RIP3 orchestrates oxidative stress and pyroptosis in doxorubicin-induced cardiotoxicity through regulation of AKT/Nrf2 signaling cascade

RIP3 orchestrates oxidative stress and pyroptosis in doxorubicin-induced cardiotoxicity through regulation of AKT/Nrf2 signaling cascade | 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 RIP3 orchestrates oxidative stress and pyroptosis in doxorubicin-induced cardiotoxicity through regulation of AKT/Nrf2 signaling cascade Zhenyi Wang, Yitong Yang, Nisha Wang, Linhe Lu, Chennian Xu, Jun Ren, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3976154/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract This study was designed to explore the role of RIP3 in DOX-induced cardiotoxicity and its underlying molecular mechanisms.Ourresults demonstrate that RIP3 exacerbates DOX-inducedcardiotoxicity through promoting oxidative stress and pyroptosis by regulating the AKT/Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Inhibition of RIP3 by using GSK-872 attenuated DOX-induced cardiac remodeling and contractile dysfunction. Moreover, by using GSK-872 in vivo , the results revealed that inhibition of RIP3 alleviated DOX-induced cardiotoxicity by the resulting inhibition of oxidative stress and pyroptosis. Besides, inhibition of RIP3 increased the protein levels of AKT and Nrf2 in DOX-treatedmouse hearts. Furthermore, the AKT inhibitor LY294002 lessened RIP3 reduction-offered protection against DOX-induced H9c2 cell injury by moderating oxidative stress and pyroptosis. Taken together, these data demonstrate that RIP3 activation orchestrates DOX-induced cardiotoxicity through elevated oxidative stress and pyroptosis in an AKT/Nrf2-dependent manner. Those findings highlight the clinical relevance and therapeutic potential of targeting RIP3for the treatment of DOX-induced cardiotoxicity. Doxorubicin Cardiotoxicity RIP3 Oxidative stress Pyroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction With the advance in diagnosis and treatment strategies, in particular application of new drugs, the survival rate has gradually improved in cancer patients. Nonetheless, cardiovascular injury evoked by chemotherapy drugs has drawn much attention[1–3]. Among various chemotherapeutic drugs, doxorubicin(DOX) is an anthracycline antibiotic with potent anti-tumor effects widely used in malignancies including hematological tumors, breast and ovarian cancer. However, clinical applicationof DOX has been greatly limited due to its cardiotoxicity. Treatment with DOX has been shown to contribute to irreversible damage to cardiomyocytes, unfavorable cardiac functional and structural changes, ultimately resulting in heart failure[4–6]. The precise mechanism behind DOX-induced cardiotoxicity is rather complex, with a prominent role for oxidative stress and pyroptosis[7–11]. Imbalance between reactive oxygen species (ROS) and endogenous antioxidant defenseevokes oxidative stress. Ample evidence has revealed an important role in DOX-induced cardiotoxicity[12–14]. Protein kinase B (PKB/AKT) is a serine/threonine protein kinase involved in the regulation of cell survival and metabolism, while it is inhibited in DOX-challenged murine hearts. Ample evidence has demonstrated that activation of AKT prevented cardiomyocyte oxidative stress in response to DOX, whereas AKT inhibition exaggerated DOX-induced cardiomyocyte oxidative stress and cardiac dysfunction[15–17]. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcriptional factor, regulates levels of antioxidant enzymes to combat with oxidative stress.AKT also played a critical role in relieving oxidative stress via influenced nuclear translocation-mediated Nrf2 nuclear export and degradation[18]and recent studies reported downregulation in both cardiac Nrf2 mRNA and protein levels in DOX-induced cardiotoxicity[19–22]. Pyroptosis, a novel form of programmed cell death, is characterized by swelling and membrane ruptures in cells, leading to release of cell contents and pro-inflammatory molecules to evoke pro-inflammatory responses[23–24]. Formation of inflammasome is a character of pyroptosis. NLR family pyrin domain containing 3 protein (NLRP3) inflammasome, a vital inflammasome of pyroptosis, is composed by NLRP3,ASC, and caspase-1. Activation of NLRP3 inflammasome upregulateslevels of IL-1β and IL-18[25–26].At present, pyroptosis is widely recognized to play a crucial role in the pathogenesis of cardiovascular diseases. Also, several studies have indicated a role for NLRP3 inflammasome in DOX-induced cardiotoxicity heart injury[27]. Therefore, regulation NLRP3 formation and inactivation may represent a novel avenue in the therapeutics of DOX-induced cardiotoxicity. Consistently, several studies indicated that Nrf2 is one of the signaling molecule regulating NLRP3 inflammasome activation[28]. Receptor-interacting Protein Kinase 3 (RIP3), an important stress sensor molecule, is thought to be a key regulator of apoptosis and necroptosis. Previous studies have shown the essential role for RIP3 in cardiomyocyte necrosis, leading to myocardial remodeling and heart failure[29]. Recent studies illustrated that RIP3 is also the essential molecule for NLRP3 inflammasome activation under stress setting[30–32]. To this end, we hypothesized that RIP3 orchestrates oxidative stress and pyroptosis in DOX-induced cardiotoxicity possibly related to AKT/Nrf2 signaling pathway. Materials and Methods Reagents The antibodies against RIP3, p- RIP3, Nrf2 were purchased from Abcam. Antibodies against NLRP3, GSDMD, Caspase-1, IL-1β, NOX2, NOX4, HO-1, AKT and P-AKT were purchased from Cell Signaling Technology. And the antibodies against GAPDH, BAX, BCL-2 and HO-1 were purchased from Proteintech. The Pierce BCA protein assay kit was purchased from Thermo Scientific. Doxorubicin and dimethylsulfoxide (DMSO) were purchased from Sigma. The GSK-872 (RIP3 inhibitor) and SC79 (AKT agonist) were purchased from Med Chem Express. LY294002 (AKT inhibitor) was purchased from Sigma. The secondary antibodies of goat anti-mouse and goat anti-rabbitwere purchased from ZSGB-Bio. The LDH enzyme-linked immune assay (ELISA) kit and lysis buffer were purchased from Beyotime Biotechnology. The terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) kit and protease and phosphatase inhibitor cocktails were purchased from Roche. ROS assay kit, DAPI (4’,6-Diamino-2-phenylindole) were purchased from Sigma-Aldrich. Experiment protocol All C57BL/6J mice used in this study were procured from the Laboratory Animal Center of the Fourth Military Medical University (Xi'an, China). All experimental protocols for this study were approved by the Ethics Committee of the Fourth Military Medical University(Approval No: [20220431]). C57BL/6J mice were randomly divide into different groups (n = 15) as follows: the control group (Control), doxorubicin group (DOX) and doxorubicin plus GSK-872 (DOX + GSK-872) group. DOX was dissolved in normal saline, GSK-872 dissolved in DMSO. Mice in DOX and DOX + GSK-872 groups were delivered 15 mg/kg DOX (one time, i.p.). GSK-872 (5 mg/kg) was intraperitoneal injected daily starting 2 hrs prior to the initial DOX challenge. Control group received equal volume of saline (i.p., daily). All drugs employed in this study were chosen based on previous findings [33–34]. Mouse survival status was monitored and recorded daily. Echocardiographic assessment All C57BL/6J mice were anaesthetized with 2% isoflurane 7 days following DOX injection. Cardiac geometry and function were evaluated in the short-axis using two-dimensional (2-D) and M- mode with a Vevo 2100 ultrasound imaging system (VisualSonics). Images were used to analyze parameters of cardiac