003c/strong\u003e. (A) MTT assay was performed to detect the H9C2 cell viability after NH treatment for 12 hours. (B) MTT assay was performed to detect the H9C2 cell viability after DOX treatment for 12hours. (C) Effect of 12-hour DOX treatment on H9C2 cell viability following 24-hour NH pretreatment. (D, E) Bright-field and H\u0026amp;E staining images of H9C2 cells after 24-hour NH pretreatment and 12-hour DOX treatment. Scale bar = 100 μm. n=3. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. ns: no significance.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8619773/v1/0c39281f128d361945162335.png"},{"id":102910589,"identity":"880756c9-7be7-43eb-8dec-a4d77430a712","added_by":"auto","created_at":"2026-02-18 10:06:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1639989,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNH attenuates DOX-induced oxidative stress in vitro and in vivo.\u003c/strong\u003e (A, B) Intracellular ROS levels in H9C2 cells and corresponding quantitative analysis. n=3. Scale bar = 50 μm. (C, D) MMP levels in H9C2 cells and corresponding quantitative analysis. n=3. Scale bar = 100 μm. (E, F) GSH levels in mouse heart tissue and serum. n=6. (G, H) Total MDA levels in mouse heart tissue and serum. n=6. (I, J) SOD levels in mouse heart tissue and serum. n=6. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. ns: no significance.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8619773/v1/fbf10dd27d2bae730d4a6954.png"},{"id":102910595,"identity":"08580e59-9ef7-4e75-9f13-3f4c14e08fc5","added_by":"auto","created_at":"2026-02-18 10:06:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1257347,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNH alleviates DOX-induced apoptosis both in vitro and in vivo. \u003c/strong\u003e(A) Gene ontology (GO) enrichment analysis of DOX-induced heart injury (DIH). (B) Representative Western blotting images of apoptosis-related proteins (Bax, Bcl-2, cleaved caspase-3, total caspase-3, and TNF-α) in H9C2 cells under different treatment conditions (CON, NH, DOX, and DOX+NH), with β-actin as an internal control. Quantification of protein expression levels of Bax (C), cleaved caspase-3 (D), Bcl-2 (E), and TNF-α (F) n=3. (G) TUNEL staining of heart tissue showing apoptotic nuclei (red) and DAPI-labeled nuclei (blue). Scale bar = 100 μm. (H) IP and bright field (BF) images demonstrate DOX-induced cytotoxicity and the protective effect of NH in H9C2 cells. Scale bar = 100 μm. (I) Quantification of relative fluorescence intensity of PI. n=3 (J) Percentage of TUNEL-positive apoptotic cells. n=6. *p \u0026lt; 0.05, **p \u0026lt; 0.01, **p \u0026lt; 0.001. ns: no significance.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8619773/v1/2256b90fb9e7142e47666c7b.png"},{"id":102963704,"identity":"f1473a8e-553e-454e-81ce-1dd292f49a19","added_by":"auto","created_at":"2026-02-19 04:20:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1814294,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNorharmane protects cardiomyocytes by activating PPARγ\u003c/strong\u003e. (A) Venn diagram showing the overlap of potential target genes between NH and DOX-induced heart injury (DIH), identifying 37 shared targets. (B) Protein–protein interaction (PPI) network analysis of the overlapping targets illustrating their functional connections, with PPARγ-related nodes highlighted. (C) Representative Western blot images of PPARγ expression. (D) Quantitative analysis of PPARγ protein levels. (E) Cell viability assays show that NH can counteract DOX-induced cytotoxicity. (F) PI-stained images of apoptotic/necrotic cells. Scale bar = 100 μm. (G) Quantitative analysis of PI-positive cells. (H) Fluorescent images of ROS. Scale bar = 100 μm. (I) Quantitative analysis of intracellular ROS levels. (J) Representative Western blot images of Bax, Bcl-2, caspase-3, and TNF-α. Quantitative analysis of Bax (K), Bcl-2 (L), TNF-α (M), and cleaved caspase-3 (N). n = 3. *p \u0026lt; 0.05, **p \u0026lt; 0.01, **p \u0026lt; 0.001. ns: no significance.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8619773/v1/c43402e7ed6d3e684c7ee3b5.png"},{"id":102910590,"identity":"d59e9125-ee1d-479d-b409-ae94cbab39b2","added_by":"auto","created_at":"2026-02-18 10:06:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":160750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNH can increase the sensitivity to DOX in A549 lung cancer cell\u003c/strong\u003e. MTT assay showing cell viability in (A) DOX-treated cells, (B) NH treated cells, and (C) DOX + NOR co-treated cells. n = 3; **p \u0026lt; 0.01, **p \u0026lt; 0.001. ns: no significance.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8619773/v1/7487bf557dc918f7a0fa7180.png"},{"id":102910585,"identity":"259d14b1-4f32-4dd2-a7da-87ff8f5876a3","added_by":"auto","created_at":"2026-02-18 10:06:07","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":447696,"visible":true,"origin":"","legend":"\u003cp\u003e\u0026nbsp;Legend not included with this version.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8619773/v1/bdbfc893634f519f2ca94edc.png"},{"id":105832436,"identity":"b2c39c17-8855-45f1-936f-cdba55f2d6e0","added_by":"auto","created_at":"2026-03-31 14:57:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10859778,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8619773/v1/29e944ca-656d-43b6-b3c9-6813ff933116.