Costunolide Protects Myocardial from Ischemia Reperfusion Injury through Nrf2 Activation

Costunolide Protects Myocardial from Ischemia Reperfusion Injury through Nrf2 Activation | 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 Costunolide Protects Myocardial from Ischemia Reperfusion Injury through Nrf2 Activation Weixin Li, Yue Luo, Zhuqi Huang, Siyuan Shen, Chengyi Dai, Sirui Shen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1857993/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 Purpose Costunolide (Cos) is a naturally occurring sesquiterpene lactone that exhibits anti-oxidative properties. In this study, we demonstrated the protective mechanism of Cos against ischemia/reperfusion (I/R)-induced heart injury. Methods C57BL/6 mice were pretreated with Cos (10 mg/kg/day) or 1% CMC-Na for 1 week. The left anterior descending coronary artery (LAD) was ligated for 30 minutes for ischemia, and followed by ligation release for 4 hours for reperfusion. H9c2 cells challenged with tert -butyl hydroperoxide (TBHP) were used for in vitro studies. Results Pretreatment of Cos significantly reduced myocardial infarct size, serum CK-MB and LDH level in I/R injured heart. Cos administration significantly attenuated the amount of reactive oxygen species (ROS) and ameliorated the apoptosis both in in vitro and in vivo studies. Further investigation revealed that Cos significantly increased the expression of heme oxygenase 1 (HO-1) and NAD(P)H [quinone] dehydrogenase 1 (NQO-1). Meanwhile Cos increased B-cell lymphoma-2(Bcl-2) and decreased the BCL2-Associated X Protein (Bax) protein level. Silence of nuclear factor erythroid 2-related factor (Nrf2) significantly reverses the protective effect of Cos in TBHP-challenged H9C2 cells. Conclusion Our data clearly showed that Cos reduced TBHP challenged H9c2 cells and attenuated myocardial I/R injury through Nrf2 activation. These results indicate that Cos might be benefit in the therapy of myocardial I/R injury. Cardiac ischemia reperfusion injury Costunolide cardiomyocytes reactive oxygen species Nrf2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Cardiovascular diseases are the primary factor of death worldwide [ 1 ], and myocardial infarction is especially harmful to the health of the people. Cardiac ischemia reduces oxygen and nutrient supply to heart tissues, leading to heart dysfunction and myocardial cell necrosis [ 2 ]. Early reperfusion treatment during myocardial ischemia is important for saving the myocardium; however, this strategy can also cause further injury in myocardial cells, known as myocardial ischemia reperfusion injury [ 3 , 4 ]. The burst of ROS in the myocardial, initiated during ischemia and exacerbated upon reperfusion, is thought to be the main cause of the heart injury [ 5 , 6 ]. Reperfusion after thrombosis induces the overproduction of ROS by mitochondria, which damages cardiomyocytes [ 7 ]; an antioxidant strategy is reported to have beneficial effects in decreasing such I/R injury. Various endogenous antioxidases such as glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) can eliminate oxidation produces and help decrease the oxidative stress-induced damage [ 8 , 9 ]. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that exist in a variety of cells. Nrf2 promotes many protective genes expression in the maintenance of cellular homeostasis. In the stable state, Nrf2 is anchored by the Keap1-Cul3 ubiquitin E3 ligase complex in the cytoplasm. Under oxidative stress, Nrf2 can quickly separate from the Keap1-Nrf2 complex, transfer to the nuclear, and eventually combine with the antioxidant response elements (AREs) to promote the synthesis of antioxidant enzymes [ 10 ]. Costunolide (Cos) is found to be extracted from various medicinal plants, including Costus speciosus [ 11 ], Saussurea lappa , and Laurus nobilis [ 12 , 13 ]. Cos is distinguished by its ability to exhibit various biological effects [ 14 ], including anti-cancer [ 15 ], antifungal, antimicrobial [ 16 ], anti-inflammatory [ 17 ], and antioxidant effects [ 18 ]. It is known that Cos exerts cytotoxic on various human cancer cells, including human prostate cancer [ 19 ], human breast carcinoma [ 20 ], renal cell carcinoma [ 15 ], and chronic myeloid leukemia [ 21 ]. It has been reported that Cos can enhance the chemotherapy effect in the cure of multidrug resistance cancer [ 22 ]. A study in our laboratory indicated that Cos can directly bounds to and inhibited the activity of thioredoxin/thioredoxin reductase 1 (TrxR1); thus, Cos administration increases ROS level and activates endoplasmic reticulum stress in colon cancer cells l [ 23 ]. In another study, Cos directly targeted AKT and inhibited colon cancer cell growth [ 24 ]. Contrarily, a C. speciosus rhizome methanolic extract can significantly clear oxygen free radical and nitric oxide (NO) produces in vitro [ 25 ]. In an in vivo experiment, it was found that gavage of Cos in a diabetes rat significantly increased heart glutathione (GSH) content and the activities of SOD [ 26 ]. In this study, Cos exhibits protective capability against myocardial I/R injury in vivo and reduces tertiary butyl hydrogen peroxide (TBHP) caused cell apoptosis in vitro . Furthermore, the underlying mechanism of antioxidation by Cos in the signaling pathways responsible for cell apoptosis was elucidated. Material And Methods Reagents and cell culture Chemicals. Tert-butyl hydroperoxide (TBHP, cat# A10777) was purchased from XIYA Reagent (Chengdu, Sichuang, China). 2,3,5-triphenyltetrazolium chloride (TTC cat# T8877) and Costunolide (cat# 553-21-9) were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against Lamin B1(cat# SC-374015), GAPDH (Cat# sc-365062), Bax (cat# SC-7480), Bcl2 (cat# SC-7382), NQO-1 (cat# SC-376023) and HO-1 (cat# SC-390991) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against Cleaved Caspase-3(cat# 9664) and Nrf2(cat#12721) were purchased from Cell Signaling Technology (Massachusetts, United States). Cell culture H9c2 embryonic rat heart-derived cardiomyocyte line was purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). H9c2 Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco/BRL life Technologies, Eggenstein, Germany) containing 4.5g/L D-glucose. The media was supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY), 100 U/mL of penicillin, and 100 mg/mL of streptomycin. Mice and animal experiments All of the experimental protocols and animal care procedures were approved by the Wenzhou Medical University Animal Policy and Welfare Committee. (Approval Document No. wydw2021-0124). Six-Seven weeks old male C57BL/6 mice weighting 20–22g were obtained from the Shanghai Laboratory Animal Research Center (Shanghai, China). All mice received humane care according to NIH Guide for the Institutional Animal Care and Use Committee (IACUC). Cos was dissolved in 1% CMC-Na and was given at a dose of 10 mg/kg/day for 7 days by gavage. Mice were given CMC-Na (1%) as vehicle. The mice were anesthetized by 1% pentobarbital sodium (50mg/kg) by intraperitoneal injection before LAD I/R surgical process. The myocardial ischemia reperfusion injury model in C57BL/6 mice was induced as described in our previous study[ 27 ]. Myocardial infarct size measurement The in vivo myocardial infarct size was evaluated using Evans Blue/TTC dual dyeing. Briefly, after retied the LAD, 1% Evans blue dye was injected into the left ventricle. Then we sliced the heart and incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma, St. Louis, MO) for 15 minutes. The sliced myocardium was dyed with blue, red and white. The images were taken by a microscopy system (Nikon TE2000-S). The infarct size was calculated use image J as described previously [ 27 ]. TUNEL staining The apoptotic cells in the heart