geometry and function, including fractional shortening (FS), ejection fraction (EF), left ventricular internal diameter at end- diastole (LVIDd) and left ventricular posterior wall thickness (LVPWd) with Vevo software. Measurement values were calculated and the average of three cardiac cycles were used. Histopathology Mice were sacrificed at the end of the experiments, the heart samples were excised and rinsed with pre-cold PBS. Heart tissues were then fixed in 4% polyformaldehyde for 48 hours followed by paraffin embedding, sections stained with hematoxylin and eosin (H&E) to evaluate heart morphology. In addition, sections were stained with Masson's trichrome and imaged with a microscope to evaluate the degree of myocardial fibrosis. Determination of LDH activity by ELISA Serum LDH activity was measured using commercial ELISA kits obtained from Beyotime Biotechnology in accordance with the manufacturer's instructions. Cell culture and treatment H9c2 cell purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China) were cultured in DMEM-high glucose supplemented with 10% FBS at 37°C in a humidifier incubator containing 5% CO 2 is used for the following in vitro experiments. H9c2 cell were randomly divided into the following groups: (1) Control: cells were treated with PBS; (2) DOX: cells were treated with DOX (5 µmol/L) for 24h to inducing cardiotoxicity; (3) DOX + GSK-872: cells were pretreated with GSK-872 (3 µmol/L) for 2h, followed by DOX (5µmol/L) for additional 24h; (4) DOX + SC79: cells were pretreated with SC79 (10µmol/L) for 2h, followed by DOX (5 µmol/L) for additional 24h; (5) DOX + GSK-872 + LY294002: cells were treated with GSK-872 (3 µmol/L) and LY294002 (10µmol/L) for 2h, followed by DOX (5 µmol/L) for additional 24h. Concentrations of pharmacological inhibitors were chosen in reference with earlier previous studies[32, 35–36]. Intracellular ROS measurement of H9c2 cells by DCFH-DA Cells were incubated with 10 µM DCFH-DA in DMEM medium withoutFBS at 37 ◦ C for 1 h. Afterward, cells were washed three times with PBS and the fluorescent images were observed with an Olympus Fluoview FV1000 microscope. The fluorescence intensity was quantified by using an Image J software (National Institutes of Health, Bethesda, MD, USA). TUNEL staining The TUNEL staining method was used to measure apoptotic rate using an in situ cell death detection kit according to the manufacturer's instructions. Images were captured using an Olympus FV1000confocal microscope (Olympus, Japan). The TUNEL-positive cardiomyocytes exhibited green nuclear staining (green) and were counted in five randomly selected fields under high-power magnification.Apoptotic rate was determined using the ratio of TUNEL-positive cells normalized to total cardiomyocyte counts. Immunofluorescence staining For immunofluorescence staining of Nrf2, H9c2 cells were permeabilized using 0.1% Triton X-100% and 1% bovine serum albumin. Then, samples were incubated with an anti-Nrf2 antibody at 4 o C overnight, followed by incubation with Alexa fluor-488 goat anti-rabbit secondary antibodies for 1 h at room temperature.Ultimately, samples were visualized with a fluorescence microscope. PI (Propidium iodide) staining assay PI was used to stain for H9c2 cells (4.5 µM PI per well) at 37°C for 20 min. Images of the cardiomyocytes were acquired immediately and examined using an Olympus Fluoview FV1000 microscope (Olympus, Japan). The percentage of positive cells was counted, and the average fluorescence intensity was evaluated using Image Pro advanced software. The results are presented as PI-positive cells (×100%). Western blotting Total protein was extracted from myocardial tissue and H9c2 cell after diverse treatments and prepared for western blotting. After separating the protein samples by 10 ~ 12% SDS-PAGE, proteins were transferred onto a PVDF membrane and incubated with 5% non-fat milk in TBST for 2 hours. The membrane was then incubated with primary antibodies at 4 o C overnight. Then washing with TBS containing 0.1% Tween 20 (TBST)for 3 times, 10 minutes at once. After washing with TBST, the membranes were incubated with HRP after diverse treatments and prepared for we in 23℃. Next, washing with TBS containing 0.1% Tween 20 (TBST) for 3 times, 10 minutes at once. Then the proteins were visualized using chemiluminescent reagents (Millipore, Billerica, MA, USA) under ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and the densities of the bands were quantified by Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA) GAPDH was used as an internal reference. Statistical analysis Results are presented as the mean ± SEM, and GraphPad Prism software was used for statistical analysis. Quantitative data were analyzed using the one-way analysis of variance (ANOVA) analysis followed by Tukey post-hoc test P < 0.05 was considered statistically significant. Results RIP3 activation and AKT/Nrf2 downregulated are involved in doxorubicin-induced cardiotoxicity Firstly, RIP3 activation and protein levels of AKT and Nrf2 were determined in mouse hearts. Our data revealed that DOX challenge activated RIP3 (Fig. 1 B-C). Furthermore, Western blot revealed that protein expression of AKT and Nrf2 were significantly downregulated following DOX challenge compared with Control group (Fig. 1 D-E). The results indicated RIP3 activation and AKT/Nrf2 downregulation in DOX-induced cardiotoxicity. RIP3 inhibition protected against DOX-induced cardiac dysfunction and pathological myocardial changes To examine the role of RIP3 in DOX-induced cardiotoxicity, GSK-872, an inhibitor of RIP3, was used in DOX-induced cardiotoxicity. With the treatment of DOX for 7 days, cardiac function indicators including FS and EF were significantly decreased, while LVIDd but not LVPWdwas elevated in DOX-treated mice, the effects of which were greatly attenuated by GSK-872 (Fig. 2 A-E). Serum LDH activity was significantly increased in DOX-treated mice (Fig. 2 F). In addition, HE and Masson's trichrome staining revealed that the myofibrillar component was disorganized and degenerated with deposition of fibrotic tissue in DOX-treated hearts (Fig. 2 G, H). Besides, survival rate was significantly decreased in the DOX-treated group (Fig. 2 I). These DOX-induced cardiac anomalies in cardiac injury marker, geometry, function, histopathology and survival were greatly attenuated by the RIP3 inhibitor GSK-872. RIP3 inhibition protected against DOX-induced oxidative stress and pyroptosis Consistent with previous studies, our results confirmed that DOX treatment resulted in significant oxidative stress and pyroptosis in mouse hearts. As indicated by the protein expression of oxidative stress including NOX2 and NOX4 were increased (Fig. 3 B-C). Similarly, the proteins associated with pyroptosis such as NLRP3, GSDMD and Caspase-1 were elevated (Fig. 3 D-F). Interestingly, these proteins level were significantly decreased by RIP3 inhibitor GSK-872 (Fig. 3 A-F). These data demonstrated that RIP3 inhibition protected against DOX-induced oxidative stress and pyroptosis in mouse hearts. RIP3 inhibition increased DOX-induced AKT/Nrf2 downregulation To detect whether AKT/Nrf2 signalling contributes to the protective effects of RIP3 inhibition against DOX-induced cardiotoxicity, we measured the protein expression of AKT and Nrf2 in mouse hearts. Western blot analysis showed robust downregulation of the protein levels of AKT and Nrf2 in DOX-treated hearts while significantly increased levels of RIP3 phosphorylation. Distinctly, these proteins expression were heavily bated by the RIP3 inhibitor GSK-872 (Fig. 4 A-D). These results indicated that AKT/Nrf2signallingwas involved in the protective