The role of HMGB1/TLR4 in pericardial fibrosis and postoperative low cardiac output syndrome in constrictive pericarditis

The role of HMGB1/TLR4 in pericardial fibrosis and postoperative low cardiac output syndrome in constrictive pericarditis | 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 The role of HMGB1/TLR4 in pericardial fibrosis and postoperative low cardiac output syndrome in constrictive pericarditis Likui Fang, Tianxiang Wang, Fangming Zhong, Guocan Yu, Weihua Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8370586/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 Background It has remained unclear about the factors that are involved in the pericardial fibrotic process and the occurrence of postoperative low cardiac output syndrome (LCOS) in constrictive pericarditis. This study aimed to analyze the role of TLR4 and HMGB1 in pericardial fibrosis, and their impact on the development of postoperative LCOS in patients with constrictive pericarditis. Methods This retrospective study enrolled 24 constrictive pericarditis patients who underwent isolated pericardiectomy at our department from May 2023 to April 2025. Pericardial tissues were subjected to immunohistochemistry to detect the expression of TLR4, HMGB1, α-SMA and collagen III. Mean Optical Density (MOD) was used for quantitative analysis of immunohistochemical staining. Pearson correlation analysis was performed to assess associations between TLR4, HMGB1 and fibrotic markers, while Receiver Operating Characteristic (ROC) curves were used to evaluate their predictive value for postoperative LCOS. Results Of the 24 patients, 7 (29.2%) developed postoperative LCOS. TLR4 was expressed in 21 (87.5%) specimens and HMGB1 was expressed in all specimens. Pearson correlation analysis showed positive correlations between TLR4 and α-SMA (r = 0.529, P = 0.008), HMGB1 and α-SMA (r = 0.516, P = 0.010), and TLR4 and HMGB1 (r = 0.844, P < 0.001). MOD values of TLR4 and HMGB1 were significantly higher in patients with postoperative LCOS (P = 0.028 and P < 0.001, respectively). ROC curve suggested that TLR4 (AUC = 0.790, 95%CI: 0.516–1.000) and HMGB1 (AUC = 0.941, 95%CI: 0.853–1.000) had potential predictive value for postoperative LCOS. Conclusion TLR4 and HMGB1 were involved in the pericardial fibrosis and were significantly associated with the occurrence of postoperative LCOS in constrictive pericarditis. constrictive pericarditis low cardiac output syndrome TLR4 HMGB1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Constrictive pericarditis is a rare but life-threatening disease, and the most common cause is tuberculosis worldwide [ 1 ] . Fibrosis is the core pathological feature of constrictive pericarditis which ultimately results in diastolic heart failure [ 2 ] . Pericardiectomy is the only definitive treatment option but is accompanied with high perioperative risk [ 3 , 4 ] . Low cardiac output syndrome (LCOS) is the main postoperative complication and a major cause of postoperative mortality [ 5 – 7 ] . At present, it has remained unclear about the factors that are involved in the pericardial fibrotic process and the occurrence of postoperative LCOS. Toll-like receptors (TLRs) are a class of transmembrane structural proteins that play a crucial role in the innate immune system [ 8 ] . Studies have shown that they may participate in the activation of fibroblasts, and among them, TLR4 has been proven to exert a significant pro-fibrotic effect [ 9 – 11 ] . In rat constrictive pericarditis model, activation of the TLR4 signaling pathway stimulates the expression of α-smooth muscle actin (α-SMA) and promotes the process of myocardial fibrosis [ 12 ] . High-Mobility Group Box 1 (HMGB1) is a highly conserved nuclear protein that is widely expressed in nearly all cell types, and a growing body of research has shown that HMGB1 is closely associated with various fibrotic diseases [ 13 , 14 ] . TLR4 is one of the most extensively studied receptors for HMGB1, and inhibiting the expression of HMGB1 could inactivate the HMGB1/TLR4 pathway to impede the development of fibrosis [ 15 ] . However, the expression patterns and functional roles of TLR4 and HMGB1 in the pericardial tissue of patients with constrictive pericarditis have remained unclear. We aimed to analyze the expression of TLR4 and HMGB1 in pericardial tissue, the associations between their expression and that of key fibrotic proteins, α-SMA and collagen III (Col-III), as well as their impact on the development of postoperative LCOS in patients with constrictive pericarditis. Methods Study population A retrospective analysis was conducted on the clinical records of patients diagnosed with constrictive pericarditis who were treated in our department between May 2023 and April 