were detected by TUNEL Apoptosis Assay Kit (cat# C1086, Beyotime, China) according to the manufacturer's instruction. For H9c2 cells, cells were fixed by 4% paraformaldehyde, incubated with 0.25% Triton-X100 in PBS, then apoptotic cells were detected with TUNEL Apoptosis Assay Kit (cat# C1086, Beyotime, China). Heart superoxide production The frozen slices of the heart were incubated with DHE for 45 min. The images were viewed under the fluorescence microscope (Nikon, Japan). The cellular hydrogen peroxide was detected using 2’,7’- dichlorodihydrofluorescein (DCF) staining. Determination of MDA and SOD H9C2 cells or heart tissue were homogenized with sample buffer, and the serums were diluted with sample buffer. The superoxide dismutase (SOD) levels in sample were determined using commercially available kits (cat# S0101S, Beyotime Biotech, Nantong, China) according to the manufacturer's instructions. The malondialdehyde (MDA) were determined using commercially available kits (cat# S0131S, Beyotime Biotech, Nantong, China). Measurement of CK-MB and LDH The serum was collected and the CK-MB, and LDH were measured using commercially available assay kits (cat# H197-1-1 and cat# A020-2-2, Jiancheng, China). Rhodamine-labeled phalloidin staining To assess cardiomyocyte hypertrophy, H9c2 cells were fixed, permeabilized, then incubated with Rhodamine-labeled phalloidin for 30 min. Nrf2 silencing by siRNA Nrf2 was silenced in H9C2 cells by siRNA transfections. siRNA for Nrf2 was designed and synthesized by Gene Pharma Co. (Shanghai, China). siRNA sequences (5’to 3’), GGGUAAGUCGAGAAGUGUUTT and AACACUUCUCGACUUACCCTT, were used. Cells were transfected using Lipofectamine™ 2000 (Invitrogen Ltd., Carlsbad, CA). Western blot analysis Lysates from cultured cells or mouse heart tissues were prepared. Samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electro-transferred to nitrocellulose membranes. Membranes were blocked for 2 hour at room temperature in Tris-buffered saline (pH 7.6) containing 0.05% Tween 20 and 5% non-fat milk. Each nitrocellulose membrane was incubated with specific antibodies at 4°C overnight. Secondary antibodies were applied for 1 h at room temperature. Immunoreactivity was visualized using enhanced chemiluminescence reagents (Bio-Rad Laboratories). The amounts of the proteins were analyzed using Image J, and normalized to their respective control. Statistical analysis All experiments were randomized and blinded, date presented in this study is representative of at least 3 independent experiments and is expressed as Mean ± SEM. Statistical analysis was calculated with GraphPad Prism 8.0 software (San Diego, CA, USA). Differences between groups were examined using the 2-tailed Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test. Details of each experiment can be found in the figure legend. Differences were considered significant at p < 0.05. Results Cos limits I/R-induced heart injury Cos, a natural sesquiterpene lactone (Fig. 1 A), has been known to show powerful antioxidative properties. To investigate whether Cos is protective in a hypoxic myocardium, we examined its effect in the in vivo cardiac ischemia reperfusion injury model. C57BL/6 mice were pretreated with Cos or 1% CMC-Na for 1 week. The model of I/R was processed with LAD ligation for 30 minutes, and followed by ligation release for reperfusion. After retied of LAD, the infarct area of the heart was assessed using Evans Blue and TTC dual staining. In Fig. 1 B, I/R increased the infarct region, whereas the Cos administration notably reduced I/R-induced myocardial infarction. There is no statistically difference in the ratio of AAR/LV among the three groups as shown in Fig. 1 C. While pretreatment of Cos for seven days significantly attenuated the infarct size compared to I/R group (Fig. 1 D). Moreover, under myocardial ischemia reperfusion injury, the serum lactate dehydrogenase (LDH) level and creatine kinase M and B isoform (CK-MB) in the mice were significantly increased. Cos downregulated the LDH and CK-MB levels (Figs. 1 E and 1 F). As presented in Fig. 1 G, compared with I/R group, Cos administration reduced the MDA content in the myocardium. Then we measured the SOD levels both in the serum and in the heart tissues. As shown in Fig. 1 H, myocardial ischemia reperfusion injury reactively increased the serum SOD activity in both the I/R model group and Cos-pretreated group. Whereas in the myocardial tissue, the Cos-treated I/R group have a higher activity of SOD compare to the sham group. These results demonstrate that myocardial I/R damages the function of the heart, and Cos treatment can ameliorate such injury. Cos attenuates I/R-induced heart apoptosis Then we detected the protective effects of Cos on apoptosis in ischemia reperfusion injured mice. As shown in Fig. 2 A- 2 B, the TUNEL-positive cells in the I/R model group were much more compare to the sham group. The apoptotic cells were less in the Cos-treated group than in the I/R model group, indicating that Cos treatment significantly reduced I/R-induced myocardial cell apoptosis. Then were further detected the expression of cleaved caspase-3, Bax, and Bcl-2 protein in each group (Figs. 2 C and 2 D). I/R mice treated with Cos showed significant downregulation of cleaved caspase-3 and Bax, and a significantly increase in Bcl-2 compared to the I/R group. Cos reduces I/R-induced hear ROS production As shown in Figs. 3 A and 3 B, ROS levels were increased in the I/R group, and Cos treatment decreased myocardial ROS levels under I/R injury. As illustrated in Figs. 3 C and 3 D, pretreatment with Cos did not increase the expression of Nrf2 protein in the I/R model myocardium, whereas it increased Nrf2 nuclear translocation. As a result, the anti-oxidant protein NQO-1 and HO-1 significantly increased (Figs. 3 E and 3 F). This further reduced ROS production in the heart of Cos treated mice. Cos attenuates TBHP-induced apoptosis and ROS levels in H9C2 cells To investigate the cytoprotective effect of Cos, the H9C2 was exposed to TBHP. As shown in Figs. 4 A and 4 B, the TUNEL staining indicated that Cos attenuated TBHP-induced cardiac cell apoptosis. In addition, Cos increased the expression of Bcl-2 (Fig. 4 C). An increase of the Bax protein was observed in TBHP-treated H9C2 cells; however, Cos significantly reduced such Bax expression. Treatment with 2.5 µM Cos significantly decreased ROS production. To further investigate the antioxidative property of Cos, H9C2 cells were stimulated with TBHP for 24 h. The SOD activity and MDA level were detected. Increasing concentrations of Cos reduced the TBHP-induced MDA level in a dose-dependent manner (Fig. 4 D) while maintaining the SOD activity (Fig. 4 E). Since the increase in ROS levels is critical in TBHP-induced cardiomyocyte injury, we determined intracellular ROS levels in TBHP-induced H9C2 cells by detecting DCF-positive cells. As evident from Figs. 4 F and 4 G, DCF staining show that ROS production significantly raised in TBHP-treated group. As shown in Figs. 4 H and 4 I, pretreatment with Cos decreased TBHP-induced cell morphology changes in H9C2 cells. The results show that Cos pretreatment reduce the ROS levels and apoptosis induced by TBHP. Cos activates Nrf2 and attenuates TBHP-induced apoptosis As shown in Figs. 5 A, the addition of Cos significantly increased Nrf2 nuclear translocation in TBHP-treated H9C2 cells. Cos also promoted the NQO-1 (Nrf2 target) protein levels in a dose-dependent manner in total cell lysate of TBHP-stimulated H9C2 cells (Fig. 5 B). In Fig. 5 C, Nrf2 levels were significantly reduced by Nrf2 siRNA transfection. Cos pretreatment decreased the level of the cleaved caspase-3, in TBHP-treated H9C2 cells, whereas Nrf2 silencing increased cleaved caspase-3 accumulation (Fig. 5 D). Further analysis of the cell apoptosis pathway revealed that Nrf2 knockdown reversed the protective effect of Cos (Fig. 5 E). Furthermore, we also analyzed that Nrf2 silencing reverses NQO-1 overexpression under Cos treatment, using western blotting (Fig. 5 F). The changes in the morphology of H9C2 cells induced by Cos were examined using rhodamine-labeled phalloidin staining. As shown in Figs. 5 G and 5 H, Nrf2 siRNA transfection increased TBHP-induced injury despite the protection by Cos. These results indicate that Cos activates Nrf2 signaling pathway and protects myocytes from TBHP-induced apoptosis in vitro . Discussion The purpose of this research was to expound the mechanism of Cos in protection of myocardial in I/R injured heart. Recent reports have emphasized that oxygen free radicals is the major cause of myocardial apoptosis in I/R injury, and that an antioxidant therapy is an effective approach to reduce cardiac injury [ 28 ]. As observed in our study, ischemia reperfusion significantly increased myocardial infarct size, serum CK-MB, ROS level and apoptosis in mice. Interestingly, treatment of Cos attenuated these abnormities, suggesting that Cos exerts myocardial protective activity in I/R mice. The protective effect of Cos was also observed in our in vitro study. Acute overgeneration of ROS products under pathophysiological states is vital in the development of reperfusion injury after ischemia for thrombolysis or thrombus removal [ 29 ]. Mounting evidence indicates that oxidative stress is critical in myocardial apoptosis in the I/R damaged heart [ 30 ]. It has been reported that Cos administration decreased doxorubicin-induced cardiorenal injury by suppressing oxidative stress [ 31 ]. In another study, Cos attenuated alcohol-induced liver injury by reducing ROS production [ 32 ]. Interestingly, Cos obviously increases the intracellular oxidative stress in human U2OS cells and promotes the generation of ROS in esophageal cancer cells, and eventually induces apoptosis of carcinoma cells [ 33 ]. Studies have shown that Cos exerts anticancer activity, the diverse mechanism such as the reduction of angiogenic factor signaling pathway [ 34 ], inhibition of telomerase activity [ 20 ] and accumulation of intracellular ROS [ 15 ]. Pretreatment with Cos inhibited TBHP-induced Bax expression and increased the content of the a Bcl-2 in H9C2 cells. It is known that elimination of oxygen free radicals is a practical strategy to prevent myocardial ischemia [ 35 ]. Our study showed that Cos decreased the ROS production induced by I/R injury or TBHP; however, the reason is unknown. As is reported that allicin active Nrf2 and protected the heart from Ang II induced cardiac hypertrophy [ 36 ]. Our previous study showed that curcumin and curcumin analogue 14p active Nrf2 pathway and attenuates I/R induced myocardial injury [ 27 ]. Nrf2 is the key transcription factor that regulates the antioxidant response, which stimulates the transcription of genes involved in many aspects of cytoprotection, such as those of glutamate cysteine ligase, SODs, HO-1, and NQO-1 [ 37 , 38 ]. Our results showed that pretreatment with Cos before I/R injury or TBHP challenge did not change total Nrf2 expression but increased Nrf2 nuclear translocation and enhanced the expression NQO1 both in vitro and in vivo . This also indicates that Nrf2 is a feasible strategic target in therapies for myocardial I/R injuries. These results are consistent with previous reports indicating that Cos exerts neuroprotective effects by activating Nrf2 in PC12 cells; however, such prior research was only conducted in vitro [ 39 ]. Conclusion To sum up, our study demonstrates the preventive role of Cos against oxidative stress and apoptosis induced by I/R or TBHP in vitro and in vivo , respectively. The protective mechanism of Cos is schematically shown in Fig. 6 . Our results strongly suggest that Cos, a Nrf2 activator, has huge therapeutic potential in the therapy of myocardial ischemia reperfusion injury. abbreviations AAR, area at risk; AREs, antioxidant response elements; CAT, catalase; CK-MB, creatine kinase M and B isoform; DHE, dihydroethidium; DCF, 2’,7’-dichlorodihydrofluorescein; GPx, glutathione peroxidase; GSH, glutathione; HO-1, heme oxygenase 1, I/R, ischemia and reperfusion; LDH, lactate-dehydrogenase; MDA, methane dicarboxylic aldehyde; NQO-1, NAD(P)H [quinone] dehydrogenase 1; NO, nitric oxide; Nrf2, NF-E2-related factor 2; ROS, reactive oxygen species; SOD, super oxygen dehydrogenases; TBHP, tert-butyl hydroperoxide; TrxR1, thioredoxin/thioredoxin reductase 1 Declarations Authors’ Contributions Guang Liang and Wu Luo contributed to the literature search and study design. Guang Liang, Wu Luo, Siyuan Shen, and Chengyi Dai participated in the drafting of the article. Weixin Li, Yue Luo, Sirui Shen, and Zhuqi Huang carried out the experiments. Guang Liang, Wu Luo, and Weixin Li revised the manuscript. Wu Luo and Weixin Li contributed to data collection and analysis. Funding This work was supported by the National Key Research Project of China (2017YFA0506000 to G.L.), National Natural Science Foundation of China (82000793 to W.L., and 81930108 to G.L.), Natural Science Foundation of Zhejiang Province (LY22H070004 to W.L.), Zhejiang Provincial Key Research Project (2018C03068 to G.L.), Wenzhou Scientific Project (Y20210213 to W.L.) and Wenzhou Key Scientific Project (2018ZY009 to G.L.). Data Availability The used and/or analyzed datasets are available from the corresponding author on reasonable request. Ethics Approval All of the experimental protocols and animal care procedures were approved by the Wenzhou Medical University Animal Policy and Welfare Committee. Conflict of Interest The authors declare no conflict of interest. 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Allicin ameliorates cardiac hypertrophy and fibrosis through enhancing of Nrf2 antioxidant signaling pathways. Cardiovasc Drugs Ther. 2012;26(6):457–65. Yarmohammadi F, Rezaee R, Karimi G. Natural compounds against doxorubicin-induced cardiotoxicity: a review on the involvement of Nrf2/ARE signaling pathway. Phytother Res. 2021;35(3):1163–75. Cui T, Lai Y, Janicki JS, Wang X. Nuclear factor erythroid-2 related factor 2 (Nrf2)-mediated protein quality control in cardiomyocytes. Front bioscience (Landmark edition). 2016;21:192. Peng S, Hou Y, Yao J, Fang J. Activation of Nrf2 by costunolide provides neuroprotective effect in PC12 cells. Food Funct. 2019;10(7):4143–52. Supplementary Files SupplymentWB.pdf 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1857993","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":122161578,"identity":"aa066c02-e411-41d2-b4b3-f111479b90ca","order_by":0,"name":"Weixin Li","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weixin","middleName":"","lastName":"Li","suffix":""},{"id":122161579,"identity":"4a45463c-c5b9-4a1e-8aa0-78336c9fc07c","order_by":1,"name":"Yue Luo","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Luo","suffix":""},{"id":122161580,"identity":"edc77248-b30b-4501-a61d-3480f26fb771","order_by":2,"name":"Zhuqi Huang","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhuqi","middleName":"","lastName":"Huang","suffix":""},{"id":122161581,"identity":"89925995-bd89-4df8-94a9-e3fa3b52b36c","order_by":3,"name":"Siyuan Shen","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Siyuan","middleName":"","lastName":"Shen","suffix":""},{"id":122161582,"identity":"8e7e7c78-c2c5-4d62-ba63-3e7af9173dd1","order_by":4,"name":"Chengyi Dai","email":"","orcid":"","institution":"Wenzhou University","correspondingAuthor":false,"prefix":"","firstName":"Chengyi","middleName":"","lastName":"Dai","suffix":""},{"id":122161583,"identity":"6bd4bd25-0d99-4e7c-a3f1-7a5eb315bbbb","order_by":5,"name":"Sirui