effects of RIP3 inhibition against DOX-induced cardiotoxicity. SC79 mitigate myocardial injury in DOX-treated H9c2 cells Next, to investigated whether AKT/Nrf2 activation protected against the DOX-induced cardiotoxicity, SC79, an agonist of AKT, was used in DOX-induced H9c2 cells. Following treated with DOX 24 hours, Western blot revealed that protein expression of Nrf2, HO-1 and BCL-2 were significantly downregulated while BAX was significantly increased (Fig. 5 A-F). Besides, LDH activity and apoptotic index greatly increased in the DOX-treated group (Fig. 5 G-J). The cardiac injury marker and apoptotic index were greatly attenuated by the AKT agonist SC79. These data indicated that protected against the DOX-induced cardiotoxicity via activating AKT/Nrf2 pathway (Fig. 5 A-J). AKT/Nrf2 inhibition blocked the protective effects of RIP3 inhibition on DOX-treated H9c2 cells Subsequently, to examinewhether AKT/Nrf2 involved in RIP3 inhibition-mediated protective effects on DOX-induced cardiotoxicity, LY294002, an inhibitor of AKT, was used in GSK-872 and DOX-induced H9c2 cells. GSK-872 alleviated DOX-induced myocardial injury in H9c2 cells. Western blot showed that the expression of NOX2, NOX4, NLRP3, GSDMD, IL-1β and Caspase-1 were significantly decreased while the expression of P-AKT and Nrf2 were significantly increased following with GSK-872 (Fig. 6 A-J). In addition, GSK-872 elevated the cell viability and decreased LDH, myocardial PI-positive cells which as evidence of cardiomyocyte pyroptosis, activity ROS production and apoptotic index (Fig. 7 A-B,F-H). Besides, immunofluorescence staining results demonstrated that the expression of Nrf2 was increased in the wake of GSK-872 (Fig. 7 C-D). The improvement of cardiac injury marker, marker for oxidative stress orpyroptosis and apoptotic index following GSK-872 were greatly abrogated by the AKT inhibitor LY294002 (Fig. 6 – 7 ). These results indicated that the effects of RIP3 inhibition on oxidative stress and pyroptosisin DOX-treated myocardial were related to AKT/Nrf2 signalling. Discussion Anthracyclines, including doxorubicin (DOX), are widely used for the treatment of a variety of cancers.It is known that anthracyclines induce irreversible cardiomyocyte injury, leading to cardiac fibrosis, cell apoptosis and left ventricular dysfunction, enroute to heart failure[37].However, the pathological mechanisms of DOX-induced cardiotoxicity have not been fully elucidated. It is confirmed cardiomyocytes apoptosis, oxidative stress and pyroptosis contribute to the development of DOX-induced cardiomyopathy. In this study, our results manifested RIP3 activation and the AKT/Nrf2 pathway inhibition in DOX-treated mouse hearts and H9c2 cells. Also, data in this experiment indicated that RIP3 inhibition reduced oxidative stress and pyroptosis through the AKT/Nrf2 pathway in mouse hearts and H9c2 cells with DOX-induced cardiotoxicity. These findings present a novel mechanism of DOX-induced myocardial injury, which suggests that RIP3 is a potential target for the prevention of DOX-induced cardiotoxicity. Increasing evidence has suggested vital roles of oxidative stress and pyroptosis in the pathogenesis and development of cardiovascular diseases. AKT is a serine/threonine protein kinase involved in the regulation of cell survival and metabolism. A growing body of studies suggested that AKT prevented cardiomyocyte oxidative stress in response to DOX, while AKT inhibition exacerbated DOX-induced cardiomyocyte oxidative stress and cardiac dysfunction[38]. Nrf2, a transcriptional factor, regulates the expression of antioxidant proteins. Activating Nrf2 to attenuate oxidative stress and apoptotic cell death, prevents DOX-induced myocardial injury, and ameliorates cardiac dysfunction. In addition, Nrf2 was reported to regulate NLRP3 inflammasome activation, influencing the degree of pyroptosis. What’s more, several studies have demonstrated that RIP3 activation is an important pathogenic factor in ischemia-and DOX-induced necroptotic cell death[39–40]. However, whether AKT/Nrf2 pathway is involved in the beneficial effects of RIP3 inhibition and the underlying mechanisms RIP3 activation in DOX-induced cardiotoxicity are still to be elucidated.To estimate the effects of RIP3 on oxidative stress and pyroptosis in DOX-induced cardiotoxicity, we measured oxidative stress and pyroptosis in DOX-challenged hearts treated with GSK-872. Consistent with previous studies, our results further confirmed that DOX-inducedpathological structural damage and cardiac dysfunction,as indicated by HE and Masson's staining, decreased EF, FS and increased LVIDd and LDH activity. While GSK-872 improved DOX-induced pathological structural damage and cardiac dysfunction, demonstrating that RIP3 inhibition could play a protective role in DOX-induced cardiotoxicity.Reactive oxygen species production and NLRP3inflammasome are involved in oxidative stress and pyroptosis respectively, bothplay a crucial role in the pathogenesis of cardiovascular diseases[12,27]. Our results also indicated that GSK-872 suppressed DOX-induced oxidative stress and pyroptosis by offsetting ROS production, formation of NLRP3inflammasomeand apoptotic rates. Meanwhile, proteinlevel of anti-oxidant protein Nrf2 was increased. However, LY294002, an inhibitor of AKT, abrogated the anti-oxidative stress and anti-pyroptosis effects of GSK-872, denoting involvement of AKT/Nrf2 signaling in the protective effects of RIP3 inhibitionagainst DOX-induced cardiotoxicity. Identically, our results showed that DOX significantly reduced AKT and Nrf2 levels. Intriguingly,GSK-872 markedly increased AKT and Nrf2 levels in DOX-treatedcardiomyocyte. However, inhibiting the AKT/Nrf2 pathway with LY294002 significantly abolished the protective effects of RIP3 inhibition against myocardial injury, oxidative stress and pyroptosisin DOX-treated cardiomyocyte. Furthermore, LY294002 reduced the protein levels of AKT and Nrf2 induced by RIP3 inhibition in DOX-treated cardiomyocyte(Fig. 8 ). In conclusion, the present study provides mechanistic evidence that RIP3 is a potential target for the treatment of DOX-induced cardiotoxicity through regulation of oxidative stress and pyroptosisin an AKT/Nrf2-dependent manner. Therefore, further studies are needed to elucidate the exact mechanism. Declarations Acknowledgements Not applicable Author contributions Zhenyi Wang and Yitong Yang conceived and designed these experiments. Nisha Wang, Linhe Lu and Chennian Xu performed these experiments. Zhenyi Wanganalyzed and interpreted the data. Zhenyi Wang wrote the manuscript. Lifang Yang and Jun Ren revised the manuscript. All the authors read and approved the final manuscript. Funding This research was supported in part by National Natural Science Foundation of China (81774415, 82174493, 92249301); Outstanding Youth Foundation of Shaanxi Province (2021JC-49); Xi’an Talent Program (XAYC210024). Data availability The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request. Conflicts of interest All authors have completed the ICMJE uniform disclosure form. All the authors declare that they have no competing interests. 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Pathogenesis of lupus nephritis: RIP3 dependent necroptosis and NLRP3 inflammasome activation. J Autoimmun. 2019 Sep;103:102286. https://doi.org/10.1016/j.jaut.2019.05.014 Zheng K, Zhang Q, Lin G, Li Y, Sheng Z, Wang J, Chen L, Lu HH. Activation of Akt by SC79 protects myocardiocytes from oxygen and glucose deprivation (OGD)/re-oxygenation. Oncotarget. 8,14978-14987. https://doi.org/10.18632/oncotarget.14785 Force, T. and Y. Wang. Mechanism-based engineering against anthracycline cardiotoxicity. Circulation 2013, 128, 98-100. 