2025. Pericardial tissues surgically resected from 24 eligible patients were selected for immunohistochemical staining. No patient had histories of autoimmune or established connective tissue diseases. The clinical and pathological data of included patients were collected from the hospital electronic medical records system. This study’s protocol received approval from the Institutional Review Board of Hangzhou Red Cross Hospital (No. 2025149-001), with the exemption of written informed consent provided by patients. Surgical procedure All enrolled patients underwent general anesthesia, followed by pericardiectomy conducted via median sternotomy with no cardiopulmonary bypass employed during the procedure. The surgical resection extent covered at least the anterolateral pericardium located between the two phrenic nerves, the basal pericardium on the diaphragmatic surface, the pericardium on the great arteries, and the pericardium extending from the superior vena cava-right atrium junction to the inferior vena cava-right atrium junction. Postoperative Outcomes Postoperative outcomes of all enrolled patients were documented, with postoperative LCOS designated as the primary outcome event. LCOS presented with myocardial dysfunction alongside a cardiac index (CI) < 2.0 L/min/m², systolic blood pressure < 90 mmHg, and confirmed tissue hypoperfusion in patients without hypovolemia [ 16 ] . Immunohistochemistry Pericardial tissue sections were stained with Hematoxylin-Eosin (HE). The paraffin-embedded tissue sections were firstly deparaffinized, followed by antigen retrieval and blocking. The sections were incubated with primary antibody against α-SMA (dilution ratio: 1:1500; #14395-1-AP, ProteinTech), Col-III (dilution ratio: 1:500; #22734-1-AP, ProteinTech), TLR4 (dilution ratio: 1:400; #19811-1-AP, ProteinTech) and HMGB1 (dilution ratio: 1:200; #10829-1-AP, ProteinTech), followed by incubation with a secondary antibody, color development and counterstaining. The procedures were conducted via the Roche Automated Multifunctional Histopathological Detection System (BenchMark XT). The stained sections were examined by two pathologists. All tissue sections after immunohistochemical staining were subjected to image acquisition under the same microscope and camera settings. For each sample, 3 non-overlapping fields of view were randomly selected for analysis. Statistical Analysis The Mean Optical Density (MOD) was used for quantitative analysis of the immunohistochemically stained images. Image-pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was employed, with the same brown-yellow color set as the uniform criterion for determining positivity in all images. For each image, the Integrated Optical Density (IOD) of positive areas and the pixel area (AREA) of the tissue were measured. The MOD value was then calculated using the formula: MOD = IOD/AREA. Categorical data were compared using the chi-square test, the corrected chi-square test or the Fisher exact test, and continuous data using the t test or Mann–Whitney U-test depending on the actual condition. Pearson correlation analysis was performed to evaluate the correlations between TLR4, HMGB1 and key fibrosis-related proteins, respectively. Receiver Operating Characteristic (ROC) curve analysis and the Area Under the Curve (AUC) were used to assess the potential diagnostic value of TLR4 and HMGB1 for postoperative LCOS. Statistical analyses were conducted using SPSS 27.0 software (IBM SPSS Inc., Chicago, IL, USA) and R 4.4.0 software (The R Project for Statistical Computing). All statistical tests were two-tailed, and a P value < 0.05 was considered statistically significant. Results Clinical characteristics A total of 24 patients receiving isolated pericardiectomy were included in this study, and 7 (29.2%) cases had postoperative LCOS. The clinical characteristics were presented in the Table 1. Of the 24 patients, 22 (91.7%) had tuberculosis as the etiological factor, while the remaining 2 (8.3%) were considered to have idiopathic constrictive pericarditis. Pathological and Immunohistochemical Features All patients exhibited fibrotic changes in their pericardial tissue. Among them, abundant collagen fibrous tissue was observed in the tissue sections of patients with idiopathic constrictive pericarditis, whereas massive inflammatory cell infiltration, granulomatous components and coagulative necrosis were noted in those of patients with tuberculous constrictive pericarditis (Figure 1). Immunohistochemical staining showed that α-SMA and Col-III were expressed in the pericardial tissue of all patients. Among these patients, significant differences were observed in the expression levels of α-SMA in pericardial tissue (Figure 2). In contrast, Col-III was abundantly