Shen","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sirui","middleName":"","lastName":"Shen","suffix":""},{"id":122161584,"identity":"6eeee535-1278-4f64-9ee6-a5a877c34399","order_by":6,"name":"Guang Liang","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guang","middleName":"","lastName":"Liang","suffix":""},{"id":122161585,"identity":"19249180-9974-4aa8-b068-a29ead45de40","order_by":7,"name":"Wu Luo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApklEQVRIiWNgGAWjYFAC5gYGhgoIU4JILYxALWdI1sLYRooWgxuJbRIf5x2ONjjAfPA2D4NdHlFaJGduO5y74QBbsjUPQ3IxQS1mQC3SvNtuA7XwmEnzMBxIbCBOyxyQFv5vpGhpANvCRpwW+zMPmy1nHPufO/Mwm7HlHINkwlok25MP3vhQk5bbd7z54Y03FXaEtSAAM4gwIF79KBgFo2AUjAI8AAC6xj6F0azmnQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3827-1159","institution":"Wenzhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Wu","middleName":"","lastName":"Luo","suffix":""}],"badges":[],"createdAt":"2022-07-14 12:38:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1857993/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1857993/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":24364691,"identity":"9d63aa2a-a160-417e-aa5b-c6030a69a957","added_by":"auto","created_at":"2022-07-26 18:52:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":989894,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCos limits ischemia reperfusion induced heart injury\u003c/strong\u003e. (A) Chemical structure of Cos. (A-I) C57BL/6 mice were pretreated with Cos (10 mg/kg/Day) or CMC-Na (1%) for 1 week, then mice were processed with LAD ligation for 30 minutes for ischemia. (B-D) At 24 hours after ligation released for reperfusion, the LAD was ligated again and the heart was retrograde perfusion with 1% Evans blue dyeing and 1% TTC staining was used to reveal the infarct area. (B) Representative images of myocardial infarct size. (C) Quantitative analysis of the ratio of area at risk (AAR) to the percentage of LV. (D) Changes in infarct size. (E-I) At 4 hours after ligation released for reperfusion, the animal was sacrificed, the heart tissues and serum were harvested. (E) The serum LDH activity, (F) The serum CK-MB activity and (G) the myocardial MDA content was detected. (H)The serum SOD activity and (I) myocardial SOD activity was detected. Mean ± SEM; n = 7-8 per group; *p\u0026lt; 0.05, **p\u0026lt; 0.01, ***p\u0026lt; 0.001 compared to I/R group; ns = not significantly different.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1857993/v1/2810e2a9829374a9d1c54928.jpg"},{"id":24364037,"identity":"d4d8cd3c-10ec-4396-90a0-438db588cc15","added_by":"auto","created_at":"2022-07-26 18:47:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":721833,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCos attenuates I/R induced myocardial apoptosis\u003c/strong\u003e. C57BL/6 mice were processed with LAD ligation for 30 minutes for ischemia, and then ligation was released for reperfusion for 4 hours. (A-B) TUNEL staining. Arrows showing TUNEL-positive cells. [Scale bar = 50μm]. (C-D) Immunoblot of Cleaved- Caspase3, Bax and Bcl-2 in the myocardial. GAPDH was used as loading control. Mean ± SEM; n = 3 per group; *p\u0026lt; 0.05, **p\u0026lt; 0.01 compared to the I/R group.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1857993/v1/1aa979cac1491c8c1587a729.jpg"},{"id":24364038,"identity":"58b2cc54-db05-4166-ab30-410d152886e2","added_by":"auto","created_at":"2022-07-26 18:47:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1180248,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCos attenuates I/R induced myocardial ROS production.\u003c/strong\u003e Myocardial ischemia reperfusion injury was produced as described in Figure2.\u0026nbsp;(A-B) Representative images of DHE staining from frozen section showing ROS (red). DAPI was used to counterstain (blue). Fluorescence intensity levels are shown in right panel. Scale bar =50μm; Mean ± SEM; n = 5; **p\u0026lt; 0.01 compared to the I/R group. (C) Total-Nrf2, protein level was detected by immunoblotting in the heart tissues. GAPDH was used as loading control. Mean ± SEM; n = 3; ns = not significantly different. (D) The nuclear Nrf2 protein and cytoplasm Nrf2 protein were isolated respectively in I/R myocardium, Western blot assay was used for the determination of Nrf2. Lamin B was used as loading control for nuclear proteins, GAPDH was used as loading control for cytoplasm proteins. Mean ± SEM; n = 3; *p\u0026lt; 0.05, **p\u0026lt; 0.01 compared to the I/R group. (E-F) Immunoblots showing levels of the HO-1 and NQO-1 protein in the heart tissues. Mean ± SEM; n = 3; *p \u0026lt; 0.05 compared to the I/R group.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1857993/v1/6ca041eb67b3d8e36eefaae8.jpg"},{"id":24364043,"identity":"4c99a22d-0afe-48c4-a31a-bcce33c20a62","added_by":"auto","created_at":"2022-07-26 18:47:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1298476,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCos attenuates TBHP-induced apoptosis and oxidative stress in H9C2 cells.\u003c/strong\u003e \u003c/p\u003e\u003cp\u003e(A-E) H9C2 cells were pretreated with Cos (0.625, 1.25, 2.5μM) for 2 hours, and then incubated with TBHP (100μM) for 24 h. (A-B) TUNEL staining was detected by kits. Arrows showing TUNEL-positive cells. [Scale bar = 100μm]. Mean ± SEM; n = 3; *p\u0026lt; 0.05 compared to the TBHP group. (C) Immunoblots showing levels of Bcl2 and Bax. GAPDH was used as loading control. Mean ± SEM; n = 3; *p\u0026lt; 0.05, **p\u0026lt; 0.01 compared to the TBHP group. The intracellular MDA content (D) and the SOD activity (E) were detected by kits. Mean ± SEM; n = 3; *p\u0026lt; 0.05, **p\u0026lt; 0.01, ***p\u0026lt; 0.001 compared to the TBHP group. (F-G) H9c2 cells were exposed to TBHP (100μM) for 12 hours. DCF staining was used to show intercellular ROS production. [Scale bar = 200μm]. Mean ± SEM; n = 3; *p\u0026lt; 0.05, compared to the TBHP group. (H-I) H9c2 cells were exposed to TBHP (100μM) for 12 hours. Representative images for Rhodamine-phalloidin staining are shown. [Scale bar = 100μm]. \u0026nbsp;Mean ± SEM, n = 3 per group, ***p\u0026lt; 0.001 compared to the TBHP group.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1857993/v1/2a8fc43fb9e1bb3a10015544.jpg"},{"id":24364039,"identity":"dba3bec0-8e5f-421b-bd20-1e279c9a97e3","added_by":"auto","created_at":"2022-07-26 18:47:16","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1242548,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCos protects H9C2 cells from the injury of TBHP by activates Nrf2.\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eH9C2 cells were pretreated with Cos (0.625, 1.25, 2.5μM) for 2 hours, and then exposed to TBHP (100μM). (A) Stimulated by TBHP for 1 hour, immunoblots showing nuclear content of Nrf2. Lamin B was used as loading control for nuclear proteins. Mean ± SEM; n = 3; *p\u0026lt; 0.05, compared to the TBHP group. (B) Stimulated by TBHP for 1hour, cytoplasmic content of NQO-1 was determined by western blotting. GAPDH was used as loading control. Mean ± SEM; n = 3; *p\u0026lt; 0.05 compared to the TBHP group. (C) Representative immunoblots showing levels of Nrf2 in H9C2 cells that transfected with Nrf2 siRNA as stimulated with TBHP (100μM) for 24h. GAPDH was used as loading control. Levels of (D) Cleaved-Caspase3 protein, (E) Bax and Bcl2 protein and (F) NQO-1 induced by 24 hours TBHP (100μM) exposure of H9C2 cells with or without Nrf2 siRNA. GAPDH was used as loading control. Mean ± SEM; n = 3; *p\u0026lt; 0.05, **p\u0026lt; 0.01 compared to the TBHP group. (G-H) Representative images for Rhodamine-phalloidin staining of H9C2 cells induced by 2hours TBHP (100μM) exposure of H9C2 cells with or without Nrf2 siRNA and Cos (2.5μM). [Scale bar = 50μm]. \u0026nbsp;Mean ± SEM, n = 3 per group, *p\u0026lt; 0.05, **p\u0026lt; 0.01 compared to the TBHP group.