383. https://doi.org/10.1161/CIRCULATIONAHA.113.003688 Das J, Ghosh J, Manna P, Sil PC. Taurine suppresses doxorubicin-triggered oxidative stress and cardiac apoptosis in rat via up-regulation of PI3-K/Akt and inhibition of p53, p38-JNK. BiochemPharmacol. 81, 891-909. https://doi.org/10.1016/j.bcp.2011.01.008 Li, X.,J.Geng,J.Zhao, Q. Ni, C.Zhao, Y.Zheng,X. Chen, and L. Wang. Trimethylamine N-Oxide Exacerbates Cardiac Fibrosis444via Activating the NLRP3 Inflammasome. Front Physiol 2019, 10, 866 445. https://doi.org/10.3389/fphys.2019.00866 Wang C, Hu L, Guo S, Yao Q, Liu X, Zhang B, Meng X, Yang X. Phosphocreatine attenuates doxorubicin-induced cardiotoxicity by inhibiting oxidative stress and activating TAK1 to promote myocardial survival in vivo and in vitro. Toxicology. 460, 152881. https://doi.org/10.1016/j.tox.2021.152881 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 24 Mar, 2024 Reviews received at journal 06 Mar, 2024 Reviewers agreed at journal 29 Feb, 2024 Reviewers invited by journal 28 Feb, 2024 Editor assigned by journal 26 Feb, 2024 Submission checks completed at journal 22 Feb, 2024 First submitted to journal 21 Feb, 2024 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-3976154","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":274941026,"identity":"f0688c7b-679b-4e92-8726-d8c95fe64eb3","order_by":0,"name":"Zhenyi Wang","email":"","orcid":"","institution":"Children's Hospital Affiliated to Xi 'an Jiao tong University","correspondingAuthor":false,"prefix":"","firstName":"Zhenyi","middleName":"","lastName":"Wang","suffix":""},{"id":274941028,"identity":"e7819026-c0cc-4ef3-816e-21bdcd185610","order_by":1,"name":"Yitong Yang","email":"","orcid":"","institution":"Second Affiliated Hospital of Shaanxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yitong","middleName":"","lastName":"Yang","suffix":""},{"id":274941029,"identity":"77a49107-a862-4a4e-93ef-80e47eb27235","order_by":2,"name":"Nisha Wang","email":"","orcid":"","institution":"Children's Hospital Affiliated to Xi 'an Jiao tong University","correspondingAuthor":false,"prefix":"","firstName":"Nisha","middleName":"","lastName":"Wang","suffix":""},{"id":274941031,"identity":"55976924-3023-4d2c-8cb3-1b022c50719d","order_by":3,"name":"Linhe Lu","email":"","orcid":"","institution":"Xijing Hospital, Air Force Medical University","correspondingAuthor":false,"prefix":"","firstName":"Linhe","middleName":"","lastName":"Lu","suffix":""},{"id":274941032,"identity":"906e472f-3051-4d71-80c8-8811044a6d22","order_by":4,"name":"Chennian Xu","email":"","orcid":"","institution":"Air Force Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chennian","middleName":"","lastName":"Xu","suffix":""},{"id":274941033,"identity":"ce4e203b-d27b-4240-b14a-ecfba0e2d316","order_by":5,"name":"Jun Ren","email":"","orcid":"","institution":"Zhongshan Hospital Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Ren","suffix":""},{"id":274941034,"identity":"d12949b4-a9b7-468f-ab20-27d90ed88c4f","order_by":6,"name":"Lifang Yang","email":"data:image/png;base64,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","orcid":"","institution":"Children's Hospital Affiliated to Xi 'an Jiao tong University","correspondingAuthor":true,"prefix":"","firstName":"Lifang","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-02-21 15:44:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3976154/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3976154/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51750120,"identity":"472a7465-d3f5-4d0b-b1a5-87ec36d402a2","added_by":"auto","created_at":"2024-02-28 12:15:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":365542,"visible":true,"origin":"","legend":"\u003cp\u003eRIP3 activity and the expression of AKT/Nrf2 in DOX-treated mouse hearts. \u003cstrong\u003eA\u003c/strong\u003eSchematic representation of the experimental procedure. \u003cstrong\u003eB\u003c/strong\u003e Representative immunoblots showing P-RIP3, RIP3, P-AKT, AKT, Nrf2 and GAPDH (internal control). \u003cstrong\u003eC– E\u003c/strong\u003e Statistical analysis of P-RIP3, RIP3, P-AKT, AKT, Nrf2each protein was normalized to GAPDH. The data are presented as the mean ± SEM, n=6. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 versus Control.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3976154/v1/68709a2f9bc393acca18cbc7.png"},{"id":51750122,"identity":"91f84279-1c83-442f-9bf3-690f586d6033","added_by":"auto","created_at":"2024-02-28 12:15:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3756788,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of RIP3 inhibition withGSK-872 on cardiac function and structure in DOX-treated mouse hearts. \u003cstrong\u003eA\u003c/strong\u003eRepresentative images of echocardiograms of hearts. \u003cstrong\u003eB–E\u003c/strong\u003eGroup results of EF, FS, LVIDd and LVPWd. \u003cstrong\u003eF\u003c/strong\u003e The representation of LDH activity. \u003cstrong\u003eG\u003c/strong\u003eQuantitative analysis of collagen volume. \u003cstrong\u003eH\u003c/strong\u003eThe left panel shows images with HE staining; the right panel shows images with Masson's staining (Scale bar: 100 μm). \u003cstrong\u003eI\u003c/strong\u003eSurvival curves. The data are presented as the mean ± SEM, n=6. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus Control,\u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus DOX.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3976154/v1/7242fa46877437a9f504bc7c.png"},{"id":51750121,"identity":"f21c13a6-c1c4-477f-a2eb-4846eaee7b35","added_by":"auto","created_at":"2024-02-28 12:15:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":334335,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of RIP3 inhibition with GSK-872 on oxidative stress and pyroptosis in DOX-treated mouse hearts. \u003cstrong\u003eA \u003c/strong\u003eRepresentative immunoblots of NOX2, NOX4, GSDMD, NLRP3, Caspase-1 and GAPDH (internal control). \u003cstrong\u003eB-F\u003c/strong\u003e Statistical analysis of NOX2, NOX4, GSDMD, NLRP3, Caspase-1. Each protein was normalized to GAPDH. The data are presented as the mean ±SEM, n=6.**\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus Control, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus DOX, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01versusDOX.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3976154/v1/bd5dd5fda669bad079f8480d.png"},{"id":51750124,"identity":"282f4214-c7ae-4ef0-8828-a60fb77bd1d2","added_by":"auto","created_at":"2024-02-28 12:15:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":374035,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of RIP3 inhibition with GSK-872 on AKT/Nrf2 in DOX-treated mouse hearts. \u003cstrong\u003eA\u003c/strong\u003e Representative immunoblots of P-RIP3, RIP3, P-AKT, AKT, Nrf2 and GAPDH (internal control). \u003cstrong\u003eB–D\u003c/strong\u003eStatistical analysis of P-RIP3/RIP3, P-AKT/AKT and Nrf2.Each protein was normalized to GAPDH. The data are presented as the mean ± SEM, n = 6. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 versus Control,\u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus DOX,\u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus DOX.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3976154/v1/c177c5b5433db8a417741b42.png"},{"id":51750128,"identity":"410d21ce-88a6-4d41-9e0c-5e4e6651fb5e","added_by":"auto","created_at":"2024-02-28 12:15:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":818129,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of SC79 on AKT/Nrf2 in DOX-treated H9c2.