expressed in the pericardial tissue of all patients, with no significant differences noted (Figure 3). Additionally, TLR4 was expressed in 21 (87.5%) specimens, and 2 specimens from patients with idiopathic constrictive pericarditis showed negative expression. Figure 4 showed the expression of TLR4 in different specimens. HMGB1 expression was observed in the pericardial tissue specimens of all patients, and Figure 5 presented the expression of HMGB1 in different specimens. Association Between TLR4, HMGB1 and α-SMA To determine the potential association between the expression of TLR4, HMGB1 and fibrosis, a correlation analysis was further performed to examine the relationship between the expression of TLR4, HMGB1 and key fibrosis-related proteins. Since Col-III was significantly expressed in the pericardial specimens of all patients, the association between TLR4, HMGB1 and α-SMA was analyzed. As shown in Figure 6, the correlation coefficient between TLR4 and α-SMA was 0.529 (P=0.008), while the correlation coefficient between HMGB1 and α-SMA was 0.516 (P=0.010). Additionally, a significant correlation was observed between TLR4 and HMGB1, with a correlation coefficient of 0.844 (P<0.001). Association Between TLR4, HMGB1 and Postoperative LCOS The MOD values of TLR4 and HMGB1 in pericardial tissue were significantly higher in patients who developed postoperative LCOS than in those who did not, with P-values of 0.028 and <0.001, respectively (Figure 7). Further analysis using ROC curves showed that the expression of TLR4 and HMGB1 had potential predictive value for the development of postoperative LCOS. The AUC of TLR4 for predicting postoperative LCOS was 0.790, with 95%CI of 0.516-1.000 (Figure 8A); the AUC of HMGB1 for predicting postoperative LCOS was 0.941, with 95%CI of 0.853-1.000 (Figure 8B). Discussion This study explored the associations of TLR4 and HMGB1 with pericardial fibrosis and the occurrence of postoperative LCOS through immunohistochemical analysis of pericardial tissues and clinical data analysis in 24 patients with constrictive pericarditis. It provided new biological evidence for understanding the disease progression of constrictive pericarditis and the mechanisms underlying postoperative adverse outcomes. Most current studies on constrictive pericarditis have focused on clinical diagnosis and treatment, while basic research on the pathogenesis and progression of the disease remains significantly scarce. Our study confirmed via immunohistochemistry that all patients exhibited marked expression of Col-III and α-SMA in pericardial tissues, with particularly prominent Col-III expression. This indicated that the fibrotic process was widespread in the pericardial tissues of these patients. We found that both TLR4 and HMGB1 were positively correlated with α-SMA expression which was a marker reflecting the activity of fibrosis, suggesting that TLR4 and HMGB1 may be involved in the pericardial fibrotic process in patients with constrictive pericarditis. However, there was no literature reporting which proteins expressed in the pericardial tissue affected cardiac function and postoperative outcomes in constrictive pericarditis. Based on this, we analyzed the associations between TLR4, HMGB1, and relevant clinical events. The expression levels of TLR4 and HMGB1 in pericardial tissues were significantly higher in patients who developed postoperative LCOS than in those who did not, and they showed potential predictive value for the occurrence of postoperative LCOS. Studies have shown that the pericardium plays a unique role in cardiac development: some cells in the pericardium may detach, invade the myocardium, and differentiate into cardiac vascular smooth muscle cells and fibroblasts, thereby playing a crucial role in maintaining myocardial integrity [ 17 ] . As the heart matures, the function of the pericardium stabilizes. However, animal model studies have suggested that myocardial injury could reactivate the pericardium, enabling it to participate in myocardium repair and fibrotic process [ 18 ] . During acute cardiac ischemic injury, activation of the Wnt/β-catenin signaling pathway is observed in the pericardium, and inhibition of this pathway further suppresses myocardial infarction induced fibrosis [ 19 ] . Therefore, the pericardium plays a potentially important role in cardiac development, repair and disease progression, and the crosstalk between the pericardium and myocardium also affects cardiac function to a certain extent [ 20 ] . TLR4 is a core regulator of innate immunity, and a prospective cohort study has found that TLR4 is independently associated with active tuberculosis infection and may be involved in the pathogenesis and progression of tuberculosis [ 21 ] . In our study, TLR4 