Dexmedetomidine Ameliorates X-Ray-Induced Myocardial Injury Via Alleviating Cardiomyocyte Apoptosis and Autophagy

Dexmedetomidine Ameliorates X-Ray-Induced Myocardial Injury Via Alleviating Cardiomyocyte Apoptosis and Autophagy | 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 Dexmedetomidine Ameliorates X-Ray-Induced Myocardial Injury Via Alleviating Cardiomyocyte Apoptosis and Autophagy Runze Zhang, Kangjie Xie, Yanhong Lian, Shufang Hong, Yuntian Zhu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3489562/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background Radiotherapy is one of the major local treatments for tumors. However, some complications may occur during the treatment, which includes radiation-induced heart disease (RIHD). However, there is no uniform standard for the prevention of RIHD currently. Dexmedetomidine is reported to have cardio protection effects, while its role in radiation-induced myocardial injury is unknown. In the current study, we aimed to evaluate the radioprotective effect of dexmedetomidine in X-ray radiation-treated mice. Methods 9 male mice were randomized into 3 groups: control, 16Gy, and 16Gy + Dex. The 16Gy group was exposed to a single dose of 16Gy X-ray radiation. 16Gy + Dex group was pretreated with dexmedetomidine before X-ray radiation. The control group was treated with saline and did not receive X-ray radiation. The myocardial tissues were collected 16 weeks after X-ray radiation and subjected to hematoxylin-eosin (HE) staining, TUNEL staining, and immunohistochemistry (IHC) staining. Besides, we established a radiation-injured cardiomyocyte model. Cell viability was assessed with CCK-8 assay and cell apoptosis was assessed using flow cytometry. Protein expression of Bcl-2, Bax, LC3 I/II, Beclin-1, and p62 was detected through western blot assay. Results The results showed that 16Gy X-ray radiation resulted in significant changes in myocardial tissues, increased myocardial apoptosis, and activated autophagy. Pretreatment with dexmedetomidine significantly protects mice against 16Gy X-ray radiation-induced myocardial injury by inhibiting apoptosis and autophagy. Conclusion In summary, our study confirmed the radioprotective effect of dexmedetomidine against 16Gy X-ray radiation-induced cardiomyocyte apoptosis and autophagy activation. X-ray-induced myocardial injury dexmedetomidine apoptosis autophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Radiotherapy is an important adjuvant therapy for patients with cancer, with up to two third of all cancer patients with solid tumors receiving radiotherapy for treatment [ 1 ]. However, besides damaging the tumor cells, radiotherapy can also damage normal tissues or organs [ 2 ]. Thoracic radiotherapy-induced damage to myocardium tissue and heart valves is called radiation-induced heart disease (RIDH) [ 3 , 4 ]. The first case of radiotherapy-induced cardiac death was reported in 1963 [ 5 ]. In recent years, many clinical studies confirm that radiation therapy increases the risk of heart disease-related deaths [ 6 ]. However, up to now, there are still no clear treatment options to prevent or avoid the development of RIHD [ 7 ]. Therefore, the prevention and management of RIHD have become urgent clinical problems. Oxidative stress is the main mechanism in the RIHD [ 4 ]. Previous studies demonstrated that reactive oxygen species generated after radiation lead to inflammatory responses, apoptosis, and autophagy [ 8 – 10 ]. Among them, autophagy is an internal waste reduction and recycling system in cells, which is also important in maintaining cell homeostasis and adaption to physiological stress [ 11 ]. Due to the different cell types and environments, autophagy shows different functions: protective autophagy and autophagic cell death. On the one hand, autophagy protects tumor cells against radiation-induced cell injury, on the other hand, autophagy induces cell death to improve the radiosensitivity of tumor cells [ 12 ]. In myocardial injury, autophagy also shows a critical role. Activated autophagy facilitates myocardial injury in ischemia/reperfusion (I/R)-induced myocardial injury [ 13 ]. In sepsis myocardial injury, activated autophagy is reported to induce inflammation and tissue apoptosis [ 14 ]. However, autophagy in radiation-induced myocardial injury has not been reported yet. Dexmedetomidine (Dex), a highly selective alpha 2-adrenergic receptor agonist, is a widely used agent in clinical anesthesia [ 15 ]. More and more studies show that dexmedetomidine has protective effects on organ damage caused by radiotherapy. For example, dexmedetomidine decreases tubular epithelial apoptosis to show a protective effect in X-ray-induced testicular damage [ 16 ]. In X-ray radiation-induced parotid damage, dexmedetomidine acts as a promising antioxidant agent [ 17 ]. In addition, the protective effects of dexmedetomidine are also seen in myocardial injury [ 18 – 20 ]. Dexmedetomidine has also been found to be a regulator of autophagy. Through inhibiting autophagy, dexmedetomidine showed neuroprotective effects in neurological injuries [ 21 ]. While Yu et al. found that dexmedetomidine enhanced autophagy to alleviate the inflammatory responses in liver injury [ 22 ]. Thus, we speculated that dexmedetomidine shows a protective role in radiation-induced myocardial injury, and whether this protective role is related to dexmedetomidine-mediated autophagy needs more studies. To verify our speculation, we established the cardiomyocyte radiation injury model in this study. Through in vivo and in vitro experiments, we aimed to confirm the role of dexmedetomidine in alleviating radiation-induced myocardial injury and regulating autophagy. Methods Mice and experimental groups Male C57BL/6 mice (8–9 weeks, 20 ± 2g) were purchased from Shanghai Model Organisms Center, Inc. (Shanghai, China), allowed to access food and water ad libitum and maintained under a 12h dark/light cycle at 22°C to 25°C. Then, these mice were randomly divided into 3 groups (n = 6 for each group): control, 16Gy, and 16Gy + Dex. Mice in the control group did not receive X-ray radiation or intraperitoneal injection of dexmedetomidine. Mice in the 16Gy group were exposed to 6 MV X-ray beam energy with 16Gy dose (radiation area: the chest includes the heart region; radiation field: 10.6mm × 15mm) once on the first day. Mice in 16Gy + Dex were given dexmedetomidine (30µg/kg, intraperitoneal injection) 30 minutes before the 16Gy X-ray radiation. Besides, mice in the control and 16Gy group were given saline in a volume equivalent to that given to mice in the 16Gy + Dex group. 16 weeks later, the mice were anesthetized and sacrificed by cervical dislocation. Their hearts were isolated and fixed in a 10% formaldehyde solution. 