\u003cstrong\u003eA\u003c/strong\u003eRepresentative immunoblots of P-AKT, AKT, Nrf2, HO-1, BAX, BCL-2 and GAPDH (internal control). \u003cstrong\u003eB-F\u003c/strong\u003eStatistical analysis of P-AKT, AKT, Nrf2, HO-1, BAX and BCL-2.Eachprotein was normalized to GAPDH. \u003cstrong\u003eG\u003c/strong\u003eThe representation of LDH activity. \u003cstrong\u003eH\u003c/strong\u003e Representative TUNEL staining images in the different groups (Scale bar: 50 μm). \u003cstrong\u003eI\u003c/strong\u003e Cell activity. \u003cstrong\u003eJ\u003c/strong\u003e Apoptotic rate. The data are presented as the mean ± SEM, n = 6. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus Control, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus DOX, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus DOX.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3976154/v1/5e4956cafa51e1bafdbc7dab.png"},{"id":51750123,"identity":"bb1e9587-e7e6-4101-9990-f7abe4eedb04","added_by":"auto","created_at":"2024-02-28 12:15:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":503192,"visible":true,"origin":"","legend":"\u003cp\u003eLY294002blocked the effects of RIP3 inhibition on AKT/Nrf2 in DOX-treated H9c2. \u003cstrong\u003eA\u003c/strong\u003eRepresentative immunoblots of P-RIP3, RIP3, P-AKT, AKT, Nrf2, NOX2, NOX4, GSDMD, NLRP3, Caspase-1, IL-1βand GAPDH (internal control). \u003cstrong\u003eB–J\u003c/strong\u003e Statistical analysis of P-RIP3, RIP3, P-AKT, AKT, Nrf2, NOX2, NOX4, GSDMD, NLRP3, Caspase-1, IL-1β. Each protein was normalized to GAPDH. The data are presented as the mean ± SEM, n = 3. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus Control, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus DOX, \u003csup\u003e■\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus DOX+GSK-872,\u003csup\u003e■■\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus DOX+GSK-872.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3976154/v1/ecd923192fe8bc81285cd793.png"},{"id":51750409,"identity":"8209893d-950e-41c8-88a3-58d887e5e110","added_by":"auto","created_at":"2024-02-28 12:23:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2147596,"visible":true,"origin":"","legend":"\u003cp\u003eLY294002 blocked the protective effects of RIP3 inhibition on oxidative stress and pyroptosis in DOX-treated H9c2. \u003cstrong\u003eA\u003c/strong\u003e Representative DCFH-DA and TUNEL staining images in the different groups (Scale bar: 50 μm). \u003cstrong\u003eB\u003c/strong\u003ePI-Positive cells. \u003cstrong\u003eC\u003c/strong\u003eThe immunofluorescence staining result of Nrf2. \u003cstrong\u003eD\u003c/strong\u003eImmunofluorescence staining intensity of Nrf2. \u003cstrong\u003eE\u003c/strong\u003eCell activity. \u003cstrong\u003eF\u003c/strong\u003eThe representation of LDH activity. \u003cstrong\u003eG\u003c/strong\u003eApoptotic. \u003cstrong\u003eH\u003c/strong\u003ePI-Positive index. Each protein was normalized to GAPDH. The data are presented as the mean ± SEM, n = 3. **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 versus Control,\u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus DOX,\u003csup\u003e■\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus DOX+GSK-872.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3976154/v1/8afdd854eb95b235c80e9789.png"},{"id":51750126,"identity":"795d00e6-a0e6-46db-a400-f1005cf746ff","added_by":"auto","created_at":"2024-02-28 12:15:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":383720,"visible":true,"origin":"","legend":"\u003cp\u003eMechanistic diagram of RIP3 orchestrates oxidative stress and pyroptosis in doxorubicin-induced cardiotoxicity.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3976154/v1/e1cc2545ebb2d9a766c5f19f.png"},{"id":51750677,"identity":"fb889881-08b3-4564-8d67-701e2b352f15","added_by":"auto","created_at":"2024-02-28 12:31:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2546613,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3976154/v1/12df4688-f681-4f3d-b099-787d7bf2a458.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"RIP3 orchestrates oxidative stress and pyroptosis in doxorubicin-induced cardiotoxicity through regulation of AKT/Nrf2 signaling cascade","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWith the advance in diagnosis and treatment strategies, in particular application of new drugs, the survival rate has gradually improved in cancer patients. Nonetheless, cardiovascular injury evoked by chemotherapy drugs has drawn much attention[1\u0026ndash;3]. Among various chemotherapeutic drugs, doxorubicin(DOX) is an anthracycline antibiotic with potent anti-tumor effects widely used in malignancies including hematological tumors, breast and ovarian cancer. However, clinical applicationof DOX has been greatly limited due to its cardiotoxicity. Treatment with DOX has been shown to contribute to irreversible damage to cardiomyocytes, unfavorable cardiac functional and structural changes, ultimately resulting in heart failure[4\u0026ndash;6]. The precise mechanism behind DOX-induced cardiotoxicity is rather complex, with a prominent role for oxidative stress and pyroptosis[7\u0026ndash;11].\u003c/p\u003e \u003cp\u003eImbalance between reactive oxygen species (ROS) and endogenous antioxidant defenseevokes oxidative stress. Ample evidence has revealed an important role in DOX-induced cardiotoxicity[12\u0026ndash;14]. Protein kinase B (PKB/AKT) is a serine/threonine protein kinase involved in the regulation of cell survival and metabolism, while it is inhibited in DOX-challenged murine hearts. Ample evidence has demonstrated that activation of AKT prevented cardiomyocyte oxidative stress in response to DOX, whereas AKT inhibition exaggerated DOX-induced cardiomyocyte oxidative stress and cardiac dysfunction[15\u0026ndash;17]. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcriptional factor, regulates levels of antioxidant enzymes to combat with oxidative stress.AKT also played a critical role in relieving oxidative stress via influenced nuclear translocation-mediated Nrf2 nuclear export and degradation[18]and recent studies reported downregulation in both cardiac Nrf2 mRNA and protein levels in DOX-induced cardiotoxicity[19\u0026ndash;22]. Pyroptosis, a novel form of programmed cell death, is characterized by swelling and membrane ruptures in cells, leading to release of cell contents and pro-inflammatory molecules to evoke pro-inflammatory responses[23\u0026ndash;24]. Formation of inflammasome is a character of pyroptosis. NLR family pyrin domain containing 3 protein (NLRP3) inflammasome, a vital inflammasome of pyroptosis, is composed by NLRP3,ASC, and caspase-1. Activation of NLRP3 inflammasome upregulateslevels of IL-1β and IL-18[25\u0026ndash;26].At present, pyroptosis is widely recognized to play a crucial role in the pathogenesis of cardiovascular diseases. Also, several studies have indicated a role for NLRP3 inflammasome in DOX-induced cardiotoxicity heart injury[27]. Therefore, regulation NLRP3 formation and inactivation may represent a novel avenue in the therapeutics of DOX-induced cardiotoxicity. Consistently, several studies indicated that Nrf2 is one of the signaling molecule regulating NLRP3 inflammasome activation[28].\u003c/p\u003e \u003cp\u003eReceptor-interacting Protein Kinase 3 (RIP3), an important stress sensor molecule, is thought to be a key regulator of apoptosis and necroptosis. Previous studies have shown the essential role for RIP3 in cardiomyocyte necrosis, leading to myocardial remodeling and heart failure[29]. Recent studies illustrated that RIP3 is also the essential molecule for NLRP3 inflammasome activation under stress setting[30\u0026ndash;32]. To this end, we hypothesized that RIP3 orchestrates oxidative stress and pyroptosis in DOX-induced cardiotoxicity possibly related to AKT/Nrf2 signaling pathway.