Cardiac irradiation combined with an anti-Programmed cell death protein 1 antibody induces time-dependent myocardial injury by regulating the HMGB1/NF-κB pathway

Cardiac irradiation combined with an anti-Programmed cell death protein 1 antibody induces time-dependent myocardial injury by regulating the HMGB1/NF-κB pathway | 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 Cardiac irradiation combined with an anti-Programmed cell death protein 1 antibody induces time-dependent myocardial injury by regulating the HMGB1/NF-κB pathway Yao Liu, Bibo Wu, Yu Wang, Jie Bai, Gang Wang, Shasha Zhao, Bing Lu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4382702/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 Programmed cell death protein 1 ( PD-1) inhibitors may further increase the risk of cardiotoxicity of radiotherapy while improving the outcomes of locally advanced lung cancer. However, few studies have focused on cardiac injury caused by radiotherapy plus anti-PD-1 therapy, and the underlying mechanism is still under exploration. This study aimed to explore this mechanism. Methods Six- to eight-week-old C57BL/6 mice were treated with either an anti-PD-1 antibody or phosphate-buffered saline (PBS) with or without 15 Gray (Gy) cardiac irradiation (IR). Five mice were sacrificed at 1 month, and the remaining mice were sacrificed at 3 months. Histological analysis was performed to determine the structural and morphological alterations and cardiac fibrosis. The infiltration of cardiac T cells was analysed via flow cytometry, and western blotting and qPCR were used to detect the protein and mRNA expression levels of HMGB1-related pathway. Results Group D (IR + anti-PD-1) demonstrated more severe injury, fibrosis, and apoptosis compared to groups A (control), B (anti-PD-1), and C (IR). Furthermore, the injury observed in Group D was significantly more severe, with higher values of apoptotic index (AI) and fibrotic area at 3 months compared to 1 month (P < 0.05). At 1 month, there were no significant differences in cardiac damage or AI or CVF values between groups A and B, but these differences emerged at 3 months (P < 0.05). Group D exhibited greater infiltration of T lymphocytes and increased expression of high mobility group box-1 protein (HMGB1), Toll-like receptor 4 (TLR4), and nuclear factor kappa-B (NF-κB P65) at both 1 and 3 months compared to the other three groups. Conclusion In combination with radiation, PD-1 inhibitors exacerbated myocardial injury by modulating the HMGB1/NF-κB signalling pathway. HMGB1 radiation-induced myocardial injury PD-1 inhibitor T lymphocyte fibrosis apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Radiotherapy is one of the most important regimens for thoracic tumors, with numerous studies verifying its strong link to cardiac dysfunction [ 1 , 2 ]. Accidental exposure of the heart during radiotherapy may lead to acute or chronic cardiovascular consequences with the latter being more common. The latency period for these consequences range from years to decades, so patients with superior survival also are confronted with an increased risk of radiation-induced cardiac dysfunction [ 3 – 5 ]. Current studies on the incidence of radiation-induced heart disease (RIHD) primarily concentrated on breast cancer and Hodgkin's lymphoma because of the superior 5-year survival rates. However, the true incidence of RIHD of other thoracic tumors with poor survival is underestimated [ 6 ]. Over the last decade, immunotherapy has emerged as an important strategy for suppressing malignant tumors. Nevertheless, the effectiveness of immune checkpoint inhibitors (ICIs) is hindered by inherent or acquired resistance in patients. This resistance is observed in up to 60–70% of melanoma patients, and its incidence is even higher in individuals with other types of cancer [ 7 , 8 ]. Research has indicated that PD-1 inhibitors not only enhance the local antitumor effects of radiotherapy but also shrink tumors beyond the radiation area, which is referred to as the "abscopal effect" [ 9 ]. The administration of PD-1 inhibitors is accompanied by a 72% incidence of grade 1–5 irAEs, and the incidence of ICI-associated myocarditis ranges from 0.27–1.14% [ 10 ]. Preclinical studies in mice have shown that PD-1 blockade combined with cardiac irradiation increases the risk of myocarditis and mortality by recruiting cluster of differentiation 8 (CD8 + T) lymphocyte infiltration and elevating tumor necrosis factor-α (TNF-α) expression in the heart, and anti-CD8 treatment reverses acute mortality, indicating that CD8 + T cells are involved in cardiac toxicity [ 11 ]. However, the underlying mechanism remains unclear. HMGB1, a high mobility group protein box 1, serves as a warning signal and operates as an extracellular signaling molecule in various processes, including tissue regeneration, infection, cell differentiation, tumorigenesis, and development. In conditions of disease such as cardiovascular disease and myocardial ischemia-reperfusion injury, HMGB1 is released by necrotic cells and contributes to the inflammatory response as an inflammatory mediator. Additionally, its receptor TLR4 plays a role in this process. TLR4 regulate apoptosis by modulating the expression of TNF-α and interleukin-6 (IL-6) through pathways like oxygen free radicals, neutrophil aggregation, p38 mitogen-activated protein kinase p38, and NF-κB. [ 12 ]. Currently, the precise role of the HMGB1/TLR4 signaling pathway in myocardial injury resulting from the combination of PD-1 inhibitors and radiotherapy remains uncertain. We established a mouse model of radiomyocardial injury to assess the potential adverse effects of PD-1 blockade on myocardial inflammation and to explore the potential involvement of the HMGB1/TLR4 pathway. Materials and methods Animals Male C57BL/6 mice (6 to 8 weeks old, 20–25 g) were obtained from Beijing Viton River Laboratory Animal Science and Technology Co., Ltd. All mice were housed in a specific pathogen-free (SPF) animal facility with food and water provided without restriction. All mouse experiments were approved by the Animal Care and Use Committee of Guizhou Medical University (Certificate No.1901009) and were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. In vivo treatment of an anti-PD-1 antibody combined with cardiac irradiation Mice were anesthetized, immobilized in 1 cm wax models, and positioned under a linear accelerator. The heart's placement at the center of the radiation field was determined by monitoring the heartbeat's visibility on the mice's body surface. The mice in the IR and IR + anti-PD1 groups received cardiac radiation (6 MV X-ray) at a total dose of 15 Gy, 0° single-field vertical chest irradiation, a 2.0 × 2.0 cm irradiation area, 100 cm of skin radiation, and a dose rate of 600 cGy/min. The mice were randomly assigned into 4 groups (n = 10): 1 group with anti-PD1 antibody (anti-PD1), 1 group with irradiation (IR), 1 group with radiation and anti-PD1 antibody (IR + anti-PD1), and the control group (control). The anti-PD-1 group and the IR + anti-PD-1 group received intraperitoneal injections of 200 µg of anti-PD-1 antibody 1 week and 30 minutes before cardiac irradiation, respectively, and 100 µg of anti-PD-1 antibody (Bio X cell, Catalogue #BE0146) on days 7, 14, and 21 after irradiation. The control and IR groups received intraperitoneal injections of the same volume of PBS at the same time. Histopathology In subsequent investigations, a total of 5 mice were subjected to anesthesia and sacrificed after being exposed to cardiac irradiation for durations of 1 and 3 months. The cardiac tissues were then embedded in paraffin. Subsequently, sections measuring 5 µm