24h later, the samples were taken and 1.5mm thick myocardial tissue was cut cross-sectional using a rotary microtome (RM 2016, Leica Biosystems, Germany). After being fixed in 10% formaldehyde solution for 12h, the sections were embedded in paraffin for histopathological examination, immunohistochemistry staining, TUNEL staining and western blot analysis. All procedures in this study were approved by the Ethics Committee of Zhejiang Cancer Hospital, and according to standard institutional guidelines. Histopathological examination For the histopathological examination, hematoxylin-eosin (HE) staining was used. Briefly, paraffin-embedded heart tissues were sectioned at 5µm thickness. Next, the sections were stained with Mayer’s Hematoxylin (Sigma, St. Louis, USA) for 5 minutes then counterstained with Eosin for 5 minutes (Sigma). After being dehydrated and sealed, the sections were observed under a microscope (BX53, Olympus, Tokyo, Japan). Immunohistochemical (IHC) analysis For immunohistochemistry, paraffin-embedded heart tissues were cut into 4µm sections, deparaffinized, rehydrated, and subjected to antigen retrieval. Next, the sections were incubated with bovine serum albumin, followed by incubation with anti-CD34 (1:100, ab81289, Abcam, Cambridge, USA) and anti-vWF (1: 100, ab287962, Abcam,) overnight at 4°C. After further washing, the sections were incubated with the secondary antibody (Abcam) and diaminobenzidine reagent (DAKO, Glostrup, Denmark), and counterstained with hematoxylin. Finally, the sections were observed under a microscope (BX53, Olympus, Tokyo, Japan). TUNEL staining To identify myocardial apoptosis, a TUNEL assay was performed. The apoptotic cardiomyocytes were labeled using the colorimetric TUNEL system (Promega, Madison, USA) according to the manufacturer’s protocol. Cell cultures and treatment Mouse cardiomyocyte HL-1 was obtained from Procell (Wuhan, China) and maintained in minimum essential medium (MEM, containing non-essential amino acids, Procell) supplemented with 10% fetal bovine serum (FBS, Procell) and 1% penicillin-streptomycin (Procell) at 37°C with 5% CO 2 . To simulate radiation-induced myocardial injury, HL-1 cells were irradiated with a single dose of 16Gy X-ray. To verify the radioprotective of dexmedetomidine in vitro , 5µM dexmedetomidine was added to the culture medium before irradiation. After 48h, the cells were collected for subsequent experiments. Cell viability assay The cell viability was analyzed using a cell counting kit-8 (CCK-8) assay kit (MedChemExpress, Monmouth Junction, USA). Briefly, cells after treatment were added with 10µL CCK-8 solution. After 2h incubation, cell absorbance was detected at 450nm with a microplate reader (Multiskan MK3, Thermo Fisher Scientific, Waltham, USA). Cell apoptosis assay The cell apoptosis was analyzed using an Annexin V-FITC apoptosis detection kit (Beyotime, Shanghai, China). Briefly, cells after transfection were stained with Annexin V-FITC and propidium iodide solution. After 20 minutes, cell apoptosis was detected using flow cytometry (CytoFLEX, Beckman Coulter, Miami, USA). Western blot analysis The proteins were extracted from tissues and cells using RIPA lysis buffer (Beyotime, Shanghai, China). Then, 40µg proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, and separately incubated with the following primary antibodies: Bax (AF0120, 1:1000, Affinity, USA), Bcl-2 (AF6139, 1:1000, Affinity), Beclin-1 (AF5128, 1:1000, Affinity), p62 (AF5384, 1:1000, Affinity), LC3 (AF5402, 1:1000, Affinity), and GAPDH (ab9485, 1:1000, Abcam). The membranes were washed and incubated with the corresponding secondary antibody (goat anti-rabbit IgG HRP, S0001, 1:5000, Affinity) for 2h at 37°C. Finally, proteins were visualized using an enhanced chemiluminescence reagent (Millipore, USA) and analyzed using Image J software. Statistical Analysis The data were expressed as mean ± standard deviation. Significance analysis was performed using a one-way analysis of variance in GraphPad software (version 9.5). P < 0.05 was considered as statistical significance. Results Effects of dexmedetomidine on myocardial pathological injuries of X-ray-induced myocardial injury mice. As shown in Fig. 1 , HE staining showed that 16Gy X-ray radiation caused the thickening of the epicardium, disorder of myocardial fiber arrangement, and elastic fiber injury. The results also showed that dexmedetomidine protected against X-ray-induced myocardial injury. As compared with the mice in the 16Gy group, the mice in the 16Gy + Dex group showed reduced thickening of epicardium and improved histological structures of myocardial tissue. Effects of dexmedetomidine on vascular endothelial cells injury of X-ray-induced myocardial injury mice. The expression of CD34 and vWF, molecular markers of vascular endothelial cells injury, in myocardial tissue sections was ascertained by IHC assay. It was found that the expression of CD34 and vWF increased in the 16Gy group compared with that from the control group, suggesting that 16Gy radiation could induce vascular endothelial cell injury. While in the 16Gy + Dex group, the expression of CD34 and vWF decreased as compared with that in the 16Gy group, suggesting that dexmedetomidine exerts a protective effect on X-ray-induced vascular endothelial cells injury (Fig. 2 A). Effects of dexmedetomidine on myocardial apoptosis of X-ray-induced myocardial injury mice. Next, we detected the protective effect of dexmedetomidine on myocardial apoptosis. TUNEL staining showed that the 16Gy X-ray radiation insult resulted in increased TUNEL-positive cells in myocardial tissue sections, suggesting the increasement in myocardial apoptosis. However, compared with mice in the 16Gy group, the mice in the 16Gy + Dex group showed decreased TUNEL-positive cells in myocardial tissue sections (Fig. 3 A). Western blot detection revealed that the expression of the proapoptotic protein Bax increased, while the expression of the antiapoptotic protein Bcl-2 decreased, in myocardial tissue samples from 16Gy X-ray radiation-treated mice. Furthermore, dexmedetomidine pretreatment attenuated the radiation-induced increase in Bax expression and decrease in Bcl-2 expression (Fig. 3 B). These results indicated that the radioprotective effects of dexmedetomidine were through the inhibition of pro-apoptosis signaling. Effects of dexmedetomidine on autophagy in X-ray-induced myocardial injury mice. As previously reported, autophagy is crucially involved in the protective effect of dexmedetomidine [ 23 ]. In this study, we detected the expression of autophagy-related proteins LC3 I/II, Beclin-1, and p62 through western blot assay. We found that the protein expression of Beclin-1 and LC3 II to I ratio increased, while the protein expression of p62 decreased, in myocardial tissue samples from 16Gy X-ray radiation-treated mice (Fig. 4 ). These results suggested that autophagy is activated during the process of 16Gy X-ray