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1857993/v1/f578850217a6a124fb093034.jpg"},{"id":24365096,"identity":"981c4c85-a8c6-47ae-a955-43e21ed35814","added_by":"auto","created_at":"2022-07-26 18:57:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1263133,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMyocardial protective model of Cos. \u003c/strong\u003e\u003c/p\u003e\u003cp\u003eAREs, antioxidant response elements; HO-1, heme oxygenase 1; NQO-1, NAD(P)H [quinone] dehydrogenase 1; Nrf2, NF-E2-related factor 2; ROS, reactive oxygen species.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1857993/v1/81ed86c8cafdd1caa9a562d9.jpg"},{"id":24692011,"identity":"567d00e9-4b97-4979-becc-92c6624453b2","added_by":"auto","created_at":"2022-08-03 01:00:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1178104,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1857993/v1/fa20b8ef-6fa5-4109-900b-9d5ce9865de6.pdf"},{"id":24364041,"identity":"ca1ac897-1f8e-4f8a-9c37-bf6a283e7da4","added_by":"auto","created_at":"2022-07-26 18:47:16","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":551565,"visible":true,"origin":"","legend":"","description":"","filename":"SupplymentWB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1857993/v1/24b18ef3501a37b0732afda6.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eCostunolide Protects Myocardial from Ischemia Reperfusion Injury through Nrf2 Activation\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular diseases are the primary factor of death worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], and myocardial infarction is especially harmful to the health of the people. Cardiac ischemia reduces oxygen and nutrient supply to heart tissues, leading to heart dysfunction and myocardial cell necrosis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Early reperfusion treatment during myocardial ischemia is important for saving the myocardium; however, this strategy can also cause further injury in myocardial cells, known as myocardial ischemia reperfusion injury [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe burst of ROS in the myocardial, initiated during ischemia and exacerbated upon reperfusion, is thought to be the main cause of the heart injury [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Reperfusion after thrombosis induces the overproduction of ROS by mitochondria, which damages cardiomyocytes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]; an antioxidant strategy is reported to have beneficial effects in decreasing such I/R injury. Various endogenous antioxidases such as glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) can eliminate oxidation produces and help decrease the oxidative stress-induced damage [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that exist in a variety of cells. Nrf2 promotes many protective genes expression in the maintenance of cellular homeostasis. In the stable state, Nrf2 is anchored by the Keap1-Cul3 ubiquitin E3 ligase complex in the cytoplasm. Under oxidative stress, Nrf2 can quickly separate from the Keap1-Nrf2 complex, transfer to the nuclear, and eventually combine with the antioxidant response elements (AREs) to promote the synthesis of antioxidant enzymes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCostunolide (Cos) is found to be extracted from various medicinal plants, including \u003cem\u003eCostus speciosus\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], \u003cem\u003eSaussurea lappa\u003c/em\u003e, and \u003cem\u003eLaurus nobilis\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Cos is distinguished by its ability to exhibit various biological effects [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], including anti-cancer [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], antifungal, antimicrobial [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], anti-inflammatory [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and antioxidant effects [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It is known that Cos exerts cytotoxic on various human cancer cells, including human prostate cancer [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], human breast carcinoma [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], renal cell carcinoma [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and chronic myeloid leukemia [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It has been reported that Cos can enhance the chemotherapy effect in the cure of multidrug resistance cancer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A study in our laboratory indicated that Cos can directly bounds to and inhibited the activity of thioredoxin/thioredoxin reductase 1 (TrxR1); thus, Cos administration increases ROS level and activates endoplasmic reticulum stress in colon cancer cells l [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In another study, Cos directly targeted AKT and inhibited colon cancer cell growth [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Contrarily, a \u003cem\u003eC. speciosus\u003c/em\u003e rhizome methanolic extract can significantly clear oxygen free radical and nitric oxide (NO) produces \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In an \u003cem\u003ein vivo\u003c/em\u003e experiment, it was found that gavage of Cos in a diabetes rat significantly increased heart glutathione (GSH) content and the activities of SOD [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, Cos exhibits protective capability against myocardial I/R injury \u003cem\u003ein vivo\u003c/em\u003e and reduces tertiary butyl hydrogen peroxide (TBHP) caused cell apoptosis \u003cem\u003ein vitro\u003c/em\u003e. Furthermore, the underlying mechanism of antioxidation by Cos in the signaling pathways responsible for cell apoptosis was elucidated.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents and cell culture\u003c/h2\u003e \u003cp\u003e \u003cb\u003eChemicals.\u003c/b\u003e Tert-butyl hydroperoxide (TBHP, cat# A10777) was purchased from XIYA Reagent (Chengdu, Sichuang, China). 2,3,5-triphenyltetrazolium chloride (TTC cat# T8877) and Costunolide (cat# 553-21-9) were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against Lamin B1(cat# SC-374015), GAPDH (Cat# sc-365062), Bax (cat# SC-7480), Bcl2 (cat# SC-7382), NQO-1 (cat# SC-376023) and HO-1 (cat# SC-390991) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against Cleaved Caspase-3(cat# 9664) and Nrf2(cat#12721) were purchased from Cell Signaling Technology (Massachusetts, United States).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eH9c2 embryonic rat heart-derived cardiomyocyte line was purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). H9c2 Cells were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, Gibco/BRL life Technologies, Eggenstein, Germany) containing 4.5g/L D-glucose. The media was supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY), 100 U/mL of penicillin, and 100 mg/mL of streptomycin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMice and animal experiments\u003c/h2\u003e \u003cp\u003e All of the experimental protocols and animal care procedures were approved by the Wenzhou Medical University Animal Policy and Welfare Committee. (Approval Document No. wydw2021-0124). Six-Seven weeks old male C57BL/6 mice weighting 20\u0026ndash;22g were obtained from the Shanghai Laboratory Animal Research Center (Shanghai, China). All mice received humane care according to NIH Guide for the Institutional Animal Care and Use Committee (IACUC). Cos was dissolved in 1% CMC-Na and was given at a dose of 10 mg/kg/day for 7 days by gavage. Mice were given CMC-Na (1%) as vehicle. The mice were anesthetized by 