in thickness were mounted on slides, while the remaining tissue was frozen at -80°C. Hematoxylin-eosin (HE) staining was performed according to the manufacturer's instructions (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Tissue sections were deparaffinized, stained with hematoxylin and eosin, dehydrated in alcohol, treated with xylene for transparency, and sealed with neutral gum. Masson’s trichrome (Masson) staining was performed according to the manufacturer's protocol (Beijing Solarbio Science & Technology Co. Ltd. Beijing, China). Optical microscopy was employed for photographs capture. Five areas were randomly selected from each section, and semiquantitative analysis of the myocardial fibrotic area was performed via ImageJ_V15.0 software. Terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labelling (TUNEL) assay Apoptosis was detected by the TUNEL (Beyotime Co. Ltd. Shanghai, China) method, and cell staining was observed under a fluorescence microscope. FITC-stained apoptotic cells emitted green light, and DAPI-stained nuclei of all cells emitted blue light. The apoptotic index (AI) was analysed semiquantitatively by ImageJ_V15.0 software. Flow cytometry analysis A single-cell suspension was prepared from thirty milligrams of myocardial tissue through enzymatic digestion, followed by labeling the cells with anti-mouse CD3, CD4, and CD8 antibodies. Staining procedures were carried out in accordance with the manufacturer's instructions (Biogem Scarl, Ariano Irpino, Italy). Statistical analysis of the data was conducted using FlowJo_V10 software. Western blot analysis Protein solutions were prepared through sonicating cardiac tissues in RIPA lysis buffer/phenylmethanesulfonyl fluoride (PMSF) at a ratio of 100:1. The concentration of the protein was determined using a bicinchoninic acid (BCA) protein assay kit. Following the electrophoretic transfer of the protein solution, the polyvinylidene fluoride (PVDF) membrane was blocked using a solution of 5% low-fat skim milk dissolved in TBST. Subsequently, the membrane was incubated with the primary antibody overnight at 4°C. An HRP-conjugated secondary antibody was then applied to the membrane and incubated for 1 hour at room temperature. The PVDF membranes were visualized using enhanced chemiluminescence (ECL) and analyzed utilizing Imag J 15.0 software. RT‒qPCR analysis Total RNA was extracted from cardiac tissue using a tissue RNA purification kit. The purity and concentration of the extracted RNA were detected using Nano DropL. Then, cDNA was synthesised according to the instructions of the Rapid All-in-One RT Kit. cDNA was used as a template to detect mRNA expression in the CFX96TM Real-Time PCR system. The cycling parameters were set as follows: predenaturation at 95°C for 5 min, cycling once; denaturation at 95°C for 15 s; annealing extension at 60°C for 30 s; and cycling 40 times. The sequences of primers used were as follows: GAPDH (forward 5′-GGTTGTTCTCCTGCGACTTCA-3′; reverse 5′-TGGTCCAGGGTTTCTTACTCC-3′), HMGB1 (forward, 5′-AGCACAAGAAGAAGCACCCG-3′; reverse, 5'-ACGAGCCTTGTCAGCCTTTG-3'); TLR4 (forward, 5'-GCCATCATTATGAGTGCCAATT-3'; reverse, 5'-AGGGATAAGAACGCTGAGAATT-3'); NF-κB p65 (forward, 5′-AGACCCAGGAGTGTTCACAGACC-3′; reverse, 5’-GTCACCAGGCGAGTTATAGCTTCAG-3′). Statistical analysis The data are presented as the means ± SEMs. Comparisons between groups were analysed by two-way analysis of variance (ANOVA), and further post hoc comparisons were made by Tukey's test. Dunnett's T3 test was used in the case of heteroscedasticity in GraphPad Prism 8.2 software. Differences were considered statistically significant at P < 0.05. Results PD-1 blockade aggravated IR-induced myocardial injury in mice We first examined whether the combination of cardiac irradiation with anti-PD-1 antibody would damage the cardiac more severe compared to irradiation or PBS or treatment with anti-PD-1 antibody alone. All treatment were performed as outlined in method. As shown in Fig. 1 a, HE staining was performed to detect pathological cardiac changes at 1 and 3 months. Mice treated with anti-PD-1 antibody alone did not arose obvious myocardial injury, but cytoplasmic lysis and lymphocyte infiltration were observed in mice subjected to cardiac irradiation alone at 1 and 3 months. Notably, the combination of radiotherapy and anti-PD-1 antibody resulted in more pronounced cardiomyocytolysis and lymphocyte infiltration compared to treatment with anti-PD-1 antibody or radiotherapy alone, further exacerbating the disarray in myocardial tissue structure. According to the findings in Fig. 1 b, a semiquantitative analysis indicated the collagen deposits in the cardiac tissue that was exposed to radiation for 1 month, resulting in a fibrotic area of 5.39 ± 0.77%. This percentage increased to 7.14 ± 0.79% ( P < 0.05) after 3 months. The fibrotic area percentages at 3 months were 1.50 ± 0.15%, 2.97 ± 0.57%, 7.14 ± 0.79%, and 10.32 ± 0.86% in the control, anti-PD-1, IR, and IR + anti-PD1 groups, respectively. A two-way ANOVA analysis revealed a significant interaction effect of time with the intervening factors (F3, 16 = 4.490, p = 0.0181). It was observed that the administration of anti-PD-1 antibody exacerbated myocardial fibrosis in mice treated with irradiation, as depicted in Fig. 1 b-c. Anti-PD-1 antibody aggravated IR-induced myocardial apoptosis The apoptosis was further investigated, and the TUNEL analysis revealed that the apoptic index (AI) showed a gradual increase in the control, anti-PD-1, IR, and IR + anti-PD-1 groups after 1 month (1.49 ± 0.41%, 2.19 ± 0.39%, 7.06 ± 0.58%, and 8.38 ± 0.88%, respectively) and 3 months (1.60 ± 0.32%, 2.68 ± 0.32%, 9.10 ± 0.40%, and 11.73 ± 0.45%, respectively). The AI was significantly increased in the PD-1 blockade combined with irradiated group compared to irradiation alone, and this trend was observed over time in the IR and IR + anti-PD-1 groups ( P < 0.05) (Figs. 2 and 3 ). Anti-PD-1 antibody promoted T lymphocyte infiltration Cardiac lymphocyte infiltration was detected to assess potential invasion by lymphocytes in the heart. The absolute counts of CD3 + T lymphocytes in the cardiac tissue were significantly higher in the other 3 groups (P < 0.05) at 1 and 3 months post-irradiation compared to the control group. Similarly, the absolute counts of CD4 + T lymphocytes were notably increased in the IR group and the IR + anti-PD-1 group compared to the control group at 1 month, although the difference among the other groups was not statistically significant. Furthermore, when comparing lymphocyte infiltration between 1 month and 3 months within each group, it was observed that CD4 + T lymphocyte infiltration remained consistent over time. At 1 and 3 months post-irradiation, there was a progressive rise in the absolute numbers of CD8 + T lymphocytes in the 4 groups ( P < 0.05), indicating that the infiltrated cardiac lymphocytes were mainly CD8 + T lymphocytes. Upon comparing lymphocyte infiltration between 1 month and 3 months within each group, it was observed that CD8 + T lymphocyte infiltration decreased with time (Fig. 4 a-c). Anti-PD-1 antibody aggravated myocardial injury via the HMGB1/NF-κB pathway The levels of HMGB1/TLR4 pathway-related proteins in myocardial tissue were determined using Western blotting. Figure 5 shows that the expression level tendency of HMGB1 and NF-κB p65 were consistent with lymphocyte infiltration. The expression of HMGB1 was highest in the combination group in either 1 or 3-month ( P < 0.0001).The expression of these genes was lower at 3 months compared to 1 month. However, there were no statistically significant differences in TLR4 expression between the groups at 1 or 3 months after irradiation (Fig. 5 c). Notably, the protein expression level across the 4 group was increased gradually. The results of the RT-qPCR assay revealed that the mRNA expression trend of the HMGB1/TLR4 pathway was consistent with the protein expression trend. There were no significant differences in TLR4 mRNA expression among the 4 groups at 1 and 3 months ( P > 0.05) (Fig. 6 d), while HMGB1 and NF-κB p65 mRNA expression were upregulated and maintained for 3 months in the other three groups (P < 0.0001). After treatment with anti-PD-1 antibody combined with cardiac irradiation, this group exhibited the highest HMGB1 and NF-κB p65 mRNA expression (Fig. 6 e, Fig. 6 f). Discussion Studies have shown that the combination of radiotherapy with PD-1 inhibitors enhances the efficacy against lung, esophageal and other thoracic tumors. However, there are concerns regarding the potential exacerbation of radiation-induced injury with PD-1 blockade. While research has primarily focused on pneumonia and esophagitis, there has been limited attention paid to cardiac toxicity [ 13 – 17 ]. In the context of concurrent thoracic radiotherapy and PD-1 blockade, inflammation is a key factor in myocardial damage. HMGB1, a significant damage-associated molecular pattern, is rapidly released following injury or infection, triggering adaptive immune responses and activating antigen-presenting cells. HMGB1 has also been linked to potent inflammatory effects in various forms of myocardiopathy. Radiation-induced cardiac injury leads to HMGB1 release, T lymphocyte activation, and inflammatory cytokine production, creating an immune microenvironment that supports inflammatory reactions and initiates fibrosis [ 19 , 20 , 10 ]. However, the relationship between HMGB1 and myocardial injury in the setting of combined radiotherapy and PD-1 inhibitors remains uncertain. To better understand the relationship between HMGB1 and cardiotoxicity induced by concurrent thoracic radiation plus anti-PD-1 therapy, we established a model in which cardiac irradiation plus anti-PD-1 treatment causes cardiomyopathy in mice. We observed that the blockade of PD-1 enhanced the infiltration of lymphocytes and increased the expression of HMGB1 and NF-κB p65 in the myocardium when combined with irradiation. However, when the anti-PD-1 antibody was administered alone, it did not cause significant myocardial injury in the early stage (1 month), and only led to mild myocardial injury and fibrosis in the late stage (3 months). Comparatively, at the late stage, there was only a slight increase in the fibrosis area compared to the control group, indicating that anti-PD-1 blockade only caused minimal damage to the heart. On the other hand, both radiation alone and radiation combined with anti-PD-1 antibody resulted in widespread injury and fibrosis, with the severity of the injury being greater when the two regimens were combined. Furthermore, the injury and fibrosis worsened over time, which aligns with the progressive deterioration of radiomyocardial fibrosis. Du et al. also reported that PD-1 inhibitors exacerbated radiomyocardial injury and impaired cardiac function in mice. Additionally, when compared to cardiac irradiation alone, the combination of PD-1 inhibitors significantly reduced cardiac output and increased mortality by 30% in mice [ 11 ]. PD-1 inhibitors aggravate immune-inflammatory responses by activating T lymphocytes. In comparison to the IR group, the IR + anti-PD-1 group exhibited a rise in CD3 + T lymphocytes and a notable infiltration of CD8 + T lymphocytes; the infiltration of CD4 + T lymphocytes was not apparent. Research has indicated that blocking CD8 + T lymphocytes alongside radiotherapy can reduce myocardial damage caused by PD-1 inhibition; nevertheless, blocking CD8 + T lymphocytes inevitably inhibits the efficacy of anticancer therapy [ 11 , 21 ]. To mitigate radiation and PD-1 inhibitor-induced myocardial injury, it is crucial to conduct comprehensive research on CD8 + T lymphocyte subsets and immunomodulatory mechanisms. After irradiation, the infiltration of T lymphocytes in the myocardium was notably decreased in both the irradiation and irradiation + anti-PD-1 groups at the 3-month mark compared to 1 month. During the initial stages, T-lymphocyte infiltration plays a significant role in intensifying the inflammatory response and worsening the radiation-induced myocardial injury. During the initial stages, T-lymphocyte infiltration plays a significant role in intensifying the inflammatory response and worsening the radiation-induced myocardial injury. HMGB1 binding to TLR4 triggers intracellular signaling that leads to degradation of the IκB protein, release of active NF-κB p65 into the nucleus, and regulation of gene transcription for inflammatory factors IL-1β, IL-6, and TNF-α. In a mouse model of autoimmune myocarditis, administration of an HMGB1 antagonist resulted in improved cardiac function and reduced myocardial fibrosis[ 22 ]. Yao et al. demonstrated that blocking the inflammatory effects induced by HMGB1 can attenuate doxorubicin-induced myocardial injury [ 23 ]. Wang et al. found that HMGB1 promotes inflammatory injury in myocardial ischemia by targeting TLR4, and knockdown of TLR4 can attenuate HMGB1-induced injury [ 24 ]. In contrast, Ma et al. reported that the application of TLR4 blockers can amplify inflammation, inhibit autophagy, and worsen adriamycin-induced heart failure and fibrosis [ 25 ]. Compared with cardiac irradiation alone, the combination of anti-PD-1 antibody increased the expression of HMGB1 and P65, whereas there was no significant change in the expression of TLR4. The inflammatory response triggered by HMGB1 could be linked to the NF-κB-p65 pathway in radiation-induced myocardial injury when combined with anti-PD-1 treatment, warranting a deeper exploration of the underlying mechanism. Conclusion Anti-PD-1 antibody alone are less toxic to the heart, and radiation induces myocardial fibrosis and injury. However, when these two treatments are combined, the PD-1 blockade results in an increase in CD8 + T lymphocyte infiltration, enhances the expression of HMGB1 and NF-κB, and worsens the radiological damage caused to the heart muscle, leading to increased injury and fibrosis. Abbreviations HMGB1 : high mobility group protein box 1; NF-κB : nuclear factor kappa-B; TLR4 : Toll-like receptor 4; HE : hematoxylin-eosin; Masson : Masson’s trichrome; TUNEL : terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling; PD-1 : programmed death 1. ANOVA : analysis of variance. PMSF : phenylmethanesulfonyl fluoride; BCA : bicinchoninic acid; PVDF: polyvinylidene fluoride Declarations Funding: This work was supported by the Science and Technology Program of Guizhou Province, China [ZK (2022)key040]; and the National Natural Science Foundation of China [No. 81960548]. Competing Interests: The authors declare that they have no conflicts of interest. Data and Material Availability: The analysed datasets generated in the present study are available from the corresponding author upon reasonable request. Code availability : Not applicable. Authors’ contributions: LY: experimental design, writing − original draft, writing − review & editing. WY: Data curation, Writing − original draft. BJ, WBB, WG: Project administration, Writing − review & editing. ZSS: Data analysis, Data curation. HYX, OYWW, GZN, WJ, LB: Irradiation technical guidance. SSF: Funding acquisition, Writing − review & editing. All the authors have read and approved the final manuscript. Ethics Approval: The institutional ethics committee of Guizhou Medical University approved this study. Consent to Participate: Not applicable. Consent for Publication: All the authors have consented to the submission of this article to the journal. References Wollschläger D, Karle H, Stockinger M et al. Radiation dose distribution in functional heart regions from tangential breast cancer radiotherapy. Radiother Oncol. 