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eThe antibodies against RIP3, p- RIP3, Nrf2 were purchased from Abcam. Antibodies against NLRP3, GSDMD, Caspase-1, IL-1β, NOX2, NOX4, HO-1, AKT and P-AKT were purchased from Cell Signaling Technology. And the antibodies against GAPDH, BAX, BCL-2 and HO-1 were purchased from Proteintech. The Pierce BCA protein assay kit was purchased from Thermo Scientific. Doxorubicin and dimethylsulfoxide (DMSO) were purchased from Sigma. The GSK-872 (RIP3 inhibitor) and SC79 (AKT agonist) were purchased from Med Chem Express. LY294002 (AKT inhibitor) was purchased from Sigma. The secondary antibodies of goat anti-mouse and goat anti-rabbitwere purchased from ZSGB-Bio. The LDH enzyme-linked immune assay (ELISA) kit and lysis buffer were purchased from Beyotime Biotechnology. The terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) kit and protease and phosphatase inhibitor cocktails were purchased from Roche. ROS assay kit, DAPI (4\u0026rsquo;,6-Diamino-2-phenylindole) were purchased from Sigma-Aldrich.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExperiment protocol\u003c/h2\u003e \u003cp\u003eAll C57BL/6J mice used in this study were procured from the Laboratory Animal Center of the Fourth Military Medical University (Xi'an, China). All experimental protocols for this study were approved by the Ethics Committee of the Fourth Military Medical University(Approval No: [20220431]). C57BL/6J mice were randomly divide into different groups (n\u0026thinsp;=\u0026thinsp;15) as follows: the control group (Control), doxorubicin group (DOX) and doxorubicin plus GSK-872 (DOX\u0026thinsp;+\u0026thinsp;GSK-872) group. DOX was dissolved in normal saline, GSK-872 dissolved in DMSO. Mice in DOX and DOX\u0026thinsp;+\u0026thinsp;GSK-872 groups were delivered 15 mg/kg DOX (one time, i.p.). GSK-872 (5 mg/kg) was intraperitoneal injected daily starting 2 hrs prior to the initial DOX challenge. Control group received equal volume of saline (i.p., daily). All drugs employed in this study were chosen based on previous findings [33\u0026ndash;34]. Mouse survival status was monitored and recorded daily.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEchocardiographic assessment\u003c/h2\u003e \u003cp\u003eAll C57BL/6J mice were anaesthetized with 2% isoflurane 7 days following DOX injection. Cardiac geometry and function were evaluated in the short-axis using two-dimensional (2-D) and M- mode with a Vevo 2100 ultrasound imaging system (VisualSonics). Images were used to analyze parameters of cardiac geometry and function, including fractional shortening (FS), ejection fraction (EF), left ventricular internal diameter at end- diastole (LVIDd) and left ventricular posterior wall thickness (LVPWd) with Vevo software. Measurement values were calculated and the average of three cardiac cycles were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology\u003c/h2\u003e \u003cp\u003eMice were sacrificed at the end of the experiments, the heart samples were excised and rinsed with pre-cold PBS. Heart tissues were then fixed in 4% polyformaldehyde for 48 hours followed by paraffin embedding, sections stained with hematoxylin and eosin (H\u0026amp;E) to evaluate heart morphology. In addition, sections were stained with Masson's trichrome and imaged with a microscope to evaluate the degree of myocardial fibrosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of LDH activity by ELISA\u003c/h2\u003e \u003cp\u003eSerum LDH activity was measured using commercial ELISA kits obtained from Beyotime Biotechnology in accordance with the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and treatment\u003c/h2\u003e \u003cp\u003eH9c2 cell purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China) were cultured in DMEM-high glucose supplemented with 10% FBS at 37\u0026deg;C in a humidifier incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e is used for the following \u003cem\u003ein vitro\u003c/em\u003e experiments. H9c2 cell were randomly divided into the following groups: (1) Control: cells were treated with PBS; (2) DOX: cells were treated with DOX (5 \u0026micro;mol/L) for 24h to inducing cardiotoxicity; (3) DOX\u0026thinsp;+\u0026thinsp;GSK-872: cells were pretreated with GSK-872 (3 \u0026micro;mol/L) for 2h, followed by DOX (5\u0026micro;mol/L) for additional 24h; (4) DOX\u0026thinsp;+\u0026thinsp;SC79: cells were pretreated with SC79 (10\u0026micro;mol/L) for 2h, followed by DOX (5 \u0026micro;mol/L) for additional 24h; (5) DOX\u0026thinsp;+\u0026thinsp;GSK-872\u0026thinsp;+\u0026thinsp;LY294002: cells were treated with GSK-872 (3 \u0026micro;mol/L) and LY294002 (10\u0026micro;mol/L) for 2h, followed by DOX (5 \u0026micro;mol/L) for additional 24h. Concentrations of pharmacological inhibitors were chosen in reference with earlier previous studies[32, 35\u0026ndash;36].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular ROS measurement of H9c2 cells by DCFH-DA\u003c/h2\u003e \u003cp\u003eCells were incubated with 10 \u0026micro;M DCFH-DA in DMEM medium withoutFBS at 37\u003csup\u003e◦\u003c/sup\u003eC for 1 h. Afterward, cells were washed three times with PBS and the fluorescent images were observed with an Olympus Fluoview FV1000 microscope. The fluorescence intensity was quantified by using an Image J software (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eTUNEL staining\u003c/h2\u003e \u003cp\u003eThe TUNEL staining method was used to measure apoptotic rate using an in situ cell death detection kit according to the manufacturer's instructions. Images were captured using an Olympus FV1000confocal microscope (Olympus, Japan). The TUNEL-positive cardiomyocytes exhibited green nuclear staining (green) and were counted in five randomly selected fields under high-power magnification.Apoptotic rate was determined using the ratio of TUNEL-positive cells normalized to total cardiomyocyte counts.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eFor immunofluorescence staining of Nrf2, H9c2 cells were permeabilized using 0.1% Triton X-100% and 1% bovine serum albumin. Then, samples were incubated with an anti-Nrf2 antibody at 4\u003csup\u003eo\u003c/sup\u003eC overnight, followed by incubation with Alexa fluor-488 goat anti-rabbit secondary antibodies for 1 h at room temperature.Ultimately, samples were visualized with a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePI (Propidium iodide) staining assay\u003c/h2\u003e \u003cp\u003ePI was used to stain for H9c2 cells (4.5 \u0026micro;M PI per well) at 37\u0026deg;C for 20 min. Images of the cardiomyocytes were acquired immediately and examined using an Olympus Fluoview FV1000 microscope (Olympus, Japan). The percentage of positive cells was counted, and the average fluorescence intensity was evaluated using Image Pro advanced software. The results are presented as PI-positive cells (\u0026times;100%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from myocardial tissue and H9c2 cell after diverse treatments and prepared for western blotting. After separating the protein samples by 10\u0026thinsp;~\u0026thinsp;12% SDS-PAGE, proteins were transferred onto a PVDF membrane and incubated with 5% non-fat milk in TBST for 2 hours. The membrane was then incubated with primary antibodies at 4\u003csup\u003eo\u003c/sup\u003eC overnight. Then washing with TBS containing 0.1% Tween 20 (TBST)for 3 times, 10 minutes at once. After washing with TBST, the membranes were incubated with HRP after diverse treatments and prepared for we in 23℃. Next, washing with TBS containing 0.1% Tween 20 (TBST) for 3 times, 10 minutes at once. Then the proteins were visualized using chemiluminescent reagents (Millipore, Billerica, MA, USA) under ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and the densities of the bands were quantified by Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA) GAPDH was used as an internal reference.