1% pentobarbital sodium (50mg/kg) by intraperitoneal injection before LAD I/R surgical process. The myocardial ischemia reperfusion injury model in C57BL/6 mice was induced as described in our previous study[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMyocardial infarct size measurement\u003c/h2\u003e \u003cp\u003eThe in vivo myocardial infarct size was evaluated using Evans Blue/TTC dual dyeing. Briefly, after retied the LAD, 1% Evans blue dye was injected into the left ventricle. Then we sliced the heart and incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma, St. Louis, MO) for 15 minutes. The sliced myocardium was dyed with blue, red and white. The images were taken by a microscopy system (Nikon TE2000-S). The infarct size was calculated use image J as described previously [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTUNEL staining\u003c/h2\u003e \u003cp\u003eThe apoptotic cells in the heart were detected by TUNEL Apoptosis Assay Kit (cat# C1086, Beyotime, China) according to the manufacturer's instruction. For H9c2 cells, cells were fixed by 4% paraformaldehyde, incubated with 0.25% Triton-X100 in PBS, then apoptotic cells were detected with TUNEL Apoptosis Assay Kit (cat# C1086, Beyotime, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHeart superoxide production\u003c/h2\u003e \u003cp\u003eThe frozen slices of the heart were incubated with DHE for 45 min. The images were viewed under the fluorescence microscope (Nikon, Japan). The cellular hydrogen peroxide was detected using 2\u0026rsquo;,7\u0026rsquo;- dichlorodihydrofluorescein (DCF) staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of MDA and SOD\u003c/h2\u003e \u003cp\u003eH9C2 cells or heart tissue were homogenized with sample buffer, and the serums were diluted with sample buffer. The superoxide dismutase (SOD) levels in sample were determined using commercially available kits (cat# S0101S, Beyotime Biotech, Nantong, China) according to the manufacturer's instructions. The malondialdehyde (MDA) were determined using commercially available kits (cat# S0131S, Beyotime Biotech, Nantong, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of CK-MB and LDH\u003c/h2\u003e \u003cp\u003eThe serum was collected and the CK-MB, and LDH were measured using commercially available assay kits (cat# H197-1-1 and cat# A020-2-2, Jiancheng, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRhodamine-labeled phalloidin staining\u003c/h2\u003e \u003cp\u003eTo assess cardiomyocyte hypertrophy, H9c2 cells were fixed, permeabilized, then incubated with Rhodamine-labeled phalloidin for 30 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eNrf2 silencing by siRNA\u003c/h2\u003e \u003cp\u003eNrf2 was silenced in H9C2 cells by siRNA transfections. siRNA for Nrf2 was designed and synthesized by Gene Pharma Co. (Shanghai, China). siRNA sequences (5\u0026rsquo;to 3\u0026rsquo;), GGGUAAGUCGAGAAGUGUUTT and AACACUUCUCGACUUACCCTT, were used. Cells were transfected using Lipofectamine\u0026trade; 2000 (Invitrogen Ltd., Carlsbad, CA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eLysates from cultured cells or mouse heart tissues were prepared. Samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electro-transferred to nitrocellulose membranes. Membranes were blocked for 2 hour at room temperature in Tris-buffered saline (pH 7.6) containing 0.05% Tween 20 and 5% non-fat milk. Each nitrocellulose membrane was incubated with specific antibodies at 4\u0026deg;C overnight. Secondary antibodies were applied for 1 h at room temperature. Immunoreactivity was visualized using enhanced chemiluminescence reagents (Bio-Rad Laboratories). The amounts of the proteins were analyzed using Image J, and normalized to their respective control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were randomized and blinded, date presented in this study is representative of at least 3 independent experiments and is expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical analysis was calculated with GraphPad Prism 8.0 software (San Diego, CA, USA). Differences between groups were examined using the 2-tailed Student\u0026rsquo;s t-test or one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. Details of each experiment can be found in the figure legend. Differences were considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eCos limits I/R-induced heart injury\u003c/h2\u003e\n\u003cp\u003eCos, a natural sesquiterpene lactone (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), has been known to show powerful antioxidative properties. To investigate whether Cos is protective in a hypoxic myocardium, we examined its effect in the \u003cem\u003ein vivo\u003c/em\u003e cardiac ischemia reperfusion injury model. C57BL/6 mice were pretreated with Cos or 1% CMC-Na for 1 week. The model of I/R was processed with LAD ligation for 30 minutes, and followed by ligation release for reperfusion. After retied of LAD, the infarct area of the heart was assessed using Evans Blue and TTC dual staining. In Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, I/R increased the infarct region, whereas the Cos administration notably reduced I/R-induced myocardial infarction. There is no statistically difference in the ratio of AAR/LV among the three groups as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC. While pretreatment of Cos for seven days significantly attenuated the infarct size compared to I/R group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). Moreover, under myocardial ischemia reperfusion injury, the serum lactate dehydrogenase (LDH) level and creatine kinase M and B isoform (CK-MB) in the mice were significantly increased. Cos downregulated the LDH and CK-MB levels (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). As presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG, compared with I/R group, Cos administration reduced the MDA content in the myocardium. Then we measured the SOD levels both in the serum and in the heart tissues. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eH, myocardial ischemia reperfusion injury reactively increased the serum SOD activity in both the I/R model group and Cos-pretreated group. Whereas in the myocardial tissue, the Cos-treated I/R group have a higher activity of SOD compare to the sham group. These results demonstrate that myocardial I/R damages the function of the heart, and Cos treatment can ameliorate such injury.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eCos attenuates I/R-induced heart apoptosis\u003c/h2\u003e\n\u003cp\u003eThen we detected the protective effects of Cos on apoptosis in ischemia reperfusion injured mice. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, the TUNEL-positive cells in the I/R model group were much more compare to the sham group. The apoptotic cells were less in the Cos-treated group than in the I/R model group, indicating that Cos treatment significantly reduced I/R-induced myocardial cell apoptosis. Then were further detected the expression of cleaved caspase-3, Bax, and Bcl-2 protein in each group (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). I/R mice treated with Cos showed significant downregulation of cleaved caspase-3 and Bax, and a significantly increase in Bcl-2 compared to the I/R group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eCos reduces I/R-induced hear ROS production\u003c/h2\u003e\n\u003cp\u003eAs shown in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, ROS levels were increased in the I/R group, and Cos treatment decreased myocardial ROS levels under I/R injury. As illustrated in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD, pretreatment with Cos did not increase the expression of Nrf2 protein in the I/R model myocardium, whereas it increased Nrf2 nuclear translocation. As a result, the anti-oxidant protein NQO-1 and HO-1 significantly increased (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF). This further reduced ROS production in the heart of Cos treated mice.