2016;119(1):65-70. doi:10.1016/j.radonc.2016.01.020. Hardy D, Liu CC, Cormier JN, Xia R, Du XL. Cardiac toxicity in association with chemotherapy and radiation therapy in a large cohort of older patients with non-small-cell lung cancer. Ann Oncol. 2010;21(9):1825-33. doi:10.1093/annonc/mdq042. Lee Chuy K, Nahhas O, Dominic P et al. Cardiovascular Complications Associated with Mediastinal Radiation. Curr Treat Options Cardiovasc Med. 2019;21(7):31. doi:10.1007/s11936-019-0737-0. Jaworski C, Mariani JA, Wheeler G, Kaye DM. Cardiac complications of thoracic irradiation. 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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-4382702","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309929103,"identity":"d1e347c2-2e42-4402-990f-5e71c3560b7b","order_by":0,"name":"Yao Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACfvbGxgcfKmp47A8zHyBOi2TP4cOGM84ck2M43pZAnBaDG2lpwrxtzMYMZ84YEOmyAzlmjDPb2BIbZ+R8vPGGwU5Ot4GADsaGM2YPPpyTSWyWyN1sOYch2djsAAEtzIw95oYzytgS2yRyt0nzMBxI3EZICxszj5k0DxtzYo9EzjPitPCwsaVJ8wC9L8Fzho04LRI8zJBANmBvM7acY0CEX+zvP4REpQEz88Mbbyrs5AhqQbOS2KhB0kKqjlEwCkbBKBgRAADV8EQ/UqdrAgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-8398-7175","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yao","middleName":"","lastName":"Liu","suffix":""},{"id":309929104,"identity":"1848609e-8cd5-4af3-915e-033cefd20aa2","order_by":1,"name":"Bibo Wu","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical 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University","correspondingAuthor":false,"prefix":"","firstName":"Gang","middleName":"","lastName":"Wang","suffix":""},{"id":309929108,"identity":"422527ae-1fd2-47dc-9e66-6e450a6ef7bb","order_by":5,"name":"Shasha Zhao","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shasha","middleName":"","lastName":"Zhao","suffix":""},{"id":309929109,"identity":"88285fe5-0b9e-4ed3-834d-a91d15e79358","order_by":6,"name":"Bing Lu","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Lu","suffix":""},{"id":309929110,"identity":"9d440fe2-1464-473a-8ad5-6cedf4b8ab52","order_by":7,"name":"Yinxiang Hu","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yinxiang","middleName":"","lastName":"Hu","suffix":""},{"id":309929111,"identity":"81e2a35e-94da-44be-a542-4e23e7096826","order_by":8,"name":"Weiwei OuYang","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"OuYang","suffix":""},{"id":309929112,"identity":"2bc8fafe-7c05-42da-a882-6fdf5a85905e","order_by":9,"name":"Zhenneng Guo","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhenneng","middleName":"","lastName":"Guo","suffix":""},{"id":309929113,"identity":"be2f903e-ec0a-49d5-add0-93fee3847610","order_by":10,"name":"Jun Wan","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Wan","suffix":""},{"id":309929114,"identity":"9a6e8660-a235-4563-bf2d-9351f8d65183","order_by":11,"name":"Rong Hu","email":"","orcid":"","institution":"Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Hu","suffix":""},{"id":309929115,"identity":"3d76afe8-9440-49bd-8e62-00850ca09fa2","order_by":12,"name":"Shengfa Su","email":"","orcid":"https://orcid.org/0000-0002-6429-0081","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shengfa","middleName":"","lastName":"Su","suffix":""}],"badges":[],"createdAt":"2024-05-07 11:35:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4382702/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4382702/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58359369,"identity":"f4474346-1d99-4d08-81c2-a26adfcdebdb","added_by":"auto","created_at":"2024-06-14 10:49:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2782838,"visible":true,"origin":"","legend":"\u003cp\u003ePathological changes in heart tissue at 1 and 3 months. a: HE staining of heart tissue (×400). b: Masson staining of myocardial fibres (red) and collagen fibres (blue) 1 and 3 months after heart irradiation (×100). c: Semiquantitative analysis of the cardiac fibrosis area. A: Control, B: anti-PD-1, C: IR, D: IR + anti-PD-1. (ns\u003cem\u003e P\u003c/em\u003e \u0026gt; 0.05, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, * *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, * * *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, * * * *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4382702/v1/3946cd563dbca4f7a0857279.png"},{"id":58358236,"identity":"a31adf86-1f08-4800-ac04-71fb6f46b373","added_by":"auto","created_at":"2024-06-14 10:33:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7343372,"visible":true,"origin":"","legend":"\u003cp\u003eTUNEL staining of cardiac tissue at 1 and 3 months. A: Control, B: anti-PD-1, C: IR, D: IR + anti-PD-1\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4382702/v1/63864551e892284913cf3cc7.png"},{"id":58358241,"identity":"191a492c-6302-4a0d-bf17-5eb7eac1abaa","added_by":"auto","created_at":"2024-06-14 10:33:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":82318,"visible":true,"origin":"","legend":"\u003cp\u003eSemiquantitative analysis of the AI. ns \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, * * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, * * * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, * * * * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4382702/v1/b86c5462c63515c465e0d30f.png"},{"id":58358912,"identity":"257a4ee1-5ecd-42c6-9108-3b90b0908702","added_by":"auto","created_at":"2024-06-14 10:41:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":227791,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of T lymphocytes in the myocardium of mice. a-c: Absolute counts of CD3+, CD4+, and CD8+T lymphocytes in the myocardium of 4 groups of mice at 1 and 3 months after irradiation. (ns \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, * * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, * * *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, * * * * \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4382702/v1/5a58b0d5425ba34a7496ce16.png"},{"id":58358238,"identity":"5ab6ebdd-1adf-45d4-81b3-a6499cddd506","added_by":"auto","created_at":"2024-06-14 10:33:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":330383,"visible":true,"origin":"","legend":"\u003cp\u003eProtein expression of the myocardial HMGB1/TLR4 signalling pathway after irradiation in mice in each group. A: Control, B: anti-PD-1, C: IR, D: IR+anti-PD-1. (ns \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, * * \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, * * *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, * * * * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4382702/v1/be5dbc606e42b88aa7df48eb.png"},{"id":58358240,"identity":"027675df-a07e-4c1d-87ec-297875fe41c9","added_by":"auto","created_at":"2024-06-14 10:33:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":274444,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blotting and RT‒qPCR were performed to detect the myocardial tissues of the irradiated mice. a-c: Relative expression of HMGB1/TLR4 signalling pathway proteins in myocardial tissues. d-f: Quantification of HMGB1, TLR4, and NF-κB p65 mRNA levels. (ns \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, * * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, * * *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, * * * * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4382702/v1/c6d7016261c633c0f54b9444.png"},{"id":58519190,"identity":"f2cdff1a-26df-41de-bd4a-fd864bb46fc3","added_by":"auto","created_at":"2024-06-17 