radiation-induced myocardial injury. When pretreatment with dexmedetomidine, it was found that the radiation-induced increase in LC3 II to I ratio and Beclin-1 expression and decrease in p62 expression was attenuated (Fig. 4 ). These results indicated that the radioprotective effects of dexmedetomidine were partially due to autophagy inhibition. Effects of dexmedetomidine on cell viability, apoptosis, and autophagy in X-ray-treated cardiomyocyte We then verified the radioprotective effects of dexmedetomidine in vitro . The cell viability was detected through a CCK-8 assay. As shown in Fig. 5 A, cell viability was suppressed in irradiated HL-1 cells, and dexmedetomidine pretreatment attenuated the radiation-induced suppression in cell viability. Conversely, the cell apoptosis rate increased in irradiated HL-1 cells, and this promoting effect was suppressed with dexmedetomidine pretreatment (Fig. 5 B). Western blot analysis revealed that Beclin-1 protein expression and LC3 II to I ratio were significantly higher and p62 protein expression was significantly lower in cardiomyocytes from the 16Gy group compared to the control group. Furthermore, Beclin-1 protein expression and LC3 II to I ratio significantly decreased and p62 protein expression clearly increased in cardiomyocytes from the 16Gy + Dex group (Fig. 5 C). These findings confirmed that the radioprotective effects of dexmedetomidine were through regulating apoptosis and autophagy in vitro . Discussion This study was conducted to investigate the role of dexmedetomidine in treating X-ray radiation-induced myocardial injury. Through animal and cell experiments, we found that dexmedetomidine pretreatment suppressed cardiomyocyte apoptosis and ameliorated the X-ray radiation-induced myocardial injury. Furthermore, we found that the radioprotective role of dexmedetomidine was related to the inhibition of autophagy. In recent years, radiotherapy has become an important approach to cancer treatment. However, when the chest is exposed to radiation, vascular endothelial cells, and cardiomyocytes may be damaged, and this damage is related to the radiation type and dose [ 24 ]. Clinical studies revealed that 1-4Gy radiation promotes the development of cardiovascular diseases and inflammation, 4-8Gy radiation increases the possibility of myocardial infarction, and > 8Gy radiation causes myocardial fibrosis [ 25 – 28 ]. In an animal study, it is found that cardiac radiation exposure (10 or 20Gy) resulted in cardiomyocyte hypertrophy, left ventricular diastolic dysfunction, and myocardial fibrosis [ 29 ]. In consist with previous studies, our results showed that 16Gy X-ray radiation resulted in the thickening of the epicardium, disorder of myocardial fiber arrangement, and vascular endothelial cells injury. The heart is an organ with high oxygen consumption, and cardiomyocytes contain a large number of mitochondria [ 29 ]. In the process of radiation-induced damage, mitochondria are particularly susceptible [ 30 ]. A previous study revealed that radiation-induced mitochondria damage could induce cell apoptosis, which is an important pathological mechanism in radiation-induced tissue damage [ 31 , 32 ]. Here, through TUNEL staining, we confirmed that 16Gy X-ray radiation facilitated apoptosis in mice and cardiomyocytes. Besides, mitochondrial damage is related to changes in levels of Bax and Bcl-2 [ 33 ]. Bax and Bcl-2 are two important genes in regulating apoptosis. The ratio between Bax/Bcl-2 proteins was a key factor in promoting apoptosis [ 34 ]. In this study, we found that 16Gy X-ray radiation promoted Bax expression while inhibiting Bcl-2 expression, which was consistent with the previous research [ 35 ]. Besides promoting apoptosis, mitochondrial damage may also lead to autophagy activation [ 4 ]. The function of autophagy in myocardial injury has been controversial. Chen et al. found that autophagy is activated in myocardial I/R injury [ 13 ]. Xing et al. found that autophagy is blocked in myocardial I/R injury, and autophagy restoring rescues heart function [ 36 ]. The reason for this contradiction may be the two sides of autophagy. Under normal circumstances, autophagy can degrade damaged organelles and harmful proteins to recover nutrients and generate energy, thus promoting cell and tissue survival. However, with the increase of the stimulation intensity or time, the level of basal autophagy will gradually increase to form excessive autophagy, which leads to impaired cell function and autophagic death [ 37 ]. In this study, under 16Gy X-ray radiation, protein expression of Beclin-1 and LC3 II to I ratio increased, while the protein expression of p62 decreased. These results suggested that autophagy was active in 16Gy X-ray radiation-induced myocardial injury. Dexmedetomidine is currently used for its excellent sedation and analgesia with minimal cardiovascular effects [ 38 ]. Previous researches have clarified the important role of dexmedetomidine in myocardial injury. Through reducing ferroptosis, dexmedetomidine alleviates sepsis‑induced myocardial cellular injury [ 39 ]. Another study presented that dexmedetomidine pretreatment attenuates myocardial I/R injury by relieving endoplasmic reticulum stress [ 40 ]. Wu et al. found that dexmedetomidine protects against myocardial I/R injury via ameliorating oxidative stress and cell apoptosis [ 41 ]. However, no previous studies have examined the effectiveness of dexmedetomidine in protecting radiation-induced myocardial injury, which this study demonstrates. By pretreatment with dexmedetomidine, the myocardial injury and apoptosis induced by 16Gy X-ray were attenuated. The effect of dexmedetomidine on autophagy is controversial. Recent studies on myocardial injury supported that dexmedetomidine inhibits autophagy to protect against myocardial injury [ 42 , 43 ], which is consistent with our findings. Some limitations of the current study should be admitted. First, only 3 mice were used in each group because of insufficient expenditure. More mice in each group are needed to make the results more are needed. Besides, additional markers of myocardial injury could be measured to clarify the protective role of dexmedetomidine in radiation-induced myocardial injury. Second, we did not add groups to investigate whether the protective effects of dexmedetomidine are related to its dose. Third, we did not proceed to molecular studies. It needs future investigation that which signaling pathway dexmedetomidine protects against radiation-induced myocardial injury. Conclusion our study demonstrated that dexmedetomidine protected against 16Gy X-ray radiation-induced myocardial injury by inhibiting apoptosis and autophagy. Our results may provide a new approach to the prevention and treatment of radiation-induced heart disease. Abbreviations CCK-8: cell counting kit-8; Dex: Dexmedetomidine; FBS: fetal bovine serum; HE: hematoxylin-eosin; IHC: immunohistochemistry; I/R: ischemia/reperfusion; MEM: minimum essential medium; RIDH: radiation-induced heart disease; RIHD: radiation-induced heart disease Declarations Ethics approval and consent to participate The design and development of animal experiments involved in this study were conducted according to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and approved by the Institutional Animal Care and Use Committee of Zhejiang Cancer Hospital Research Ethics Committee (approval no. zjzlsd-2020-07-017). Consent for publication Not applicable. Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by Zhejiang Province Medical Science and Technology Project (Grant numbers 2020KY485 and 2021KY089) and Zhejiang Traditional Chinese Medicine Science and Technology Plan (Grant numbers 2023ZL021 and 2023ZL302). Authors' contributions All authors contributed to the study conception and design. Study design and data collection were performed by RZZ, KX, YHL and YTZ. Data analysis was performed by RZZ, KX and SFH. The first draft of the manuscript was written by RZZ. Acknowledgements Not applicable. References Ruckert M, Flohr AS, Hecht M, Gaipl US. Radiotherapy and the immune system: More than just immune suppression. Stem Cells. 2021;39(9):1155–65. Banfill K, Giuliani M, Aznar M, Franks K, McWilliam A, Schmitt M, Sun F, Vozenin MC. Faivre Finn C, committee IART: Cardiac Toxicity of Thoracic Radiotherapy: Existing Evidence and Future Directions. J Thorac Oncol. 2021;16(2):216–27. Heidenreich PA, Kapoor JR. 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Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 22 May, 2024 Reviews received at journal 22 May, 2024 Reviewers agreed at journal 15 May, 2024 Reviews received at journal 07 May, 2024 Reviewers agreed at journal 03 May, 2024 Reviewers invited by journal 25 Jan, 2024 Editor assigned by journal 25 Jan, 2024 Editor invited by journal 17 Jan, 2024 Submission checks completed at journal 17 Jan, 2024 First submitted to journal 25 Oct, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3489562","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267690194,"identity":"ae422119-1309-4554-9462-6b910c530ca4","order_by":0,"name":"Runze Zhang","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Runze","middleName":"","lastName":"Zhang","suffix":""},{"id":267690195,"identity":"44d9d67f-dd1c-489a-98b8-070eb0baeb53","order_by":1,"name":"Kangjie Xie","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kangjie","middleName":"","lastName":"Xie","suffix":""},{"id":267690196,"identity":"885b2667-b971-4a6a-98a5-838438136ad8","order_by":2,"name":"Yanhong Lian","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yanhong","middleName":"","lastName":"Lian","suffix":""},{"id":267690197,"identity":"398445bf-4c31-40a3-aad1-b0ca9baa4f6c","order_by":3,"name":"Shufang Hong","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shufang","middleName":"","lastName":"Hong","suffix":""},{"id":267690198,"identity":"c30fb5ea-7724-4710-ba8b-848ea261a63c","order_by":4,"name":"Yuntian Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYDACCRBRwcBMopYDZ0jWcrCNFHfJz+4xk/44r46dv/0A4+OKXwzy5oS0MM45YyZxcBsbs8SZBGbDs30MhjsbCGhhlsgBaeFhNpBgYJNs7GFIMDhAQAsbWMscCRK08IC1NBhAtDT8IEKLhERascWZYwlAvyQ2GzY2SBhuIKRFfkbyxhsVNXXJ/O2HDz5s+GMjT9AWIGABRU0yMPAaGBjbJAirBwLmD0DCDsL+Q5SOUTAKRsEoGGEAANCzOF4XyZJLAAAAAElFTkSuQmCC","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yuntian","middleName":"","lastName":"Zhu","suffix":""}],"badges":[],"createdAt":"2023-10-25 08:59:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3489562/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3489562/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49895868,"identity":"c5ce824a-a34d-41e9-87d9-b8674cfd2797","added_by":"auto","created_at":"2024-01-19 21:45:23","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1001266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative images of HE staining results (n = 6 for each group)\u003c/strong\u003e. The epicardium was shown by red arrows. Dex: dexmedetomidine. Scale bar: 20 μm.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3489562/v1/d105974546d9b19934a3c683.jpg"},{"id":49894978,"identity":"9bd747e7-ca6f-484d-b188-ddeb0ea00c6d","added_by":"auto","created_at":"2024-01-19 21:37:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1173199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative images of IHC staining results (n = 6 for each group)\u003c/strong\u003e. Dex: dexmedetomidine. Scale bar: 20 μm.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3489562/v1/a3b84858e294a193a2f4a1a2.jpg"},{"id":49895869,"identity":"06ff82b6-e165-405e-8f66-45ba70a8be0f","added_by":"auto","created_at":"2024-01-19 21:45:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":695484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of dexmedetomidine on regulating myocardial apoptosis.\u003c/strong\u003e (A) Representative images of IHC staining results (n =6 for each group). The TUNEL-positive cells were shown by red arrows. (B) Western blot analysis of the protein expression of Bcl-2 and Bax expression. ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. control; ## \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. 16Gy.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3489562/v1/504a77acd3d6f4c4efd97b23.jpg"},{"id":49894983,"identity":"25a1531f-a2ca-4a08-b264-8242bbc08c17","added_by":"auto","created_at":"2024-01-19 21:37:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":203712,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestern blot analysis of the protein expression of Beclin-1, LC3 I/II, and p62 expression.\u003c/strong\u003e** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. control; ## \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. 16Gy.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3489562/v1/e360853d01db5fb4c10bd4bb.jpg"},{"id":49894980,"identity":"cfa2f1cb-1ca7-47ee-a37a-43c6db56d333","added_by":"auto","created_at":"2024-01-19 21:37:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":420607,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of dexmedetomidine in X-ray-treated cardiomyocytes.\u003c/strong\u003e (A) CCK-8 assay was applied to detect cell viability. (B) Flow cytometry was used to detect cell apoptosis. (C) Western blot analysis of the protein expression of Beclin-1, LC3 I/II and p62 expression. ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. control; ## \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. 16Gy.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3489562/v1/804244b93afabc6d252b5601.jpg"},{"id":49896160,"identity":"e76f7052-4792-495b-84c7-8e3caa315e68","added_by":"auto","created_at":"2024-01-19 21:53:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1114392,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3489562/v1/0c25fe08-0075-4b40-a81d-2ee4b0019284.