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Norharmane Mitigates Doxorubicin-Induced Heart Failure via PPARγ- Mediated Suppression of Oxidative Stress and Apoptosis","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eHeart failure is a complex and multifactorial disease categorized by the heart\u0026rsquo;s inability to adequately perfuse meet the metabolic requirements of the body, emerge in reduced cardiac output including organ perfusion (Khan, Shahid et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A wide range of pathologies can interrupt this balance, lead to heart failure. Important risk factors include myocardial infarction, chronic hypertension, valvular heart disease, diabetes, obesity, and cardiotoxicity caused by chemotherapeutic agents such as doxorubicin (DOX)(Azer, AlSwaidan et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) .\u003c/p\u003e \u003cp\u003eRegardless of advances in medical therapy, heart failure remains a significant contributor to global morbidity and mortality, drastically reducing patients quality of life and placing a substantial burden on health care system (Arnold \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Sapna, Raveena et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Recent pharmacological approaches for the treatment of heart failure contain Angiotensin-1 receptor antagonists (like losartan and candesartan), angiotensin-converting enzyme (ACE) inhibitors (like enalapril and captopril), beta-blockers, diuretics, and angiotensin receptor blockers (like losartan and candesartan LCZ696) (Beghini, Sammartino et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These agents mainly focus neurohormonal pathways to moderate the heart's workload, regulate blood pressure, and expand cardiac function. Nevertheless, their long-term effectiveness is restricted, and many patients endure from side effects such as cough, angioedema, renal impairment, and hypotension, leading to poor adherence and suboptimal outcomes (Flather, Yusuf et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Edwards, Price et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDoxorubicin (DOX) is a legitimate chemotherapeutic agent mainly used to treat various cancers, comprise breast, lung, and ovarian cancer(Varghese, Ambrose et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Hassan, Zulkifli et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) However, its clinical implementation is severely bounded by its dose-dependent cardiotoxic effects, which can trigger heart failure and other cardiovascular complications (Lipshultz, Colan et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1991\u003c/span\u003e, Szab\u0026oacute;, Volk et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Andreev, Balakin et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This cardiotoxicity not only reduces patient survival but also significantly decrease the value of life of cancer survivors (Kappel, Tumlinson et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The main mechanisms of DOX-induced cardiotoxicity embrace reactive oxygen species (ROS) initiation, mitochondrial dysfunction, apoptosis, and inflammatory responses(Woodward \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Linders, Dias et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Vitale, Marzocco et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). DOX produces an excessive amount of ROS in cardiomyocytes, which overwhelms the heart's antioxidant defenses and leads to lipid peroxidation, protein modification, and mitochondrial dysfunction. DOX activates pro-inflammatory pathways, including the NF-κB and NLRP3 inflammasome pathways(Zhang, Hu et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This causes the body to release cytokines including TNF-α, IL-1β, and IL-6, which make heart inflammation worse and encourage fibrosis. This inflammatory response causes more oxidative stress and kills heart cells, which is a major cause of heart failure and loss of heart muscle(Riehle and Bauersachs \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Cardiomyocyte death in DOX-treated hearts is largely determined by the intrinsic apoptotic pathway, which is typified by caspase activation, cytochrome c release, and loss of mitochondrial membrane potential(Kim, Kim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNorharman (NH) is a naturally occurring β-carboline alkaloid widely found in medicinal plants such as Eurycoma longifolia, Peganum harmala and Banisteriopsis caapi. Structurally, NH belongs to the indole alkaloid family and is characterized by a tricyclic pyrido[3,4-b] indole scaffold, which contributes to its diverse biological activities (Yu, Shen et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In recent years, NH has attracted considerable pharmacological interest due to its antioxidant, anti-inflammatory, antimicrobial, anticancer and neuroprotective properties (Xie, Cao et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Several studies have shown that NH can effectively scavenge reactive oxygen species (ROS), upregulate endogenous antioxidant mechanisms and modulate redox-sensitive signaling pathways, including the Nrf2/HO-1 axis (Liu, Han et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, its ability to inhibit proinflammatory mediators such as NF-κB, TNF-α and IL-6 highlights its