17:37:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18046386,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4382702/v1/27550f5d-fbf7-4ac9-ad51-3df9d133df83.pdf"}],"financialInterests":"","formattedTitle":"Cardiac irradiation combined with an anti-Programmed cell death protein 1 antibody induces time-dependent myocardial injury by regulating the HMGB1/NF-κB pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRadiotherapy is one of the most important regimens for thoracic tumors, with numerous studies verifying its strong link to cardiac dysfunction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Accidental exposure of the heart during radiotherapy may lead to acute or chronic cardiovascular consequences with the latter being more common. The latency period for these consequences range from years to decades, so patients with superior survival also are confronted with an increased risk of radiation-induced cardiac dysfunction [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Current studies on the incidence of radiation-induced heart disease (RIHD) primarily concentrated on breast cancer and Hodgkin's lymphoma because of the superior 5-year survival rates. However, the true incidence of RIHD of other thoracic tumors with poor survival is underestimated [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOver the last decade, immunotherapy has emerged as an important strategy for suppressing malignant tumors. Nevertheless, the effectiveness of immune checkpoint inhibitors (ICIs) is hindered by inherent or acquired resistance in patients. This resistance is observed in up to 60\u0026ndash;70% of melanoma patients, and its incidence is even higher in individuals with other types of cancer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Research has indicated that PD-1 inhibitors not only enhance the local antitumor effects of radiotherapy but also shrink tumors beyond the radiation area, which is referred to as the \"abscopal effect\" [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe administration of PD-1 inhibitors is accompanied by a 72% incidence of grade 1\u0026ndash;5 irAEs, and the incidence of ICI-associated myocarditis ranges from 0.27\u0026ndash;1.14% [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Preclinical studies in mice have shown that PD-1 blockade combined with cardiac irradiation increases the risk of myocarditis and mortality by recruiting cluster of differentiation 8 (CD8\u003csup\u003e+\u003c/sup\u003eT) lymphocyte infiltration and elevating tumor necrosis factor-α (TNF-α) expression in the heart, and anti-CD8 treatment reverses acute mortality, indicating that CD8\u003csup\u003e+\u003c/sup\u003eT cells are involved in cardiac toxicity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the underlying mechanism remains unclear.\u003c/p\u003e \u003cp\u003eHMGB1, a high mobility group protein box 1, serves as a warning signal and operates as an extracellular signaling molecule in various processes, including tissue regeneration, infection, cell differentiation, tumorigenesis, and development. In conditions of disease such as cardiovascular disease and myocardial ischemia-reperfusion injury, HMGB1 is released by necrotic cells and contributes to the inflammatory response as an inflammatory mediator. Additionally, its receptor TLR4 plays a role in this process. TLR4 regulate apoptosis by modulating the expression of TNF-α and interleukin-6 (IL-6) through pathways like oxygen free radicals, neutrophil aggregation, p38 mitogen-activated protein kinase p38, and NF-κB. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, the precise role of the HMGB1/TLR4 signaling pathway in myocardial injury resulting from the combination of PD-1 inhibitors and radiotherapy remains uncertain. We established a mouse model of radiomyocardial injury to assess the potential adverse effects of PD-1 blockade on myocardial inflammation and to explore the potential involvement of the HMGB1/TLR4 pathway.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice (6 to 8 weeks old, 20\u0026ndash;25 g) were obtained from Beijing Viton River Laboratory Animal Science and Technology Co., Ltd. All mice were housed in a specific pathogen-free (SPF) animal facility with food and water provided without restriction. All mouse experiments were approved by the Animal Care and Use Committee of Guizhou Medical University (Certificate No.1901009) and were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo treatment of an anti-PD-1 antibody combined with cardiac irradiation\u003c/h2\u003e \u003cp\u003eMice were anesthetized, immobilized in 1 cm wax models, and positioned under a linear accelerator. The heart's placement at the center of the radiation field was determined by monitoring the heartbeat's visibility on the mice's body surface. The mice in the IR and IR\u0026thinsp;+\u0026thinsp;anti-PD1 groups received cardiac radiation (6 MV X-ray) at a total dose of 15 Gy, 0\u0026deg; single-field vertical chest irradiation, a 2.0 \u0026times; 2.0 cm irradiation area, 100 cm of skin radiation, and a dose rate of 600 cGy/min.\u003c/p\u003e \u003cp\u003eThe mice were randomly assigned into 4 groups (n\u0026thinsp;=\u0026thinsp;10): 1 group with anti-PD1 antibody (anti-PD1), 1 group with irradiation (IR), 1 group with radiation and anti-PD1 antibody (IR\u0026thinsp;+\u0026thinsp;anti-PD1), and the control group (control). The anti-PD-1 group and the IR\u0026thinsp;+\u0026thinsp;anti-PD-1 group received intraperitoneal injections of 200 \u0026micro;g of anti-PD-1 antibody 1 week and 30 minutes before cardiac irradiation, respectively, and 100 \u0026micro;g of anti-PD-1 antibody (Bio X cell, Catalogue #BE0146) on days 7, 14, and 21 after irradiation. The control and IR groups received intraperitoneal injections of the same volume of PBS at the same time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology\u003c/h2\u003e \u003cp\u003eIn subsequent investigations, a total of 5 mice were subjected to anesthesia and sacrificed after being exposed to cardiac irradiation for durations of 1 and 3 months. The cardiac tissues were then embedded in paraffin. Subsequently, sections measuring 5 \u0026micro;m in thickness were mounted on slides, while the remaining tissue was frozen at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003eHematoxylin-eosin (HE) staining was performed according to the manufacturer's instructions (Beijing Solarbio Science \u0026amp; Technology Co., Ltd., Beijing, China). Tissue sections were deparaffinized, stained with hematoxylin and eosin, dehydrated in alcohol, treated with xylene for transparency, and sealed with neutral gum.\u003c/p\u003e \u003cp\u003eMasson\u0026rsquo;s trichrome (Masson) staining was performed according to the manufacturer's protocol (Beijing Solarbio Science \u0026amp; Technology Co. Ltd. Beijing, China). Optical microscopy was employed for photographs capture. Five areas were randomly selected from each section, and semiquantitative analysis of the myocardial fibrotic area was performed via ImageJ_V15.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTerminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labelling (TUNEL) assay\u003c/h2\u003e \u003cp\u003eApoptosis was detected by the TUNEL (Beyotime Co. Ltd. Shanghai, China) method, and cell staining was observed under a fluorescence microscope. FITC-stained apoptotic cells emitted green light, and DAPI-stained nuclei of all cells emitted blue light. The apoptotic index (AI) was analysed semiquantitatively by ImageJ_V15.