expression was detected in almost all specimens of tuberculous pericarditis. There was a single-cell RNA sequencing study revealing that activation of TLR4 in fibroblasts was a major driver of cardiac fibrosis, confirming the direct role of TLR4 in regulating fibroblasts [ 22 ] . Additionally, as a hub molecule for inflammatory signaling, TLR4 plays an important role in pericardial-myocardial crosstalk. A study has shown that specific activation of pericardial TLR4 significantly upregulated the expression and secretion of pro-inflammatory cytokines via the NF-κB pathway, and these cytokines not only accumulate in the pericardial cavity but also diffuse to adjacent myocardium, forming a local inflammatory microenvironment [ 23 ] . Induction of myocardial inflammation could promote fibrosis, leading to deterioration of contractile function and other functional impairments [ 24 ] . In our study, the association between TLR4 expression in pericardial tissue and postoperative LCOS might also be attributed to TLR4 mediating pericardial-myocardial crosstalk under inflammatory stimulation, which induced myocardial fibrosis and thereby affected the occurrence of postoperative LCOS. Furthermore, HMGB1 is an important endogenous ligand of TLR4. The signaling pathway initiated by their binding could drive the activation of cardiac fibroblasts and the fibrotic process, and the inhibition of HMGB1 could reduce TLR4 expression and block pro-fibrotic effects [ 25 ] . However, most studies on HMGB1 are limited to cell and animal models. We found that HMGB1 was widely expressed in pericardial specimens from constrictive pericarditis patients, with positive staining observed in the nucleus, cytoplasm and extracellular space. Moreover, HMGB1 expression was significantly positively correlated with TLR4 expression, suggesting that HMGB1 and TLR4 might synergistically participate in pericardial fibrosis and pericardial-myocardial crosstalk, thereby influencing the occurrence of postoperative LCOS. Although this study innovatively analyzed the expression of TLR4 and HMGB1 in the pericardium and their associations with postoperative LCOS, it had several limitations. First, the sample size was small and the study was single-centered, leading to unavoidable biases. Second, while immunohistochemistry could reflect protein expression level, it could not clarify functional mechanisms. Future studies are needed to further verify the specific mechanisms of these proteins in pericardial fibrosis through cell and animal experiments. Additionally, this study did not obtain myocardial specimens, so it was unable to directly confirm the pericardial-myocardial crosstalk in constrictive pericarditis and the specific mechanisms by which TLR4 and HMGB1 expression in the pericardium affected postoperative LCOS. Conclusion This study suggested that TLR4 and HMGB1 might be involved in the process of pericardial fibrosis, and the expressions of TLR4 and HMGB1 were significantly associated with the occurrence of postoperative LCOS and exhibited certain predictive value. These findings offered new insights into understanding the pathological mechanisms of constrictive pericarditis, and also provided potential biomarkers for postoperative risk assessment. Abbreviations LCOS Low cardiac output syndrome TLRs Toll-like receptors α-SMA α-smooth muscle actin HMGB1 High-Mobility Group Box 1 Col-III Collagen III CI cardiac index HE Hematoxylin-Eosin MOD Mean Optical Density IOD Integrated Optical Density Declarations Conflict of interest The authors declare that they have no conflict of interest. Data availability The data supporting the findings of this study are available from the first or the corresponding author upon reasonable request. Funding No funding Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Likui Fang, Tianxiang Wang, Guocan Yu, Fangming Zhong and Weihua Li. The first draft of the manuscript was written by Likui Fang and Weihua Li. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Ethics approval and consent to participate The study protocol was approved by the Institutional Review Board of Hangzhou Red Cross Hospital (No. 2025149-001). Because of the retrospective nature of the study and without any specific intervention, the informed consent has been agreed to be waived. The data were maintained with confidentiality. The present study complied with the Declaration of Helsinki. Clinical trial number Not applicable. Consent for publication Not applicable. References Gillombardo CB, Hoit BD. Constrictive pericarditis in the new millennium. J Cardiol. 2023;S0914–5087(23):00225–3. Hoit BD. Pathophysiology of the Pericardium. Prog Cardiovasc Dis. 2017;59(4):341–8. Wang TKM, Klein AL, Cremer PC, et al. 2025 Concise Clinical Guidance: An ACC Expert Consensus Statement on the Diagnosis and Management of Pericarditis: A Report of the American College of Cardiology Solution Set Oversight Committee. J Am Coll Cardiol. 