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dexmedetomidine Ameliorates X-Ray-Induced Myocardial Injury Via Alleviating Cardiomyocyte Apoptosis and Autophagy","fulltext":[{"header":"Background","content":"\u003cp\u003eRadiotherapy is an important adjuvant therapy for patients with cancer, with up to two third of all cancer patients with solid tumors receiving radiotherapy for treatment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, besides damaging the tumor cells, radiotherapy can also damage normal tissues or organs [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Thoracic radiotherapy-induced damage to myocardium tissue and heart valves is called radiation-induced heart disease (RIDH) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The first case of radiotherapy-induced cardiac death was reported in 1963 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In recent years, many clinical studies confirm that radiation therapy increases the risk of heart disease-related deaths [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, up to now, there are still no clear treatment options to prevent or avoid the development of RIHD [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, the prevention and management of RIHD have become urgent clinical problems.\u003c/p\u003e \u003cp\u003eOxidative stress is the main mechanism in the RIHD [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Previous studies demonstrated that reactive oxygen species generated after radiation lead to inflammatory responses, apoptosis, and autophagy [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among them, autophagy is an internal waste reduction and recycling system in cells, which is also important in maintaining cell homeostasis and adaption to physiological stress [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Due to the different cell types and environments, autophagy shows different functions: protective autophagy and autophagic cell death. On the one hand, autophagy protects tumor cells against radiation-induced cell injury, on the other hand, autophagy induces cell death to improve the radiosensitivity of tumor cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In myocardial injury, autophagy also shows a critical role. Activated autophagy facilitates myocardial injury in ischemia/reperfusion (I/R)-induced myocardial injury [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In sepsis myocardial injury, activated autophagy is reported to induce inflammation and tissue apoptosis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, autophagy in radiation-induced myocardial injury has not been reported yet.\u003c/p\u003e \u003cp\u003eDexmedetomidine (Dex), a highly selective alpha 2-adrenergic receptor agonist, is a widely used agent in clinical anesthesia [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. More and more studies show that dexmedetomidine has protective effects on organ damage caused by radiotherapy. For example, dexmedetomidine decreases tubular epithelial apoptosis to show a protective effect in X-ray-induced testicular damage [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In X-ray radiation-induced parotid damage, dexmedetomidine acts as a promising antioxidant agent [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In addition, the protective effects of dexmedetomidine are also seen in myocardial injury [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Dexmedetomidine has also been found to be a regulator of autophagy. Through inhibiting autophagy, dexmedetomidine showed neuroprotective effects in neurological injuries [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. While Yu \u003cem\u003eet al.\u003c/em\u003e found that dexmedetomidine enhanced autophagy to alleviate the inflammatory responses in liver injury [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Thus, we speculated that dexmedetomidine shows a protective role in radiation-induced myocardial injury, and whether this protective role is related to dexmedetomidine-mediated autophagy needs more studies.\u003c/p\u003e \u003cp\u003eTo verify our speculation, we established the cardiomyocyte radiation injury model in this study. Through \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments, we aimed to confirm the role of dexmedetomidine in alleviating radiation-induced myocardial injury and regulating autophagy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice and experimental groups\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice (8\u0026ndash;9 weeks, 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2g) were purchased from Shanghai Model Organisms Center, Inc. (Shanghai, China), allowed to access food and water ad libitum and maintained under a 12h dark/light cycle at 22\u0026deg;C to 25\u0026deg;C. Then, these mice were randomly divided into 3 groups (n\u0026thinsp;=\u0026thinsp;6 for each group): control, 16Gy, and 16Gy\u0026thinsp;+\u0026thinsp;Dex. Mice in the control group did not receive X-ray radiation or intraperitoneal injection of dexmedetomidine. Mice in the 16Gy group were exposed to 6 MV X-ray beam energy with 16Gy dose (radiation area: the chest includes the heart region; radiation field: 10.6mm \u0026times; 15mm) once on the first day. Mice in 16Gy\u0026thinsp;+\u0026thinsp;Dex were given dexmedetomidine (30\u0026micro;g/kg, intraperitoneal injection) 30 minutes before the 16Gy X-ray radiation. Besides, mice in the control and 16Gy group were given saline in a volume equivalent to that given to mice in the 16Gy\u0026thinsp;+\u0026thinsp;Dex group. 16 weeks later, the mice were anesthetized and sacrificed by cervical dislocation. Their hearts were isolated and fixed in a 10% formaldehyde solution. 24h later, the samples were taken and 1.5mm thick myocardial tissue was cut cross-sectional using a rotary microtome (RM 2016, Leica Biosystems, Germany). After being fixed in 10% formaldehyde solution for 12h, the sections were embedded in paraffin for histopathological examination, immunohistochemistry staining, TUNEL staining and western blot analysis. All procedures in this study were approved by the Ethics Committee of Zhejiang Cancer Hospital, and according to standard institutional guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological examination\u003c/h2\u003e \u003cp\u003eFor the histopathological examination, hematoxylin-eosin (HE) staining was used. Briefly, paraffin-embedded heart tissues were sectioned at 5\u0026micro;m thickness. Next, the sections were stained with Mayer\u0026rsquo;s Hematoxylin (Sigma, St. Louis, USA) for 5 minutes then counterstained with Eosin for 5 minutes (Sigma). After being dehydrated and sealed, the sections were observed under a microscope (BX53, Olympus, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical (IHC) analysis\u003c/h2\u003e \u003cp\u003eFor immunohistochemistry, paraffin-embedded heart tissues were cut into 4\u0026micro;m sections, deparaffinized, rehydrated, and subjected to antigen retrieval. Next, the sections were incubated with bovine serum albumin, followed by incubation with anti-CD34 (1:100, ab81289, Abcam, Cambridge, USA) and anti-vWF (1: 100, ab287962, Abcam,) overnight at 4\u0026deg;C. After further washing, the sections were incubated with the secondary antibody (Abcam) and diaminobenzidine reagent (DAKO, Glostrup, Denmark), and counterstained with hematoxylin. Finally, the sections were observed under a microscope (BX53, Olympus, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTUNEL staining\u003c/h2\u003e \u003cp\u003eTo identify myocardial apoptosis, a TUNEL assay was performed. The apoptotic cardiomyocytes were labeled using the colorimetric TUNEL system (Promega, Madison, USA) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell cultures and treatment\u003c/h2\u003e \u003cp\u003eMouse cardiomyocyte HL-1 was obtained from Procell (Wuhan, China) and maintained in minimum essential medium (MEM, containing non-essential amino acids, Procell) supplemented with 10% fetal bovine serum (FBS, Procell) and 1% penicillin-streptomycin (Procell) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eTo simulate radiation-induced myocardial injury, HL-1 cells were irradiated with a single dose of 16Gy X-ray. To verify the radioprotective of dexmedetomidine \u003cem\u003ein vitro\u003c/em\u003e, 5\u0026micro;M dexmedetomidine was added to the culture medium before irradiation. After 48h, the cells were collected for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay\u003c/h2\u003e \u003cp\u003eThe cell viability was analyzed using a cell counting kit-8 (CCK-8) assay kit (MedChemExpress, Monmouth Junction, USA). Briefly, cells after treatment were added with 10\u0026micro;L CCK-8 solution. After 2h incubation, cell absorbance was detected at 450nm with a microplate reader (Multiskan MK3, Thermo Fisher Scientific, Waltham, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell apoptosis assay\u003c/h2\u003e \u003cp\u003eThe cell apoptosis was analyzed using an Annexin V-FITC apoptosis detection kit (Beyotime, Shanghai, China). Briefly, cells after transfection were stained with Annexin V-FITC and propidium iodide solution. After 20 minutes, cell apoptosis was detected using flow cytometry (CytoFLEX, Beckman Coulter, Miami, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eThe proteins were extracted from tissues and cells using RIPA lysis buffer (Beyotime, Shanghai, China). Then, 40\u0026micro;g proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, and separately incubated with the following primary antibodies: Bax (AF0120, 1:1000, Affinity, USA), Bcl-2 (AF6139, 1:1000, Affinity), Beclin-1 (AF5128, 1:1000, Affinity), p62 (AF5384, 1:1000, Affinity), LC3 (AF5402, 1:1000, Affinity), and GAPDH (ab9485, 1:1000, Abcam). The membranes were washed and incubated with the corresponding secondary antibody (goat anti-rabbit IgG HRP, S0001, 1:5000, Affinity) for 2h at 37\u0026deg;C. Finally, proteins were visualized using an enhanced chemiluminescence reagent (Millipore, USA) and analyzed using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Significance analysis was performed using a one-way analysis of variance in GraphPad software (version 9.5). \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffects of dexmedetomidine on myocardial pathological injuries of X-ray-induced myocardial injury mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, HE staining showed that 16Gy X-ray radiation caused the thickening of the epicardium, disorder of myocardial fiber arrangement, and elastic fiber injury. The results also showed that dexmedetomidine protected against X-ray-induced myocardial injury. As compared with the mice in the 16Gy group, the mice in the 16Gy\u0026thinsp;+\u0026thinsp;Dex group showed reduced thickening of epicardium and improved histological structures of myocardial tissue.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of dexmedetomidine on vascular endothelial cells injury of X-ray-induced myocardial injury mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe expression of CD34 and vWF, molecular markers of vascular endothelial cells injury, in myocardial tissue sections was ascertained by IHC assay. It was found that the expression of CD34 and vWF increased in the 16Gy group compared with that from the control group, suggesting that 16Gy radiation could induce vascular endothelial cell injury. While in the 16Gy\u0026thinsp;+\u0026thinsp;Dex group, the expression of CD34 and vWF decreased as compared with that in the 16Gy group, suggesting that dexmedetomidine exerts a protective effect on X-ray-induced vascular endothelial cells injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of dexmedetomidine on myocardial apoptosis of X-ray-induced myocardial injury mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we detected the protective effect of dexmedetomidine on myocardial apoptosis. TUNEL staining showed that the 16Gy X-ray radiation insult resulted in increased TUNEL-positive cells in myocardial tissue sections, suggesting the increasement in myocardial apoptosis. However, compared with mice in the 16Gy group, the mice in the 16Gy\u0026thinsp;+\u0026thinsp;Dex group showed decreased TUNEL-positive cells in myocardial tissue sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Western blot detection revealed that the expression of the proapoptotic protein Bax increased, while the expression of the antiapoptotic protein Bcl-2 decreased, in myocardial tissue samples from 16Gy X-ray radiation-treated mice. Furthermore, dexmedetomidine pretreatment attenuated the radiation-induced increase in Bax expression and decrease in Bcl-2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These results indicated that the radioprotective effects of dexmedetomidine were through the inhibition of pro-apoptosis signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of dexmedetomidine on autophagy in X-ray-induced myocardial injury mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs previously reported, autophagy is crucially involved in the protective effect of dexmedetomidine [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this study, we detected the expression of autophagy-related proteins LC3 I/II, Beclin-1, and p62 through western blot assay. We found that the protein expression of Beclin-1 and LC3 II to I ratio increased, while the protein expression of p62 decreased, in myocardial tissue samples from 16Gy X-ray radiation-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results suggested that autophagy is activated during the process of 16Gy X-ray radiation-induced myocardial injury. When pretreatment with dexmedetomidine, it was found that the radiation-induced increase in LC3 II to I ratio and Beclin-1 expression and decrease in p62 expression was attenuated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results indicated that the radioprotective effects of dexmedetomidine were partially due to autophagy inhibition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffects of dexmedetomidine on cell viability, apoptosis, and autophagy in X-ray-treated cardiomyocyte\u003c/h2\u003e \u003cp\u003eWe then verified the radioprotective effects of dexmedetomidine \u003cem\u003ein vitro\u003c/em\u003e. The cell viability was detected through a CCK-8 assay. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, cell viability was suppressed in irradiated HL-1 cells, and dexmedetomidine pretreatment attenuated the radiation-induced suppression in cell viability. Conversely, the cell apoptosis rate increased in irradiated HL-1 cells, and this promoting effect was suppressed with dexmedetomidine pretreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Western blot analysis revealed that Beclin-1 protein expression and LC3 II to I ratio were significantly higher and p62 protein expression was significantly lower in cardiomyocytes from the 16Gy group compared to the control group. Furthermore, Beclin-1 protein expression and LC3 II to I ratio significantly decreased and p62 protein expression clearly increased in cardiomyocytes from the 16Gy\u0026thinsp;+\u0026thinsp;Dex group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These findings confirmed that the radioprotective effects of dexmedetomidine were through regulating apoptosis and autophagy \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study was conducted to investigate the role of dexmedetomidine in treating X-ray radiation-induced myocardial injury. Through animal and cell experiments, we found that dexmedetomidine pretreatment suppressed cardiomyocyte apoptosis and ameliorated the X-ray radiation-induced myocardial injury. Furthermore, we found that the radioprotective role of dexmedetomidine was related to the inhibition of autophagy.\u003c/p\u003e \u003cp\u003eIn recent years, radiotherapy has become an important approach to cancer treatment. However, when the chest is exposed to radiation, vascular endothelial cells, and cardiomyocytes may be damaged, and this damage is related to the radiation type and dose [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Clinical studies revealed that 1-4Gy radiation promotes the development of cardiovascular diseases and inflammation, 4-8Gy radiation increases the possibility of myocardial infarction, and \u0026gt;\u0026thinsp;8Gy radiation causes myocardial fibrosis [\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In an animal study, it is found that cardiac radiation exposure (10 or 20Gy) resulted in cardiomyocyte hypertrophy, left ventricular diastolic dysfunction, and myocardial fibrosis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In consist with previous studies, our results showed that 16Gy X-ray radiation resulted in the thickening of the epicardium, disorder of myocardial fiber arrangement, and vascular endothelial cells injury.\u003c/p\u003e \u003cp\u003eThe heart is an organ with high oxygen consumption, and cardiomyocytes contain a large number of mitochondria [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In the process of radiation-induced damage, mitochondria are particularly susceptible [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. A previous study revealed that radiation-induced mitochondria damage could induce cell apoptosis, which is an important pathological mechanism in radiation-induced tissue damage [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Here, through TUNEL staining, we confirmed that 16Gy X-ray radiation facilitated apoptosis in mice and cardiomyocytes. Besides, mitochondrial damage is related to changes in levels of Bax and Bcl-2 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Bax and Bcl-2 are two important genes in regulating apoptosis. The ratio between Bax/Bcl-2 proteins was a key factor in promoting apoptosis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this study, we found that 16Gy X-ray radiation promoted Bax expression while inhibiting Bcl-2 expression, which was consistent with the previous research [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBesides promoting apoptosis, mitochondrial damage may also lead to autophagy activation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The function of autophagy in myocardial injury has been controversial. Chen \u003cem\u003eet al.\u003c/em\u003e found that autophagy is activated in myocardial I/R injury [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Xing \u003cem\u003eet al.\u003c/em\u003e found that autophagy is blocked in myocardial I/R injury, and autophagy restoring rescues heart function [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The reason for this contradiction may be the two sides of autophagy. Under normal circumstances, autophagy can degrade damaged organelles and harmful proteins to recover nutrients and generate energy, thus promoting cell and tissue survival. However, with the increase of the stimulation intensity or time, the level of basal autophagy will gradually increase to form excessive autophagy, which leads to impaired cell function and autophagic death [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, under 16Gy X-ray radiation, protein expression of Beclin-1 and LC3 II to I ratio increased, while the protein expression of p62 decreased. These results suggested that autophagy was active in 16Gy X-ray radiation-induced myocardial injury.\u003c/p\u003e \u003cp\u003eDexmedetomidine is currently used for its excellent sedation and analgesia with minimal cardiovascular effects [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Previous researches have clarified the important role of dexmedetomidine in myocardial injury. Through reducing ferroptosis, dexmedetomidine alleviates sepsis‑induced myocardial cellular injury [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Another study presented that dexmedetomidine pretreatment attenuates myocardial I/R injury by relieving endoplasmic reticulum stress [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Wu \u003cem\u003eet al.\u003c/em\u003e found that dexmedetomidine protects against myocardial I/R injury via ameliorating oxidative stress and cell apoptosis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, no previous studies have examined the effectiveness of dexmedetomidine in protecting radiation-induced myocardial injury, which this study demonstrates. By pretreatment with dexmedetomidine, the myocardial injury and apoptosis induced by 16Gy X-ray were attenuated.