therapeutic potential in diseases associated with oxidative and inflammatory damage (Roh and Sohn \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Since oxidative stress, inflammation, and apoptosis are the main mechanisms of DOX-induced cardiac injury (Sheibani, Azizi et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), incorporation of NH into a model of DOX-induced cardiotoxicity will allow a mechanistic assessment of its ability to preserve cardiac structure and function during chemotherapy\u003c/p\u003e"},{"header":"2. MATERIAL METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Reagents\u003c/h2\u003e \u003cp\u003eNorharmane (NH) (Cat.N0. HY-W008566) and Doxorubicin (DOX) (S17092) was provided by Yuanye Biotechnology (Shanghai, China). Creatine kinase (CK) (A032-1-1), lactate dehydrogenase (LDH) assay kits (Cat.NoA020-1-2) were purchased from Nanjing Jincheng (Nanjing, China). Malondialdehyde (MDA) kit (Cat.NO.S0131S) and Masson Trichrome staining kit were purchased from Solarbio (Beijing, China) (CAT. No. G1340). 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Cat.No.M8180 and Picrosirius Red were obtained from Solarbio(S8060) (Beijing, China). TUNEL detection kit (Cat.No.C1089), Reactive Oxygen Species Assay (ROS) Kit (S0033), GSH Assay Kit (Cat.NO.S0053), Mitochondrial Membrane Potential Assay Kit with TMRE (C2001S) and Total Superoxide Dismutase Assay Kit (S0101) with WST-8 (SOD) were purchased from Beyotime Institute of Biotechnology (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Animal design\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eEight-weeks-old male C57BL/6J mice(weighing 22-25g) were obtained from the animal center of Dalian Medical University. All experimental procedure were approved by the Institutional Animal Care and Use Committee of the Dalian Medical University and conducted in accordance with established ethical guidelines. The mice were randomly allocated into four groups (n\u0026thinsp;=\u0026thinsp;6 per group): Control group, received intragastric corn oil and an intraperitoneal injection of saline (100\u0026micro;l/mice). NH group received oral administration of (NH) at 10 mg/kg/day and intraperitoneal saline. DOX model group received intraperitoneal injection of doxorubicin (DOX) at 2 mg/kg/2days for 20 days. DOX plus NH group, Received oral administered NH (10 mg/kg/day) along with DOX injection (2 mg/kg/2day) for 20 days.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eAt the end of the treatment period, mice were anesthetized using tribromoethanol. Electrocardiograms (ECG) recordings were performed and then blood, and cardiac tissue samples were collected for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Electrocardiography (ECG) detection\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTwenty-four hours after the final DOX injection, the mice were anesthetized with intraperitoneal injection of tribromoethanol (dosage). ECG recording was conducted. Their limbs were attached to four electrode clamps, and heart function parameters were recorded using Power Lab software (ADI instruments).The following interval heart rate (HR), QRS interval, QT interval (an indirect measure of the duration between ventricular depolarization and repolarization), and RRI (the interval from the peak of one QRS complex to the peak of the next) of each mouse were recorded(Podyacheva, Kushnareva et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) .\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Histological and fibrosis Analysis\u003c/h2\u003e \u003cp\u003eAfter being preserved in 4% paraformaldehyde, the heart tissue was dehydrated, embedded in paraffin, and cut into 5 \u0026micro;m thick slices for staining with hematoxylin and eosin (H\u0026amp;E), Masson's trichrome, Picrosirius Red, and TUNEL tests. After that, a microscope (Carl Zeiss, Germany) was used to look at and film the slices(Fischer, Jacobson et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Biochemical analysis\u003c/h2\u003e \u003cp\u003eThe Lactate Dehydrogenase (LDH) assay, using an LDH assay kit from Sigma-Aldrich (USA), measured the release of LDH, which shows that the cell membrane is damaged. We used commercial test kits from Beyotime Biotechnology in China to measure the activity of superoxide dismutase (SOD) and the levels of malondialdehyde (MDA). Reduced Glutathione (GSH): We used a colorimetric test kit (Cayman Chemical, USA) to quantify GSH levels. Following the manufacturer's directions, we used a microplate reader (Enspire2300, USA) to measure the activity of CK.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cell culture and determination of cellular proliferation\u003c/h2\u003e \u003cp\u003eH9C2 cells were purchased from ATCC (Manassas, USA) and cultured in DMEM medium supplemented with 10% fetal bovine serum and 100-U/ml penicillin. Cell viability was assessed using the MTT assay (Sigma-Aldrich, USA). After 24 hours of drug treatment, 20 \u0026micro;L of MTT solution (5 mg/mL in PBS) was added to each well and incubated at 37\u0026deg;C for 4 hours. The resulting formazan crystals were dissolved in 150 \u0026micro;L of dimethyl sulfoxide (DMSO) and the absorbance was measured at 570 nm using a microplate reader (Biotech, USA). Cell viability was expressed as a percentage of the control group(Xu, Li et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Intracellular