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eResults are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, and GraphPad Prism software was used for statistical analysis. Quantitative data were analyzed using the one-way analysis of variance (ANOVA) analysis followed by Tukey post-hoc test \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRIP3 activation and AKT/Nrf2 downregulated are involved in doxorubicin-induced cardiotoxicity\u003c/h2\u003e \u003cp\u003eFirstly, RIP3 activation and protein levels of AKT and Nrf2 were determined in mouse hearts. Our data revealed that DOX challenge activated RIP3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C). Furthermore, Western blot revealed that protein expression of AKT and Nrf2 were significantly downregulated following DOX challenge compared with Control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). The results indicated RIP3 activation and AKT/Nrf2 downregulation in DOX-induced cardiotoxicity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRIP3 inhibition protected against DOX-induced cardiac dysfunction and pathological myocardial changes\u003c/h2\u003e \u003cp\u003eTo examine the role of RIP3 in DOX-induced cardiotoxicity, GSK-872, an inhibitor of RIP3, was used in DOX-induced cardiotoxicity. With the treatment of DOX for 7 days, cardiac function indicators including FS and EF were significantly decreased, while LVIDd but not LVPWdwas elevated in DOX-treated mice, the effects of which were greatly attenuated by GSK-872 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-E). Serum LDH activity was significantly increased in DOX-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). In addition, HE and Masson's trichrome staining revealed that the myofibrillar component was disorganized and degenerated with deposition of fibrotic tissue in DOX-treated hearts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, H). Besides, survival rate was significantly decreased in the DOX-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). These DOX-induced cardiac anomalies in cardiac injury marker, geometry, function, histopathology and survival were greatly attenuated by the RIP3 inhibitor GSK-872.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRIP3 inhibition protected against DOX-induced oxidative stress and pyroptosis\u003c/h2\u003e \u003cp\u003eConsistent with previous studies, our results confirmed that DOX treatment resulted in significant oxidative stress and pyroptosis in mouse hearts. As indicated by the protein expression of oxidative stress including NOX2 and NOX4 were increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C). Similarly, the proteins associated with pyroptosis such as NLRP3, GSDMD and Caspase-1 were elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). Interestingly, these proteins level were significantly decreased by RIP3 inhibitor GSK-872 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-F). These data demonstrated that RIP3 inhibition protected against DOX-induced oxidative stress and pyroptosis in mouse hearts.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRIP3 inhibition increased DOX-induced AKT/Nrf2 downregulation\u003c/h2\u003e \u003cp\u003eTo detect whether AKT/Nrf2 signalling contributes to the protective effects of RIP3 inhibition against DOX-induced cardiotoxicity, we measured the protein expression of AKT and Nrf2 in mouse hearts. Western blot analysis showed robust downregulation of the protein levels of AKT and Nrf2 in DOX-treated hearts while significantly increased levels of RIP3 phosphorylation. Distinctly, these proteins expression were heavily bated by the RIP3 inhibitor GSK-872 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). These results indicated that AKT/Nrf2signallingwas involved in the protective effects of RIP3 inhibition against DOX-induced cardiotoxicity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eSC79 mitigate myocardial injury in DOX-treated H9c2 cells\u003c/h2\u003e \u003cp\u003eNext, to investigated whether AKT/Nrf2 activation protected against the DOX-induced cardiotoxicity, SC79, an agonist of AKT, was used in DOX-induced H9c2 cells. Following treated with DOX 24 hours, Western blot revealed that protein expression of Nrf2, HO-1 and BCL-2 were significantly downregulated while BAX was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-F). Besides, LDH activity and apoptotic index greatly increased in the DOX-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-J). The cardiac injury marker and apoptotic index were greatly attenuated by the AKT agonist SC79. These data indicated that protected against the DOX-induced cardiotoxicity via activating AKT/Nrf2 pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-J).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eAKT/Nrf2 inhibition blocked the protective effects of RIP3 inhibition on DOX-treated H9c2 cells\u003c/h2\u003e \u003cp\u003eSubsequently, to examinewhether AKT/Nrf2 involved in RIP3 inhibition-mediated protective effects on DOX-induced cardiotoxicity, LY294002, an inhibitor of AKT, was used in GSK-872 and DOX-induced H9c2 cells. GSK-872 alleviated DOX-induced myocardial injury in H9c2 cells. Western blot showed that the expression of NOX2, NOX4, NLRP3, GSDMD, IL-1β and Caspase-1 were significantly decreased while the expression of P-AKT and Nrf2 were significantly increased following with GSK-872 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-J). In addition, GSK-872 elevated the cell viability and decreased LDH, myocardial PI-positive cells which as evidence of cardiomyocyte pyroptosis, activity ROS production and apoptotic index (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B,F-H). Besides, immunofluorescence staining results demonstrated that the expression of Nrf2 was increased in the wake of GSK-872 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-D). The improvement of cardiac injury marker, marker for oxidative stress orpyroptosis and apoptotic index following GSK-872 were greatly abrogated by the AKT inhibitor LY294002 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results indicated that the effects of RIP3 inhibition on oxidative stress and pyroptosisin DOX-treated myocardial were related to AKT/Nrf2 signalling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAnthracyclines, including doxorubicin (DOX), are widely used for the treatment of a variety of cancers.It is known that anthracyclines induce irreversible cardiomyocyte injury, leading to cardiac fibrosis, cell apoptosis and left ventricular dysfunction, enroute to heart failure[37].However, the pathological mechanisms of DOX-induced cardiotoxicity have not been fully elucidated. It is confirmed cardiomyocytes apoptosis, oxidative stress and pyroptosis contribute to the development of DOX-induced cardiomyopathy. In this study, our results manifested RIP3 activation and the AKT/Nrf2 pathway inhibition in DOX-treated mouse hearts and H9c2 cells. Also, data in this experiment indicated that RIP3 inhibition reduced oxidative stress and pyroptosis through the AKT/Nrf2 pathway in mouse hearts and H9c2 cells with DOX-induced cardiotoxicity. These findings present a novel mechanism of DOX-induced myocardial injury, which suggests that RIP3 is a potential target for the prevention of DOX-induced cardiotoxicity.