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eA single-cell suspension was prepared from thirty milligrams of myocardial tissue through enzymatic digestion, followed by labeling the cells with anti-mouse CD3, CD4, and CD8 antibodies. Staining procedures were carried out in accordance with the manufacturer's instructions (Biogem Scarl, Ariano Irpino, Italy). Statistical analysis of the data was conducted using FlowJo_V10 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eProtein solutions were prepared through sonicating cardiac tissues in RIPA lysis buffer/phenylmethanesulfonyl fluoride (PMSF) at a ratio of 100:1. The concentration of the protein was determined using a bicinchoninic acid (BCA) protein assay kit. Following the electrophoretic transfer of the protein solution, the polyvinylidene fluoride (PVDF) membrane was blocked using a solution of 5% low-fat skim milk dissolved in TBST. Subsequently, the membrane was incubated with the primary antibody overnight at 4\u0026deg;C. An HRP-conjugated secondary antibody was then applied to the membrane and incubated for 1 hour at room temperature. The PVDF membranes were visualized using enhanced chemiluminescence (ECL) and analyzed utilizing Imag J 15.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRT‒qPCR analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cardiac tissue using a tissue RNA purification kit. The purity and concentration of the extracted RNA were detected using Nano DropL. Then, cDNA was synthesised according to the instructions of the Rapid All-in-One RT Kit. cDNA was used as a template to detect mRNA expression in the CFX96TM Real-Time PCR system. The cycling parameters were set as follows: predenaturation at 95\u0026deg;C for 5 min, cycling once; denaturation at 95\u0026deg;C for 15 s; annealing extension at 60\u0026deg;C for 30 s; and cycling 40 times.\u003c/p\u003e \u003cp\u003eThe sequences of primers used were as follows:\u003c/p\u003e \u003cp\u003eGAPDH (forward 5\u0026prime;-GGTTGTTCTCCTGCGACTTCA-3\u0026prime;; reverse 5\u0026prime;-TGGTCCAGGGTTTCTTACTCC-3\u0026prime;),\u003c/p\u003e \u003cp\u003eHMGB1 (forward, 5\u0026prime;-AGCACAAGAAGAAGCACCCG-3\u0026prime;; reverse, 5'-ACGAGCCTTGTCAGCCTTTG-3');\u003c/p\u003e \u003cp\u003eTLR4 (forward, 5'-GCCATCATTATGAGTGCCAATT-3'; reverse, 5'-AGGGATAAGAACGCTGAGAATT-3');\u003c/p\u003e \u003cp\u003eNF-κB p65 (forward, 5\u0026prime;-AGACCCAGGAGTGTTCACAGACC-3\u0026prime;; reverse, 5\u0026rsquo;-GTCACCAGGCGAGTTATAGCTTCAG-3\u0026prime;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs. Comparisons between groups were analysed by two-way analysis of variance (ANOVA), and further post hoc comparisons were made by Tukey's test. Dunnett's T3 test was used in the case of heteroscedasticity in GraphPad Prism 8.2 software. Differences were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePD-1 blockade aggravated IR-induced myocardial injury in mice\u003c/h2\u003e \u003cp\u003eWe first examined whether the combination of cardiac irradiation with anti-PD-1 antibody would damage the cardiac more severe compared to irradiation or PBS or treatment with anti-PD-1 antibody alone. All treatment were performed as outlined in method. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, HE staining was performed to detect pathological cardiac changes at 1 and 3 months. Mice treated with anti-PD-1 antibody alone did not arose obvious myocardial injury, but cytoplasmic lysis and lymphocyte infiltration were observed in mice subjected to cardiac irradiation alone at 1 and 3 months. Notably, the combination of radiotherapy and anti-PD-1 antibody resulted in more pronounced cardiomyocytolysis and lymphocyte infiltration compared to treatment with anti-PD-1 antibody or radiotherapy alone, further exacerbating the disarray in myocardial tissue structure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to the findings in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, a semiquantitative analysis indicated the collagen deposits in the cardiac tissue that was exposed to radiation for 1 month, resulting in a fibrotic area of 5.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77%. This percentage increased to 7.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after 3 months. The fibrotic area percentages at 3 months were 1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15%, 2.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57%, 7.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79%, and 10.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86% in the control, anti-PD-1, IR, and IR\u0026thinsp;+\u0026thinsp;anti-PD1 groups, respectively. A two-way ANOVA analysis revealed a significant interaction effect of time with the intervening factors (F3, 16\u0026thinsp;=\u0026thinsp;4.490, p\u0026thinsp;=\u0026thinsp;0.0181). It was observed that the administration of anti-PD-1 antibody exacerbated myocardial fibrosis in mice treated with irradiation, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-c.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnti-PD-1 antibody aggravated IR-induced myocardial apoptosis\u003c/h2\u003e \u003cp\u003eThe apoptosis was further investigated, and the TUNEL analysis revealed that the apoptic index (AI) showed a gradual increase in the control, anti-PD-1, IR, and IR\u0026thinsp;+\u0026thinsp;anti-PD-1 groups after 1 month (1.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41%, 2.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39%, 7.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58%, and 8.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88%, respectively) and 3 months (1.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32%, 2.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32%, 9.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40%, and 11.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45%, respectively). The AI was significantly increased in the PD-1 blockade combined with irradiated group compared to irradiation alone, and this trend was observed over time in the IR and IR\u0026thinsp;+\u0026thinsp;anti-PD-1 groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnti-PD-1 antibody promoted T lymphocyte infiltration\u003c/h2\u003e \u003cp\u003eCardiac lymphocyte infiltration was detected to assess potential invasion by lymphocytes in the heart. The absolute counts of CD3\u0026thinsp;+\u0026thinsp;T lymphocytes in the cardiac tissue were significantly higher in the other 3 groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) at 1 and 3 months post-irradiation compared to the control group. Similarly, the absolute counts of CD4\u0026thinsp;+\u0026thinsp;T lymphocytes were notably increased in the IR group and the IR\u0026thinsp;+\u0026thinsp;anti-PD-1 group compared to the control group at 1 month, although the difference among the other groups was not statistically significant. Furthermore, when comparing lymphocyte infiltration between 1 month and 3 months within each group, it was observed that CD4\u0026thinsp;+\u0026thinsp;T lymphocyte infiltration remained consistent over time.\u003c/p\u003e \u003cp\u003eAt 1 and 3 months post-irradiation, there was a progressive rise in the absolute numbers of CD8\u0026thinsp;+\u0026thinsp;T lymphocytes in the 4 groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that the infiltrated cardiac lymphocytes were mainly CD8\u0026thinsp;+\u0026thinsp;T lymphocytes. Upon comparing lymphocyte infiltration between 1 month and 3 months within each group, it was observed that CD8\u0026thinsp;+\u0026thinsp;T lymphocyte infiltration decreased with time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAnti-PD-1 antibody aggravated myocardial injury via the HMGB1/NF-κB pathway\u003c/h2\u003e \u003cp\u003eThe levels of HMGB1/TLR4 pathway-related proteins in myocardial tissue were determined using Western blotting. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows that the expression level tendency of HMGB1 and NF-κB p65 were consistent with lymphocyte infiltration. The expression of HMGB1 was highest in the combination group in either 1 or 3-month (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).The expression of these genes was lower at 3 months compared to 1 month. However, there were no statistically significant differences in TLR4 expression between the groups at 1 or 3 months after irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Notably, the protein expression level across the 4 group was increased gradually.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of the RT-qPCR assay revealed that the mRNA expression trend of the HMGB1/TLR4 pathway was consistent with the protein expression trend. There were no significant differences in TLR4 mRNA expression among the 4 groups at 1 and 3 months (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed), while HMGB1 and NF-κB p65 mRNA expression were upregulated and maintained for 3 months in the other three groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). After treatment with anti-PD-1 antibody combined with cardiac irradiation, this group exhibited the highest HMGB1 and NF-κB p65 mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eStudies have shown that the combination of radiotherapy with PD-1 inhibitors enhances the efficacy against lung, esophageal and other thoracic tumors. However, there are concerns regarding the potential exacerbation of radiation-induced injury with PD-1 blockade. While research has primarily focused on pneumonia and esophagitis, there has been limited attention paid to cardiac toxicity [\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the context of concurrent thoracic radiotherapy and PD-1 blockade, inflammation is a key factor in myocardial damage. HMGB1, a significant damage-associated molecular pattern, is rapidly released following injury or infection, triggering adaptive immune responses and activating antigen-presenting cells. HMGB1 has also been linked to potent inflammatory effects in various forms of myocardiopathy. Radiation-induced cardiac injury leads to HMGB1 release, T lymphocyte activation, and inflammatory cytokine production, creating an immune microenvironment that supports inflammatory reactions and initiates fibrosis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the relationship between HMGB1 and myocardial injury in the setting of combined radiotherapy and PD-1 inhibitors remains uncertain.\u003c/p\u003e \u003cp\u003eTo better understand the relationship between HMGB1 and cardiotoxicity induced by concurrent thoracic radiation plus anti-PD-1 therapy, we established a model in which cardiac irradiation plus anti-PD-1 treatment causes cardiomyopathy in mice. We observed that the blockade of PD-1 enhanced the infiltration of lymphocytes and increased the expression of HMGB1 and NF-κB p65 in the myocardium when combined with irradiation. However, when the anti-PD-1 antibody was administered alone, it did not cause significant myocardial injury in the early stage (1 month), and only led to mild myocardial injury and fibrosis in the late stage (3 months). Comparatively, at the late stage, there was only a slight increase in the fibrosis area compared to the control group, indicating that anti-PD-1 blockade only caused minimal damage to the heart. On the other hand, both radiation alone and radiation combined with anti-PD-1 antibody resulted in widespread injury and fibrosis, with the severity of the injury being greater when the two regimens were combined. Furthermore, the injury and fibrosis worsened over time, which aligns with the progressive deterioration of radiomyocardial fibrosis. Du et al. also reported that PD-1 inhibitors exacerbated radiomyocardial injury and impaired cardiac function in mice. Additionally, when compared to cardiac irradiation alone, the combination of PD-1 inhibitors significantly reduced cardiac output and increased mortality by 30% in mice [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePD-1 inhibitors aggravate immune-inflammatory responses by activating T lymphocytes. In comparison to the IR group, the IR\u0026thinsp;+\u0026thinsp;anti-PD-1 group exhibited a rise in CD3\u0026thinsp;+\u0026thinsp;T lymphocytes and a notable infiltration of CD8\u0026thinsp;+\u0026thinsp;T lymphocytes; the infiltration of CD4\u0026thinsp;+\u0026thinsp;T lymphocytes was not apparent. Research has indicated that blocking CD8\u0026thinsp;+\u0026thinsp;T lymphocytes alongside radiotherapy can reduce myocardial damage caused by PD-1 inhibition; nevertheless, blocking CD8\u0026thinsp;+\u0026thinsp;T lymphocytes inevitably inhibits the efficacy of anticancer therapy [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo mitigate radiation and PD-1 inhibitor-induced myocardial injury, it is crucial to conduct comprehensive research on CD8\u0026thinsp;+\u0026thinsp;T lymphocyte subsets and immunomodulatory mechanisms. After irradiation, the infiltration of T lymphocytes in the myocardium was notably decreased in both the irradiation and irradiation\u0026thinsp;+\u0026thinsp;anti-PD-1 groups at the 3-month mark compared to 1 month. During the initial stages, T-lymphocyte infiltration plays a significant role in intensifying the inflammatory response and worsening the radiation-induced myocardial injury. During the initial stages, T-lymphocyte infiltration plays a significant role in intensifying the inflammatory response and worsening the radiation-induced myocardial injury.\u003c/p\u003e \u003cp\u003eHMGB1 binding to TLR4 triggers intracellular signaling that leads to degradation of the IκB protein, release of active NF-κB p65 into the nucleus, and regulation of gene transcription for inflammatory factors IL-1β, IL-6, and TNF-α. In a mouse model of autoimmune myocarditis, administration of an HMGB1 antagonist resulted in improved cardiac function and reduced myocardial fibrosis[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Yao et al. demonstrated that blocking the inflammatory effects induced by HMGB1 can attenuate doxorubicin-induced myocardial injury [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Wang et al. found that HMGB1 promotes inflammatory injury in myocardial ischemia by targeting TLR4, and knockdown of TLR4 can attenuate HMGB1-induced injury [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In contrast, Ma et al. reported that the application of TLR4 blockers can amplify inflammation, inhibit autophagy, and worsen adriamycin-induced heart failure and fibrosis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Compared with cardiac irradiation alone, the combination of anti-PD-1 antibody increased the expression of HMGB1 and P65, whereas there was no significant change in the expression of TLR4. The inflammatory response triggered by HMGB1 could be linked to the NF-κB-p65 pathway in radiation-induced myocardial injury when combined with anti-PD-1 treatment, warranting a deeper exploration of the underlying mechanism.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAnti-PD-1 antibody alone are less toxic to the heart, and radiation induces myocardial fibrosis and injury. However, when these two treatments are combined, the PD-1 blockade results in an increase in CD8\u0026thinsp;+\u0026thinsp;T lymphocyte infiltration, enhances the expression of HMGB1 and NF-κB, and worsens the radiological damage caused to the heart muscle, leading to increased injury and fibrosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eHMGB1\u003c/em\u003e: high\u0026nbsp;mobility group protein box 1; \u003cem\u003eNF-\u0026kappa;B\u003c/em\u003e: nuclear\u0026nbsp;factor kappa-B; \u003cem\u003eTLR4\u003c/em\u003e:\u0026nbsp;Toll-like receptor 4; \u003cem\u003eHE\u003c/em\u003e: hematoxylin-eosin; \u003cem\u003eMasson\u003c/em\u003e:\u0026nbsp;Masson\u0026rsquo;s trichrome;\u0026nbsp;\u003cem\u003eTUNEL\u003c/em\u003e: terminal deoxynucleotidyl transferase-mediated dUTP\u0026nbsp;nick-end labelling; \u003cem\u003ePD-1\u003c/em\u003e: programmed death\u0026nbsp;1.