ROS detection\u003c/h2\u003e \u003cp\u003eIntracellular ROS levels were measured with the Reactive Oxygen Specific Assay Kit (Shanghai Beyotime Institute of Biotechnology Institute) according to the manufacturer's instructions.). Cells were incubated with 10 \u0026micro;M DCFH-DA at 37\u0026deg;C for 30 minutes in the dark. Fluorescence intensity was measured using a fluorescence microplate reader (excitation: 488 nm, emission: 525 nm) (Biotech, USA)(Amaldoss, Pandzic et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Fluorescence\u0026rsquo;s Images were captured using an Olympus BX63 fluorescence and confocal microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.9 TMRE assay\u003c/h2\u003e \u003cp\u003eH9C2 cells were seeded in 12-well plates and culture until they reached the appropriate density mitochondrial membrane potential (MMP) was assessed using the TMRE dye. A total of 500 \u0026micro;L of TMRE working solution, (diluted1:1000 in serum-free DMEM medium), was added to each well. After incubating for 15 minutes, the cells were washed twice with DMEM and observed under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Western blotting assay\u003c/h2\u003e \u003cp\u003eH9C2 cells were lysed with RIPA buffer (Solarbio, Beijing) and the supernatant from the lysate was collected after centrifugation and stored as the protein sample. Western blotting was performed to assess protein expression of apoptosis related proteins (Caspase-3, Bax, and Bcl-2) and inflammatory marker (TNF-α). Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with specific primary antibodies (Abcam, USA). Proteins signals were detected using enhanced chemiluminescence (Bio-Rad, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Tunnel staining measurement\u003c/h2\u003e \u003cp\u003eAfter deparaffinization and rehydration, heart tissue sections were incubated with DNase-free protein K (20 \u0026micro;g/mL) at 37\u0026deg;C for 20 minutes and then washed three times with PBS for 10 minutes each. A TdT-labeled nucleotide mixture was added and incubated in the dark at 37\u0026deg;C for 1 hour. Finally, the sections were counterstained with DAPI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Cell death and apoptosis analysis\u003c/h2\u003e \u003cp\u003ePropidium Iodide (PI) Staining: PI staining was performed to assess cell membrane integrity. Cells were stained with 5 \u0026micro;g/mL PI (Sigma-Aldrich, USA) and analyzed by flow cytometry\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Data analysis\u003c/h2\u003e \u003cp\u003eAll data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM). Statistical analysis was performed using GraphPad Prism 7.0 software (San Diego, USA) and analyzed using one-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s post-hoc test when comparing multiple groups. The significant difference was considered if P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 NH Alleviates Doxorubicin-Induced Cardiac Dysfunction in Mice\u003c/h2\u003e \u003cp\u003eTo clarify the cardioprotective effect of NH, a heart failure model in doxorubicin (DOX)-induced cardiac injury was established by intravenous injection of DOX with concomitant treatment with NH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Morphological examination of whole animals and excised hearts shows that DOX-treated mice are emaciated, with visibly atrophied hearts, whereas mice in the DOX\u0026thinsp;+\u0026thinsp;NH group exhibits significant preservation of cardiac structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). Furthermore, DOX administration significantly reduced the heart weight to body weight ratio, indicating cardiac atrophy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). This reduction was substantially restored with NH treatment. To validate the model and evaluate the functional continuation of NH, ECG recordings were performed in each experimental group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). DOX treated mice exhibits a marked QT interval(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) and prolongation of QRS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) and, reflecting reduced heart rate(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), disturbed electrical activity, and impaired cardiac functions.\u003c/p\u003e \u003cp\u003eMarkers of cardiac injury are also assessed. Serum creatine kinase (CK) levels are significantly elevated in DOX-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ), indicating myocardial damage. Significantly, Norharmane substantially reduces this increase. Similarly, lactate dehydrogenase (LDH) levels increases in the DOX group and decreases in the DOX\u0026thinsp;+\u0026thinsp;NH group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). These data suggest that NH alleviates doxorubicin-induced cardiac dysfunction in mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Mitigated Doxorubicin-Induced Histopathological Cardiac Injury in Mice\u003c/h2\u003e \u003cp\u003eHistopathological analyses are performed on cardiac tissue specimens derived from experimental mice to evaluate the influence of NH on DOX-induced myocardial injury. H\u0026amp;E staining shows that the cardiomyocytes in the control (CON) and NH-only groups have typical shapes, with a well-organized cellular structure and regular alignment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, the group that got DOX shows clear pathological changes, such as disorganized cardiac fibers, more gaps between cells, and less clear cellular architecture. Interestingly, these pathological changes were significantly reversed in the DOX\u0026thinsp;+\u0026thinsp;NH group, showing that NH protects the heart tissue from damage.