\u003c/p\u003e \u003cp\u003eThe effect of dexmedetomidine on autophagy is controversial. Recent studies on myocardial injury supported that dexmedetomidine inhibits autophagy to protect against myocardial injury [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], which is consistent with our findings.\u003c/p\u003e \u003cp\u003eSome limitations of the current study should be admitted. First, only 3 mice were used in each group because of insufficient expenditure. More mice in each group are needed to make the results more are needed. Besides, additional markers of myocardial injury could be measured to clarify the protective role of dexmedetomidine in radiation-induced myocardial injury. Second, we did not add groups to investigate whether the protective effects of dexmedetomidine are related to its dose. Third, we did not proceed to molecular studies. It needs future investigation that which signaling pathway dexmedetomidine protects against radiation-induced myocardial injury.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eour study demonstrated that dexmedetomidine protected against 16Gy X-ray radiation-induced myocardial injury by inhibiting apoptosis and autophagy. Our results may provide a new approach to the prevention and treatment of radiation-induced heart disease.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCCK-8: cell counting kit-8; Dex: Dexmedetomidine; FBS: fetal bovine serum; HE: hematoxylin-eosin; IHC: immunohistochemistry; I/R: ischemia/reperfusion; MEM: minimum essential medium; RIDH: radiation-induced heart disease; RIHD: radiation-induced heart disease\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe design and development of animal experiments involved in this study were conducted according to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and approved by the Institutional Animal Care and Use Committee of Zhejiang Cancer Hospital Research Ethics Committee (approval no. zjzlsd-2020-07-017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Zhejiang Province Medical Science and Technology Project (Grant numbers 2020KY485 and 2021KY089) and Zhejiang Traditional Chinese Medicine Science and Technology Plan\u0026nbsp;(Grant numbers 2023ZL021 and 2023ZL302).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Study design and data collection were performed by RZZ, KX, YHL and YTZ. Data analysis was performed by RZZ, KX and SFH. The first draft of the manuscript was written by RZZ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRuckert M, Flohr AS, Hecht M, Gaipl US. Radiotherapy and the immune system: More than just immune suppression. Stem Cells. 2021;39(9):1155\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBanfill K, Giuliani M, Aznar M, Franks K, McWilliam A, Schmitt M, Sun F, Vozenin MC. Faivre Finn C, committee IART: Cardiac Toxicity of Thoracic Radiotherapy: Existing Evidence and Future Directions. J Thorac Oncol. 2021;16(2):216\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeidenreich PA, Kapoor JR. 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The Protective Mechanism of Dexmedetomidine in Regulating Atg14L-Beclin1-Vps34 Complex Against Myocardial Ischemia-Reperfusion Injury. J Cardiovasc Transl Res. 2021;14(6):1063\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"X-ray-induced myocardial injury, dexmedetomidine, apoptosis, autophagy","lastPublishedDoi":"10.21203/rs.3.rs-3489562/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3489562/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRadiotherapy is one of the major local treatments for tumors. However, some complications may occur during the treatment, which includes radiation-induced heart disease (RIHD). However, there is no uniform standard for the prevention of RIHD currently. Dexmedetomidine is reported to have cardio protection effects, while its role in radiation-induced myocardial injury is unknown. In the current study, we aimed to evaluate the radioprotective effect of dexmedetomidine in X-ray radiation-treated mice.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e9 male mice were randomized into 3 groups: control, 16Gy, and 16Gy\u0026thinsp;+\u0026thinsp;Dex. The 16Gy group was exposed to a single dose of 16Gy X-ray radiation. 16Gy\u0026thinsp;+\u0026thinsp;Dex group was pretreated with dexmedetomidine before X-ray radiation. The control group was treated with saline and did not receive X-ray radiation. The myocardial tissues were collected 16 weeks after X-ray radiation and subjected to hematoxylin-eosin (HE) staining, TUNEL staining, and immunohistochemistry (IHC) staining. Besides, we established a radiation-injured cardiomyocyte model. Cell viability was assessed with CCK-8 assay and cell apoptosis was assessed using flow cytometry. Protein expression of Bcl-2, Bax, LC3 I/II, Beclin-1, and p62 was detected through western blot assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results showed that 16Gy X-ray radiation resulted in significant changes in myocardial tissues, increased myocardial apoptosis, and activated autophagy. Pretreatment with dexmedetomidine significantly protects mice against 16Gy X-ray radiation-induced myocardial injury by inhibiting apoptosis and autophagy.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn summary, our study confirmed the radioprotective effect of dexmedetomidine against 16Gy X-ray radiation-induced cardiomyocyte apoptosis and autophagy activation.\u003c/p\u003e","manuscriptTitle":"Dexmedetomidine Ameliorates X-Ray-Induced Myocardial Injury Via Alleviating Cardiomyocyte Apoptosis and Autophagy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-19 21:37:18","doi":"10.21203/rs.3.rs-3489562/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-22T19:44:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-22T19:29:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111561074316767318054734918696506919617","date":"2024-05-15T17:19:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-07T18:49:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28815470622015571932533265999462328583","date":"2024-05-03T13:43:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-25T15:48:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-25T15:47:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-01-17T19:08:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-17T19:05:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cardiovascular Disorders","date":"2023-10-25T08:43:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2aabf32e-f435-4f0a-822e-08e47daca57b","owner":[],"postedDate":"January 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-19T06:26:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-19 21:37:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3489562","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3489562","identity":"rs-3489562","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}