\u003c/p\u003e \u003cp\u003eIncreasing evidence has suggested vital roles of oxidative stress and pyroptosis in the pathogenesis and development of cardiovascular diseases. AKT is a serine/threonine protein kinase involved in the regulation of cell survival and metabolism. A growing body of studies suggested that AKT prevented cardiomyocyte oxidative stress in response to DOX, while AKT inhibition exacerbated DOX-induced cardiomyocyte oxidative stress and cardiac dysfunction[38]. Nrf2, a transcriptional factor, regulates the expression of antioxidant proteins. Activating Nrf2 to attenuate oxidative stress and apoptotic cell death, prevents DOX-induced myocardial injury, and ameliorates cardiac dysfunction. In addition, Nrf2 was reported to regulate NLRP3 inflammasome activation, influencing the degree of pyroptosis. What\u0026rsquo;s more, several studies have demonstrated that RIP3 activation is an important pathogenic factor in ischemia-and DOX-induced necroptotic cell death[39\u0026ndash;40]. However, whether AKT/Nrf2 pathway is involved in the beneficial effects of RIP3 inhibition and the underlying mechanisms RIP3 activation in DOX-induced cardiotoxicity are still to be elucidated.To estimate the effects of RIP3 on oxidative stress and pyroptosis in DOX-induced cardiotoxicity, we measured oxidative stress and pyroptosis in DOX-challenged hearts treated with GSK-872. Consistent with previous studies, our results further confirmed that DOX-inducedpathological structural damage and cardiac dysfunction,as indicated by HE and Masson's staining, decreased EF, FS and increased LVIDd and LDH activity. While GSK-872 improved DOX-induced pathological structural damage and cardiac dysfunction, demonstrating that RIP3 inhibition could play a protective role in DOX-induced cardiotoxicity.Reactive oxygen species production and NLRP3inflammasome are involved in oxidative stress and pyroptosis respectively, bothplay a crucial role in the pathogenesis of cardiovascular diseases[12,27]. Our results also indicated that GSK-872 suppressed DOX-induced oxidative stress and pyroptosis by offsetting ROS production, formation of NLRP3inflammasomeand apoptotic rates. Meanwhile, proteinlevel of anti-oxidant protein Nrf2 was increased. However, LY294002, an inhibitor of AKT, abrogated the anti-oxidative stress and anti-pyroptosis effects of GSK-872, denoting involvement of AKT/Nrf2 signaling in the protective effects of RIP3 inhibitionagainst DOX-induced cardiotoxicity.\u003c/p\u003e \u003cp\u003eIdentically, our results showed that DOX significantly reduced AKT and Nrf2 levels. Intriguingly,GSK-872 markedly increased AKT and Nrf2 levels in DOX-treatedcardiomyocyte. However, inhibiting the AKT/Nrf2 pathway with LY294002 significantly abolished the protective effects of RIP3 inhibition against myocardial injury, oxidative stress and pyroptosisin DOX-treated cardiomyocyte. Furthermore, LY294002 reduced the protein levels of AKT and Nrf2 induced by RIP3 inhibition in DOX-treated cardiomyocyte(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, the present study provides mechanistic evidence that RIP3 is a potential target for the treatment of DOX-induced cardiotoxicity through regulation of oxidative stress and pyroptosisin an AKT/Nrf2-dependent manner. Therefore, further studies are needed to elucidate the exact mechanism.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003eZhenyi Wang and Yitong Yang conceived and designed these experiments. Nisha Wang, Linhe Lu and Chennian Xu performed these experiments. Zhenyi Wanganalyzed and interpreted the data. Zhenyi Wang wrote the manuscript. Lifang Yang and Jun Ren revised the manuscript. All the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003eThis research was supported in part by National Natural Science Foundation of China (81774415, 82174493, 92249301); Outstanding Youth Foundation of Shaanxi Province (2021JC-49); Xi\u0026rsquo;an Talent Program (XAYC210024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003eAll authors have completed the ICMJE uniform disclosure form. All the authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003eThe study protocol was approved by the Ethics Committee of the Fourth Military Medical University. Animal care and study were approved by the Laboratory Animal Center of the Fourth Military Medical University\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003eNot Applicable.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDeSantis CE, Siegel RL, Sauer AG, Miller KD, Fedewa SA, Alcaraz KI, Jemal A. Cancer statistics for African Americans, 2016: Progress and opportunities in reducing racial disparities. CA Cancer J Clin. 66, 290-308. https://doi.org/10.3322/caac.21340\u003c/li\u003e\n\u003cli\u003eFern\u0026aacute;ndez-Ruiz I. Cardioprotection: Cardiotoxicity of anticancer therapy. 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Toxicology. 460, 152881. https://doi.org/10.1016/j.tox.2021.152881\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Doxorubicin, Cardiotoxicity, RIP3, Oxidative stress, Pyroptosis","lastPublishedDoi":"10.21203/rs.3.rs-3976154/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3976154/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study was designed to explore the role of RIP3 in DOX-induced cardiotoxicity and its underlying molecular mechanisms.Ourresults demonstrate that RIP3 exacerbates DOX-inducedcardiotoxicity through promoting oxidative stress and pyroptosis by regulating the AKT/Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Inhibition of RIP3 by using GSK-872 attenuated DOX-induced cardiac remodeling and contractile dysfunction. Moreover, by using GSK-872 \u003cem\u003ein vivo\u003c/em\u003e, the results revealed that inhibition of RIP3 alleviated DOX-induced cardiotoxicity by the resulting inhibition of oxidative stress and pyroptosis. Besides, inhibition of RIP3 increased the protein levels of AKT and Nrf2 in DOX-treatedmouse hearts. Furthermore, the AKT inhibitor LY294002 lessened RIP3 reduction-offered protection against DOX-induced H9c2 cell injury by moderating oxidative stress and pyroptosis. Taken together, these data demonstrate that RIP3 activation orchestrates DOX-induced cardiotoxicity through elevated oxidative stress and pyroptosis in an AKT/Nrf2-dependent manner. Those findings highlight the clinical relevance and therapeutic potential of targeting RIP3for the treatment of DOX-induced cardiotoxicity.\u003c/p\u003e","manuscriptTitle":"RIP3 orchestrates oxidative stress and pyroptosis in doxorubicin-induced cardiotoxicity through regulation of AKT/Nrf2 signaling cascade","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-28 12:15:16","doi":"10.21203/rs.3.rs-3976154/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-24T18:33:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-06T06:07:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9769ab20-6155-43f5-9ee2-2ecdf66e8195","date":"2024-02-29T12:37:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-28T07:13:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-26T11:29:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-22T07:17:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular and Cellular Biochemistry","date":"2024-02-21T15:31:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"04d950c8-3569-4054-86ec-9dc1ffc744da","owner":[],"postedDate":"February 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-04T21:09:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-28 12:15:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3976154","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3976154","identity":"rs-3976154","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}