\u003c/p\u003e \u003cp\u003eTo further evaluate the cardioprotective effect of NH we used Masson staining which reveals equally pronounced fibrotic remodeling in the DOX group as in the CON group. NH administration significantly attenuates this fibrosis, resulting in collagen levels like controls ( Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003eThese findings are further confirmed by Sirius Red staining to evaluate the deposition of collagen fibers. Quantitative analysis of red-stained fibrotic areas relative to total tissue area shows minimal collagen accumulation in both the CON and NH groups, with occasional perivascular deposition observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E). In contrast, significant collagen accumulation was observed in the interstitial spaces and around blood vessels in the DOX group. NH treatment significantly reduces collagen accumulation in these regions, supporting the antifibrotic potential of collagen. These data revealed that NH mitigates DOX-induced histopathological cardiac injury in mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 NH Attenuates Doxorubicin-Induced Cardiomyocyte Toxicity In Vitro\u003c/h2\u003e \u003cp\u003eTo assess the protective effects of norharmane (NH) against DOX-induced cardiomyocyte toxicity, cell viability studies are performed under diverse treatment conditions. The protective dose of cardiomyocytes of NH (0.01\u0026ndash;0.5\u0026micro;M) and cardiomyocyte toxic dose of DOX (0.5\u0026micro;M) was confirmed by MTT assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, NH dose-dependently increases H9C2 cell viability under DOX exposure. Consistent with the MTT results, morphological results shows that NH reverses the cell death induced by DOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). Cells exhibit a significant structural damage in DOX alone group, reflecting by cell shrinkage, membrane blebs, and overall morphological deterioration. In contrast, cells treated with NH showed preserved morphology. Those findings reveal that NH attenuates DOX-induced cardiomyocyte toxicity in vitro.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4 NH Reduces Doxorubicin-Induced Oxidative Stress in Vitro and in Vivo.\u003c/h2\u003e \u003cp\u003eNext, we evaluate the role of Norharmane (NH) in DOX-induced oxidative stress in vitro. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B. DOX treatment resulted in a significant increase in intracellular ROS levels, as indicated by strong green fluorescence. In contrast, co-treatment with NH significantly reduced ROS accumulation in DOX-treated cells, suggesting strong antioxidant activity. As we known, high levels ROS accumulation impairs the mitochondrial member potential (MMP)(Gorospe, Carvalho et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Then, TMRE staining assay was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). As expected, DOX caused mitochondrial depolarization, which was shown by the drop in TMRE fluorescence. NH restored the MMP in DOX-treated cells, indicating the preservation of mitochondrial function. In vivo, DOX caused a significant decrease of GSH/GSSG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F) in heart tissues and serum. Nevertheless, NH reversed these changes. Meanwhile, NH decreased the enhanced MDA concentration induced by DOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, H). In accordance with the GSH/GSSG results, NH increases the activity of SOD after DOX exposure in heart tissues and serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, J). All these results implicated that NH reduces oxidative stress induced by DOX in vitro and in vivo.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5 NH Inhibits Doxorubicin-Induced Apoptosis in Cardiomyocytes\u003c/h2\u003e \u003cp\u003eTo investigate the effect of NH against DOX-induced cardiotoxicity, transcriptome analysis was first performed. Gene ontology (GO) enrichment revealed that DOX treatment significantly activated apoptosis-related biological processes, including the regulation of apoptotic signaling and post-translational protein modification (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Western blot analysis also confirmed that DOX treatment significantly increased the expression of the pro-apoptotic proteins Bax, cleaved caspase-3, and TNF-α, along with a decrease of the anti-apoptotic protein Bcl-2 in H9C2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-F). In contrast, co-treatment with NH significantly reversed these changes, suggesting that NH inhibits DOX-induced cell apoptosis.\u003c/p\u003e \u003cp\u003eIn confirmation of these results, TUNEL staining in heart tissue sections showed a significant increase in the number of TUNEL-positive nuclei upon DOX exposure, suggesting increased apoptosis, whereas cotreatment with NH significantly reduced the number of apoptotic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H). Consistent with these results, PI staining of H9C2 cells showed a significant increase in cell death upon DOX treatment, which was significantly attenuated by NH (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, J). Taken together, these results suggest that NH effectively attenuates DOX-induced cardiomyocyte apoptosis both in vitro and in vivo.