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003eCos attenuates TBHP-induced apoptosis and ROS levels in H9C2 cells\u003c/h2\u003e\n\u003cp\u003eTo investigate the cytoprotective effect of Cos, the H9C2 was exposed to TBHP. As shown in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, the TUNEL staining indicated that Cos attenuated TBHP-induced cardiac cell apoptosis. In addition, Cos increased the expression of Bcl-2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). An increase of the Bax protein was observed in TBHP-treated H9C2 cells; however, Cos significantly reduced such Bax expression. Treatment with 2.5 \u0026micro;M Cos significantly decreased ROS production. To further investigate the antioxidative property of Cos, H9C2 cells were stimulated with TBHP for 24 h. The SOD activity and MDA level were detected. Increasing concentrations of Cos reduced the TBHP-induced MDA level in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD) while maintaining the SOD activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). Since the increase in ROS levels is critical in TBHP-induced cardiomyocyte injury, we determined intracellular ROS levels in TBHP-induced H9C2 cells by detecting DCF-positive cells. As evident from Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG, DCF staining show that ROS production significantly raised in TBHP-treated group. As shown in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI, pretreatment with Cos decreased TBHP-induced cell morphology changes in H9C2 cells. The results show that Cos pretreatment reduce the ROS levels and apoptosis induced by TBHP.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003eCos activates Nrf2 and attenuates TBHP-induced apoptosis\u003c/h2\u003e\n\u003cp\u003eAs shown in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, the addition of Cos significantly increased Nrf2 nuclear translocation in TBHP-treated H9C2 cells. Cos also promoted the NQO-1 (Nrf2 target) protein levels in a dose-dependent manner in total cell lysate of TBHP-stimulated H9C2 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). In Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC, Nrf2 levels were significantly reduced by Nrf2 siRNA transfection. Cos pretreatment decreased the level of the cleaved caspase-3, in TBHP-treated H9C2 cells, whereas Nrf2 silencing increased cleaved caspase-3 accumulation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). Further analysis of the cell apoptosis pathway revealed that Nrf2 knockdown reversed the protective effect of Cos (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). Furthermore, we also analyzed that Nrf2 silencing reverses NQO-1 overexpression under Cos treatment, using western blotting (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF). The changes in the morphology of H9C2 cells induced by Cos were examined using rhodamine-labeled phalloidin staining. As shown in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH, Nrf2 siRNA transfection increased TBHP-induced injury despite the protection by Cos. These results indicate that Cos activates Nrf2 signaling pathway and protects myocytes from TBHP-induced apoptosis \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe purpose of this research was to expound the mechanism of Cos in protection of myocardial in I/R injured heart. Recent reports have emphasized that oxygen free radicals is the major cause of myocardial apoptosis in I/R injury, and that an antioxidant therapy is an effective approach to reduce cardiac injury [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. As observed in our study, ischemia reperfusion significantly increased myocardial infarct size, serum CK-MB, ROS level and apoptosis in mice. Interestingly, treatment of Cos attenuated these abnormities, suggesting that Cos exerts myocardial protective activity in I/R mice. The protective effect of Cos was also observed in our \u003cem\u003ein vitro\u003c/em\u003e study.\u003c/p\u003e \u003cp\u003eAcute overgeneration of ROS products under pathophysiological states is vital in the development of reperfusion injury after ischemia for thrombolysis or thrombus removal [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Mounting evidence indicates that oxidative stress is critical in myocardial apoptosis in the I/R damaged heart [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It has been reported that Cos administration decreased doxorubicin-induced cardiorenal injury by suppressing oxidative stress [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In another study, Cos attenuated alcohol-induced liver injury by reducing ROS production [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Interestingly, Cos obviously increases the intracellular oxidative stress in human U2OS cells and promotes the generation of ROS in esophageal cancer cells, and eventually induces apoptosis of carcinoma cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Studies have shown that Cos exerts anticancer activity, the diverse mechanism such as the reduction of angiogenic factor signaling pathway [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], inhibition of telomerase activity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and accumulation of intracellular ROS [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Pretreatment with Cos inhibited TBHP-induced Bax expression and increased the content of the a Bcl-2 in H9C2 cells.\u003c/p\u003e \u003cp\u003eIt is known that elimination of oxygen free radicals is a practical strategy to prevent myocardial ischemia [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Our study showed that Cos decreased the ROS production induced by I/R injury or TBHP; however, the reason is unknown. As is reported that allicin active Nrf2 and protected the heart from Ang II induced cardiac hypertrophy [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Our previous study showed that curcumin and curcumin analogue 14p active Nrf2 pathway and attenuates I/R induced myocardial injury [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Nrf2 is the key transcription factor that regulates the antioxidant response, which stimulates the transcription of genes involved in many aspects of cytoprotection, such as those of glutamate cysteine ligase, SODs, HO-1, and NQO-1 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our results showed that pretreatment with Cos before I/R injury or TBHP challenge did not change total Nrf2 expression but increased Nrf2 nuclear translocation and enhanced the expression NQO1 both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. This also indicates that Nrf2 is a feasible strategic target in therapies for myocardial I/R injuries. These results are consistent with previous reports indicating that Cos exerts neuroprotective effects by activating Nrf2 in PC12 cells; however, such prior research was only conducted \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo sum up, our study demonstrates the preventive role of Cos against oxidative stress and apoptosis induced by I/R or TBHP \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, respectively. The protective mechanism of Cos is schematically shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. Our results strongly suggest that Cos, a Nrf2 activator, has huge therapeutic potential in the therapy of myocardial ischemia reperfusion injury.