\u0026nbsp;\u003cem\u003eANOVA\u003c/em\u003e:\u0026nbsp;analysis of variance.\u0026nbsp;\u003cem\u003ePMSF\u003c/em\u003e: phenylmethanesulfonyl fluoride;\u003cem\u003e\u0026nbsp;BCA\u003c/em\u003e: bicinchoninic acid; PVDF: polyvinylidene fluoride\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by the\u0026nbsp;Science and Technology Program of Guizhou\u0026nbsp;Province, China\u0026nbsp;[ZK (2022)key040]; and the National Natural Science Foundation of China [No. 81960548].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e The authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and Material Availability:\u0026nbsp;\u003c/strong\u003eThe analysed datasets generated in the present study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eavailability\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eNot\u0026nbsp;applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eLY:\u0026nbsp;experimental\u0026nbsp;design,\u0026nbsp;writing\u0026nbsp;\u0026minus; original draft,\u0026nbsp;writing\u0026nbsp;\u0026minus; review \u0026amp; editing. WY: Data curation, Writing \u0026minus; original draft. BJ,\u0026nbsp;WBB,\u0026nbsp;WG: Project administration, Writing \u0026minus; review \u0026amp; editing. ZSS:\u0026nbsp;Data analysis, Data curation. HYX, OYWW, GZN, WJ,\u0026nbsp;LB: Irradiation technical guidance. SSF: Funding acquisition, Writing \u0026minus; review \u0026amp; editing. All the authors have read and approved the\u0026nbsp;final\u0026nbsp;manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval:\u003c/strong\u003e The institutional ethics committee of Guizhou Medical University approved this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u0026nbsp;\u003c/strong\u003eNot\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eapplicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication:\u0026nbsp;\u003c/strong\u003eAll the authors have consented to the submission of this article to the journal.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWollschl\u0026auml;ger D, Karle H, Stockinger M et al. Radiation dose distribution in functional heart regions from tangential breast cancer radiotherapy. Radiother Oncol. 2016;119(1):65-70. doi:10.1016/j.radonc.2016.01.020.\u003c/li\u003e\n\u003cli\u003eHardy D, Liu CC, Cormier JN, Xia R, Du XL. 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PD-1 Modulates Radiation-Induced Cardiac Toxicity through Cytotoxic T Lymphocytes. J Thorac Oncol. 2018;13(4):510-20. doi:10.1016/j.jtho.2017.12.002.\u003c/li\u003e\n\u003cli\u003eDing HS, Yang J, Chen P et al. The HMGB1-TLR4 axis contributes to myocardial ischemia/reperfusion injury via regulation of cardiomyocyte apoptosis. Gene. 2013;527(1):389-93. doi:10.1016/j.gene.2013.05.041.\u003c/li\u003e\n\u003cli\u003eAntonia SJ, Villegas A, Daniel D et al. Overall Survival with Durvalumab after Chemoradiotherapy in Stage III NSCLC. N Engl J Med. 2018;379(24):2342-50. doi:10.1056/NEJMoa1809697.\u003c/li\u003e\n\u003cli\u003eAnouti B, Althouse S, Durm G, Hanna N. Prognostic Variables Associated With Improved Outcomes in Patients With Stage III NSCLC Treated With Chemoradiation Followed by Consolidation Pembrolizumab: A Subset Analysis of a Phase II Study From the Hoosier Cancer Research Network LUN 14-179. 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HMGB1 blockade attenuates experimental autoimmune myocarditis and suppresses Th17-cell expansion. Eur J Immunol. 2011;41(12):3586-95. doi:10.1002/eji.201141879.\u003c/li\u003e\n\u003cli\u003eYao Y, Xu X, Zhang G et al. Role of HMGB1 in doxorubicin-induced myocardial apoptosis and its regulation pathway. Basic Res Cardiol. 2012;107(3):267. doi:10.1007/s00395-012-0267-3.\u003c/li\u003e\n\u003cli\u003eWang R, Wang P, Du G. [HMGB1 promotes myocardial ischemic injury and regulates the proportion of CD4(+), CD8(+)T cells and Th17 cells in spleen through TLR4]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2018;34(9):794-9. \u003c/li\u003e\n\u003cli\u003eMa Y, Zhang X, Bao H et al. Toll-like receptor (TLR) 2 and TLR4 differentially regulate doxorubicin induced cardiomyopathy in mice. PLoS One. 2012;7(7):e40763. doi:10.1371/journal.pone.0040763.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HMGB1, radiation-induced myocardial injury, PD-1 inhibitor, T lymphocyte, fibrosis, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-4382702/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4382702/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eProgrammed cell death protein 1 \u003cb\u003e(\u003c/b\u003ePD-1) inhibitors may further increase the risk of cardiotoxicity of radiotherapy while improving the outcomes of locally advanced lung cancer. However, few studies have focused on cardiac injury caused by radiotherapy plus anti-PD-1 therapy, and the underlying mechanism is still under exploration. This study aimed to explore this mechanism.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSix- to eight-week-old C57BL/6 mice were treated with either an anti-PD-1 antibody or phosphate-buffered saline (PBS) with or without 15 Gray (Gy) cardiac irradiation (IR). Five mice were sacrificed at 1 month, and the remaining mice were sacrificed at 3 months. Histological analysis was performed to determine the structural and morphological alterations and cardiac fibrosis. The infiltration of cardiac T cells was analysed via flow cytometry, and western blotting and qPCR were used to detect the protein and mRNA expression levels of HMGB1-related pathway.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eGroup D (IR\u0026thinsp;+\u0026thinsp;anti-PD-1) demonstrated more severe injury, fibrosis, and apoptosis compared to groups A (control), B (anti-PD-1), and C (IR). Furthermore, the injury observed in Group D was significantly more severe, with higher values of apoptotic index (AI) and fibrotic area at 3 months compared to 1 month (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At 1 month, there were no significant differences in cardiac damage or AI or CVF values between groups A and B, but these differences emerged at 3 months (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Group D exhibited greater infiltration of T lymphocytes and increased expression of high mobility group box-1 protein (HMGB1), Toll-like receptor 4 (TLR4), and nuclear factor kappa-B (NF-κB P65) at both 1 and 3 months compared to the other three groups.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn combination with radiation, PD-1 inhibitors exacerbated myocardial injury by modulating the HMGB1/NF-κB signalling pathway.\u003c/p\u003e","manuscriptTitle":"Cardiac irradiation combined with an anti-Programmed cell death protein 1 antibody induces time-dependent myocardial injury by regulating the HMGB1/NF-κB pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-14 10:33:19","doi":"10.21203/rs.3.rs-4382702/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":"e5c51f07-2ce1-486a-8891-8632856baeb3","owner":[],"postedDate":"June 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-17T17:28:57+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-14 10:33:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4382702","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4382702","identity":"rs-4382702","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}