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6 NH Attenuates Doxorubicin-Induced Cardiocytes Oxidative Stress and Apoptosis via Upregulating PPARγ\u003c/h2\u003e \u003cp\u003eTo investigate the underlying molecular mechanism of NH-mediated cardio protection, a Venn diagram was constructed to identify the common target genes between NH and DOX-induced heart injury (DIH). A total of 37 overlapping genes were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Protein\u0026ndash;protein interaction (PPI) network analysis revealed that several of these genes were functionally associated with PPARγ signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Western blotting results show that DOX markedly downregulates PPARγ expression, whereas NH treatment restores its levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). MTT analysis confirmed that NH significantly improved cell viability compared with DOX alone, and this effect was diminished by GW9662 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eApoptotic cell death, as assessed by PI staining, was significantly increased in DOX-treated cells but was attenuated by NH; however, inhibition of PPARγ reversed the anti-apoptotic effect of NH (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). Similarly, intracellular ROS accumulation, as assessed by DCFH-DA staining, increased after DOX exposure but was significantly decreased by NH treatment, an effect that was reversed by GW9662 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, H).\u003c/p\u003e \u003cp\u003eFurther analysis of apoptosis- and inflammation-related proteins showed that DOX increased the expression of pro-apoptotic Bax and cleave-caspase3 and pro-inflammatory TNF-α, while decreasing the expression of anti-apoptotic Bcl-2. Treatment with NH reversed these changes, whereas co-treatment with GW9662 abolished the protective effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eI-N). Taken together, these results indicate that NH potentiates DOX-induced apoptosis and oxidative stress in H9C2 cells through a PPARγ-dependent mechanism.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.7 NH can Enhance the Sensitivity to DOX in A549 Lung Cancer Cell\u003c/h2\u003e \u003cp\u003eNH itself has some antitumor effects, and to understand the effect of NH on the antitumor effect of DOX, we performed an MTT experiment to explore it. The results showed that when DOX and NH were used alone to treat A549 lung cancer cells, they could inhibit the viability of A549 cells, and the higher the drug concentration, the stronger the inhibitory effect; When DOX and NH were used in combination, NH could enhance the inhibitory effect of DOX on the viability of A549 cells. The results indicate that NH can enhance the sensitivity of A549 lung cancer cells to DOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;C).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eDOX, originally extracted from Streptomyces paucities, has been wildly used as an anti-cancer chemotherapeutic drug to treat solid tumors and acute leukemias(Starobin, Danford et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Although DOX has been identified as one of the most effective and safe antitumor medicines, the spectrum of its clinical application is somewhat restricted due to its high toxicity to liver, kidney and heart (Arcamone, Cassinelli et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1969\u003c/span\u003e, Thorn, Oshiro et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). DOX can induce production of a large amount of ROS and subsequent mitochondrial dysfunctions and cell damages, particularly to cardiomyocytes(Yang, Teves et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, DOX can be wildly used to induce heart failure in animal models. In current study, we used DOX-induced heart failure in mice to determine if NH treatment can protect against cardiac dysfunctions. Consistent with previous studies, we also found that DOX caused abnormal ECG, increased serum CK and LDH levels, and induced cardiac fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). More importantly, we observed that the damages to heart caused by DOX were substantially attenuated by NH treatment. The cardio protection of NH should be attributed to its inhibition of ROS production, inflammation and apoptosis.\u003c/p\u003e \u003cp\u003ePrimary electrocardiographic studies provided preliminary evidence of cardiac stress in DOX-treated mice, showing prolonged QRS and QTc intervals and reduced heart rate, which are typically associated with electrical remodeling and early cardiomyopathy(Christidi and Brunham \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Interestingly, ECG parameters and the ability to maintain electrophysiological stability were preserved with co-administration of NH This finding is consistent with previous studies demonstrating the role of natural antioxidants in stabilizing cardiac conduction during toxic stress(Van Ommen, Kessler et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) .