\u003c/p\u003e"},{"header":"abbreviations ","content":"\u003cp\u003eAAR, area at risk; AREs, antioxidant response elements; CAT, catalase; CK-MB, creatine kinase M and B isoform; DHE, dihydroethidium; DCF, 2\u0026rsquo;,7\u0026rsquo;-dichlorodihydrofluorescein; GPx, glutathione peroxidase; GSH, glutathione; HO-1, heme oxygenase 1, I/R, ischemia and reperfusion; LDH, lactate-dehydrogenase; MDA, methane dicarboxylic aldehyde; NQO-1, NAD(P)H [quinone] dehydrogenase 1; NO, nitric oxide; Nrf2, NF-E2-related factor 2; ROS, reactive oxygen species; SOD, super oxygen dehydrogenases; TBHP, tert-butyl hydroperoxide; TrxR1, thioredoxin/thioredoxin reductase 1\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e Guang Liang and Wu Luo contributed to the literature search and study design. Guang Liang, Wu Luo, Siyuan Shen, and Chengyi Dai participated in the drafting of the article. Weixin Li, Yue Luo, Sirui Shen, and Zhuqi Huang carried out the experiments. Guang Liang, Wu Luo, and Weixin Li revised the manuscript. Wu Luo and Weixin Li contributed to data collection and analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was supported by the National Key Research Project of China (2017YFA0506000 to G.L.), National Natural Science Foundation of China (82000793 to W.L., and 81930108 to G.L.), Natural Science Foundation of Zhejiang Province (LY22H070004 to W.L.), Zhejiang Provincial Key Research Project (2018C03068 to G.L.), Wenzhou Scientific Project (Y20210213 to W.L.) and Wenzhou Key Scientific Project (2018ZY009 to G.L.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e The used and/or analyzed datasets are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e All of the experimental protocols and animal care procedures were approved by the Wenzhou Medical University Animal Policy and Welfare Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYusuf S, Joseph P, Rangarajan S, Islam S, Mente A, Hystad P, et al. 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BMC complementary and alternative medicine. 2012; 12(1): 1\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Attas AA, El-Shaer NS, Mohamed GA, Ibrahim SR, Esmat A. Anti-inflammatory sesquiterpenes from Costus speciosus rhizomes. J Ethnopharmacol. 2015;176:365\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShediwah FMH, Naji KM, Gumaih HS, Alhadi FA, Al-Hammami AL. and M.R. D'Souza. Antioxidant and antihyperlipidemic activity of Costus speciosus against atherogenic diet-induced hyperlipidemia in rabbits. J Integr Med. 2019;17(3):181\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHsu J, Pan S, Ho Y, Hwang T, Kung F, Guh J. Costunolide induces apoptosis through nuclear calcium2 + overload and DNA damage response in human prostate cancer. J Urol. 2011;185(5):1967\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi S, Im E, Kang H, Lee J, Kwak H, Bae Y, et al. Inhibitory effects of costunolide on the telomerase activity in human breast carcinoma cells. Cancer Lett. 2005;227(2):153\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai H, He X, Yang C. Costunolide promotes imatinib-induced apoptosis in chronic myeloid leukemia cells via the Bcr/Abl-Stat5 pathway. Phytother Res. 2018;32(9):1764\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Kim J, Lee K, Choi J. Costunolide induces apoptosis in platinum-resistant human ovarian cancer cells by generating reactive oxygen species. Gynecol Oncol. 2011;123(3):588\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhuge W, Chen R, Vladimir K, Dong X, Zia K, Sun X, et al. Costunolide specifically binds and inhibits thioredoxin reductase 1 to induce apoptosis in colon cancer. Cancer Lett. 2018;412:46\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang H, Park S, Zhang H, Park S, Kwon W, Kim E, et al. Targeting AKT with costunolide suppresses the growth of colorectal cancer cells and induces apoptosis in vitro and in vivo. J experimental Clin cancer research: CR. 2021;40(1):114.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJha M, Alam M, Hossain M, Islam A. In vitro antioxidant and cytotoxic potential of Costus speciosus (Koen.) Smith rhizome. Int J Pharm Sci Res. 2010;1(10):138.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEliza J, Daisy P, Ignacimuthu S. Antioxidant activity of costunolide and eremanthin isolated from Costus speciosus (Koen ex. Retz) Sm. Chemico-Biol Interact. 2010;188(3):467\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi W, Wu M, Tang L, Pan Y, Liu Z, Zeng C, et al. 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Cardiovasc Drugs Ther. 2012;26(6):457\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYarmohammadi F, Rezaee R, Karimi G. Natural compounds against doxorubicin-induced cardiotoxicity: a review on the involvement of Nrf2/ARE signaling pathway. Phytother Res. 2021;35(3):1163\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui T, Lai Y, Janicki JS, Wang X. Nuclear factor erythroid-2 related factor 2 (Nrf2)-mediated protein quality control in cardiomyocytes. Front bioscience (Landmark edition). 2016;21:192.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng S, Hou Y, Yao J, Fang J. Activation of Nrf2 by costunolide provides neuroprotective effect in PC12 cells. Food Funct. 2019;10(7):4143\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\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":"Cardiac ischemia reperfusion injury, Costunolide, cardiomyocytes, reactive oxygen species, Nrf2","lastPublishedDoi":"10.21203/rs.3.rs-1857993/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1857993/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eCostunolide (Cos) is a naturally occurring sesquiterpene lactone that exhibits anti-oxidative properties. In this study, we demonstrated the protective mechanism of Cos against ischemia/reperfusion (I/R)-induced heart injury.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eC57BL/6 mice were pretreated with Cos (10 mg/kg/day) or 1% CMC-Na for 1 week. The left anterior descending coronary artery (LAD) was ligated for 30 minutes for ischemia, and followed by ligation release for 4 hours for reperfusion. H9c2 cells challenged with \u003cem\u003etert\u003c/em\u003e-butyl hydroperoxide (TBHP) were used for in vitro studies.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePretreatment of Cos significantly reduced myocardial infarct size, serum CK-MB and LDH level in I/R injured heart. Cos administration significantly attenuated the amount of reactive oxygen species (ROS) and ameliorated the apoptosis both in \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies. Further investigation revealed that Cos significantly increased the expression of heme oxygenase 1 (HO-1) and NAD(P)H [quinone] dehydrogenase 1 (NQO-1). Meanwhile Cos increased B-cell lymphoma-2(Bcl-2) and decreased the BCL2-Associated X Protein (Bax) protein level. Silence of nuclear factor erythroid 2-related factor (Nrf2) significantly reverses the protective effect of Cos in TBHP-challenged H9C2 cells.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur data clearly showed that Cos reduced TBHP challenged H9c2 cells and attenuated myocardial I/R injury through Nrf2 activation. These results indicate that Cos might be benefit in the therapy of myocardial I/R injury.\u003c/p\u003e","manuscriptTitle":"Costunolide Protects Myocardial from Ischemia Reperfusion Injury through Nrf2 Activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-26 18:47:14","doi":"10.21203/rs.3.rs-1857993/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":"2c8f52b1-a37f-400f-9ee4-236f24cab6b5","owner":[],"postedDate":"July 26th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-08-03T01:00:49+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-26 18:47:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1857993","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1857993","identity":"rs-1857993","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}