\u003c/p\u003e \u003cp\u003eAdditional support was provided by biochemical markers. CK and LDH levels in cardiac lesions were significantly increased in the DOX group, while NH administration significantly decreased these markers, mimicking myocardial protection. Histological evaluations confirmed that the HE-stained myocardial tissue in the DOX group showed significant cell disorganization and vacuolization, while the NH maintained a normal structure. These data suggest that NH may suppress fibrotic remodeling, most likely by interfering with pro-fibrotic mediators such as TGF-β1 or matrix metalloproteinases(Saadat, Noureddini et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe main contributor of oxidative stress to DOX-induced cardiotoxicity, which induces reactive oxygen species (ROS) responses and endogenous antioxidant activity, is myocyte membrane degradation (Tadokoro, Ikeda et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Excessive ROS in mitochondria induces apoptotic degradation(Liu, Han et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Cardiomyocyte apoptosis has been demonstrated to be the direct cause of DOX-induced cardiotoxicity(Songbo, Lang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). NH models significantly improved the GSH/GSSG ratio, increased total SOD activity, and decreased MDA synthesis in serum and tissues. High levels of antioxidants are essential for lipid peroxidation. Microscopic studies confirmed that ROS are not sequestered and reflect mitochondrial membrane potential (TMRE assay) and mitochondria are hemostatically and oxidatively induced.\u003c/p\u003e \u003cp\u003eThe anti-apoptotic potential of NH could not be evaluated by TUNEL and PI staining, as there were significantly lower numbers of apoptotic nuclei in the NH\u0026thinsp;+\u0026thinsp;DOX group than in the DOX group. Western blot analysis was based on the NH module and the critical regulation of apoptosis, which upregulates Bcl-2 and downregulates Bax. Caspase-3 cleavage restores the balance between pro- and anti-apoptotic signals. These results are consistent with previous studies on natural cardioprotective compounds, as it is resistant to curcumin, which further induces the effects of mitochondrial apoptosis (Koss-Mikołajczyk, Todorovic et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Our mechanistic studies showed that NH-induced cardio protection is strongly mediated by peroxisome proliferator-activated receptor gamma (PPARγ) activation. In vitro, NH restored cardiomyocyte viability, reduced ROS accumulation, and inhibited apoptosis, as evidenced by increased Bcl-2 and decreased levels of Bax, cleaved caspase-3, and TNF-α. Importantly, these effects were reversed in the presence of GW9662, a selective PPARγ antagonist. This confirms that PPARγ activation is essential for the cardioprotective effects of NH (Kim and Yang \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Its activation in cardiomyocytes is increasingly recognized as a therapeutic target to ameliorate oxidative damage and apoptosis. Therefore, our findings place NH among the natural small molecules that act as PPARγ agonists, thus providing a novel mechanistic rationale for its protective efficacy. The anti-apoptotic effects of NH, demonstrated by TMRE and PI staining, are consistent with its ability to activate PPARγ, a transcription factor known to modulate the apoptotic gene network. (Yuan, Yi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). By targeting both apoptotic and inflammatory signaling pathways in a PPARγ-dependent manner, NH disrupted the self-reinforcing cycle of oxidative stress and cardiomyocyte death that underlies anthracycline-induced cardiotoxicity(Xiang, Xin et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur findings identify NH as a potent cardioprotective agent that exerts beneficial effects primarily through activation of PPARγ signaling. The abrogation of NH effects by GW9662 provides compelling evidence for this mechanism. Since synthetic PPARγ agonists are already used clinically in metabolic diseases, our data suggest a high potential translation of NH as a natural PPARγ modulator in cancer patients at risk of cardiotoxicity. Future studies should investigate whether NH alters the antitumor efficacy of DOX and explores its long-term safety and pharmacokinetic profile, thus allowing for clinical translation.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eIn summary, our results demonstrate that (NH), a natural beta-carboline alkaloid, provides effective protection against doxorubicin-induced cardiotoxicity through activation of PPARγ signaling pathways. NH preserves cardiac structure and function by reducing oxidative stress, suppressing inflammatory responses, and inhibiting mitochondrial apoptosis.\u003c/p\u003e \u003cp\u003eInterestingly, pharmacological suppression of PPARγ with GW9662 abrogated these benefits, confirming PPARγ activation as the primary mechanism of the cardioprotective effect of NH. Our findings suggest that NH may serve as a useful adjunct to alleviate anthracycline-induced cardiac injury. Further research is needed to elucidate the pharmacokinetics, long-term safety, and potential interactions with the antitumor activity of doxorubicin to facilitate clinical implementation of NH.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBax B-cell lymphoma-2 associated X protein\u003c/p\u003e\n\u003cp\u003eBcl-2 B-cell lymphoma-2\u003c/p\u003e\n\u003cp\u003eBF Bright Field\u003c/p\u003e\n\u003cp\u003eCK-MB Creatine kinase-MB\u003c/p\u003e\n\u003cp\u003eDOX Doxorubicin\u003c/p\u003e\n\u003cp\u003eEF Ejection fraction\u003c/p\u003e\n\u003cp\u003eFS Fractional shortening\u003c/p\u003e\n\u003cp\u003eGSH Glutathione (Reduced Glutathione)\u003c/p\u003e\n\u003cp\u003eH E Hematoxylin Eosin\u003c/p\u003e\n\u003cp\u003eLDH Lactate dehydrogenase\u003c/p\u003e\n\u003cp\u003eMDA Malondialdehyde\u003c/p\u003e\n\u003cp\u003eNH Norharmane\u003c/p\u003e\n\u003cp\u003ePI. Propidium Iodide\u003c/p\u003e\n\u003cp\u003ePPAR\u0026gamma; Peroxisome proliferator-activated receptor gamma\u003c/p\u003e\n\u003cp\u003eROS Reactive oxygen species\u003c/p\u003e\n\u003cp\u003eSOD Superoxide Dismutase\u003c/p\u003e\n\u003cp\u003eTNF-\u0026alpha; Tumor necrosis factor-alpha\u003c/p\u003e\n\u003cp\u003eTMRE. Tetramethyl rhodamine Ethyl Ester\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCredit author statementSaqib Hina, Yu Hou, and Rui Wang designed and conducted the experiments. Saqib Hina wrote the manuscript. Hoaming Yin, Ronghui Hou, Jina Zhao, and Huaidong Zhang contributed to material and method preparation. Chunmei Bai, Tong Huo, Yuying Chen, Mai Wang, Hasnain Baltte and Neha Baqai analyzed the data. Jinjin Pan and Yuhui Yuan administrated the experiments and reviewed the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAmaldoss, M. J. N., E. Pandzic, P. Koshy, N. Kumar, C. C. Sorrell and A. Unnikrishnan (2022). \u0026quot;Detection and quantification of nanoparticle-induced intracellular ROS in live cells by laser scanning confocal microscopy.\u0026quot; \u003cu\u003eMethods\u003c/u\u003e \u003cstrong\u003e207\u003c/strong\u003e: 11-19.\u003c/li\u003e\n \u003cli\u003eAndreev, D., E. Balakin, A. Samoilov and V. Pustovoit (2024). \u0026quot;The Role of Doxorubicin in the Formation of Cardiotoxicity\u0026ndash;Generally Accepted Statement. Part I. Prevalence and Mechanisms of Formation (Review). Drug development \u0026amp; registration. 2024; 13 (1): 190\u0026ndash;199.\u0026quot; \u003cu\u003eRuss.) doi\u003c/u\u003e \u003cstrong\u003e10\u003c/strong\u003e: 2305-2066.\u003c/li\u003e\n \u003cli\u003eArcamone, F., G. Cassinelli, G. Fantini, A. 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Tang (2020). \u0026quot;FNDC5 alleviates oxidative stress and cardiomyocyte apoptosis in doxorubicin-induced cardiotoxicity via activating AKT.\u0026quot; \u003cu\u003eCell Death \u0026amp; Differentiation\u003c/u\u003e \u003cstrong\u003e27\u003c/strong\u003e(2): 540-555.\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":"Doxorubicin, Cardiotoxicity, Norharmane, Apoptosis, Oxidative stress, PPARγ","lastPublishedDoi":"10.21203/rs.3.rs-8619773/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8619773/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe studied the cardioprotective effects of norharman (NH), a natural alkaloid, against doxorubicin (DOX)-induced myocardial damage and its mechanism of action through PPARγ signaling. Clinical efficacy is limited by dose-dependent cardiotoxicity (which can lead to cardiomyopathy and heart failure), highlighting the need for safe cardioprotective agents. This study combined in vivo and in vitro experimental designs. C57BL/6 mice were treated with DOX alone or in combination with NH. Cardiac function was analyzed by echocardiography. Serum creatine kinase and lactate dehydrogenase levels were also measured as indicators of myocardial damage. Myocardial fibrosis, inflammation, and oxidative stress were assessed by histological and biochemical analyses. Cultured cardiomyocytes were exposed to DOX\u0026thinsp;\u0026plusmn;\u0026thinsp;NH to evaluate cell viability, reactive oxygen species formation, and apoptosis-associated protein expression. The selective PPARγ inhibitor GW9662 was used to determine pathway specificity. DOX significantly reduced myocardial ejection fraction and fractional shortening, increased oxidative stress and damage biomarkers, and induced myocardial fibrosis. NH co-treatment protected cardiac function, reduced oxidative damage, and attenuated fibrosis and inflammation. In cardiomyocytes, NH increased cell viability, inhibited reactive oxygen species production, upregulated Bcl-2, and downregulated Bax, cleaved caspase-3, and TNF-α. GW9662 reversed NH\u0026rsquo;s protective effects, confirming PPARγ involvement. These results indicate that NH, as a novel PPARγ activator, effectively attenuates DOX-induced oxidative stress and apoptosis, highlighting its potential as a natural therapeutic agent for preventing anthracycline-associated cardiotoxicity.\u003c/p\u003e","manuscriptTitle":"Norharmane Mitigates Doxorubicin-Induced Heart Failure via PPARγ- Mediated Suppression of Oxidative Stress and Apoptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-18 10:05:55","doi":"10.21203/rs.3.rs-8619773/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":"d4e6523d-6f67-4222-a09b-dec96250924a","owner":[],"postedDate":"February 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-31T14:56:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-18 10:05:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8619773","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8619773","identity":"rs-8619773","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}