2025;S0735–1097(25):06503–9. Al-Kazaz M, Klein AL, Oh JK, et al. Pericardial Diseases and Best Practices for Pericardiectomy: JACC State-of-the-Art Review. J Am Coll Cardiol. 2024;84(6):561–80. Mutyaba AK, Balkaran S, Cloete R, et al. Constrictive pericarditis requiring pericardiectomy at Groote Schuur Hospital, Cape Town, South Africa: causes and perioperative outcomes in the HIV era (1990–2012). J Thorac Cardiovasc Surg. 2014;148(6):3058–65. Choi MS, Jeong DS, Oh JK, et al. Long-term results of radical pericardiectomy for constrictive pericarditis in Korean population. J Cardiothorac Surg. 2019;14(1):32. Wang J, Zhang X, Liu X, et al. Predictors of low cardiac output after isolated pericardiectomy: an observational study. Perioper Med (Lond). 2022;11(1):34. O'Neill LA, Golenbock D, Bowie AG. The history of Toll-like receptors - redefining innate immunity. Nat Rev Immunol. 2013;13(6):453–60. Yiu WH, Lin M, Tang SC. Toll-like receptor activation: from renal inflammation to fibrosis. Kidney Int Suppl (2011). 2014;4(1):20 – 5. Bolourani S, Brenner M, Wang P. The interplay of DAMPs, TLR4, and proinflammatory cytokines in pulmonary fibrosis. J Mol Med. 2021;99(10):1373–84. Plikus MV, Wang X, Sinha S, et al. Fibroblasts: Origins, definitions, and functions in health and disease. Cell. 2021;184(15):3852–72. Xiao Y, Qiao W, Wang X, et al. MiR-146a mediates TLR-4 signaling pathway to affect myocardial fibrosis in rat constrictive pericarditis model. J Thorac disease. 2021;13(2):935–45. Li LC, Gao J, Li J. Emerging role of HMGB1 in fibrotic diseases. J Cell Mol Med. 2014;18(12):2331–9. Tang D, Kang R, Zeh HJ, et al. The multifunctional protein HMGB1: 50 years of discovery. Nat Rev Immunol. 2023;23(12):824–41. Jing X, Zhou G, Zhu A, et al. RG-I pectin-like polysaccharide from Rosa chinensis inhibits inflammation and fibrosis associated to HMGB1/TLR4/NF-kappaB signaling pathway to improve non-alcoholic steatohepatitis. Carbohydr Polym. 2024;337:122139. Lomivorotov VV, Efremov SM, Kirov MY, et al. Low-Cardiac-Output Syndrome After Cardiac Surgery. J Cardiothorac Vasc Anesth. 2017;31(1):291–308. Zhou B, Ma Q, Rajagopal S, et al. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature. 2008;454(7200):109–13. Cao J, Poss KD. The epicardium as a hub for heart regeneration. Nat reviews Cardiol. 2018;15(10):631–47. Duan J, Gherghe C, Liu D, et al. Wnt1/betacatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair. EMBO J. 2012;31(2):429–42. Fang M, Xiang FL, Braitsch CM, et al. Epicardium-derived fibroblasts in heart development and disease. J Mol Cell Cardiol. 2016;91:23–7. Cubillos-Angulo JM, Arriaga MB, Silva EC, et al. Polymorphisms in TLR4 and TNFA and Risk of Mycobacterium tuberculosis Infection and Development of Active Disease in Contacts of Tuberculosis Cases in Brazil: A Prospective Cohort Study. Clin Infect Dis. 2019;69(6):1027–35. Zhang H, Thai PN, Shivnaraine RV, et al. Multiscale drug screening for cardiac fibrosis identifies MD2 as a therapeutic target. Cell. 2024;187(25):7143–e6322. Lin FJ, Li SJ, Lu YY, et al. Toll-like receptor 4 activation modulates pericardium-myocardium interactions in lipopolysaccharide-induced atrial arrhythmogenesis. Europace. 2021;23(11):1837–46. Kaya Z, Goser S, Buss SJ, et al. Identification of cardiac troponin I sequence motifs leading to heart failure by induction of myocardial inflammation and fibrosis. Circulation. 2008;118(20):2063–72. Ni SY, Zhong XL, Li ZH, et al. Puerarin Alleviates Lipopolysaccharide-Induced Myocardial Fibrosis by Inhibiting PARP-1 to Prevent HMGB1-Mediated TLR4-NF-kappaB Signaling Pathway. Cardiovasc Toxicol. 2020;20(5):482–91. Table Table 1. Clinical characteristics of the study population Variables N=24 Age，years 65.5 (18.0-83.0) Sex Male Female 18 (75.0%) 6 (25.0%) Etiology Tuberculosis Idiopathic 22 (91.7%) 2 (8.3%) NYHA functional class I II III IV 0 (0%) 10 (41.7%) 11 (45.8%) 3 (12.5%) BMI, kg/m2 21.7 (15.6-31.5) CVP, cmH2O 24.0 (16.0-36.4) Pericardial thickness, mm 8.6 (3.5-18.4) LVEF, % 58.3 (42.0-68.6) Postoperative LCOS 7 (29.2%) Notes: Values presented as N (percentage) for categorical variables and median (range) for continuous variables. Abbreviations: NYHA, New York Heart Association; BMI, body mass index; CVP, central venous pressure; LVEF, left ventricular ejection fraction (measured on echocardiogram). Additional Declarations No competing interests reported. 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14:45:25","extension":"xml","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65254,"visible":true,"origin":"","legend":"","description":"","filename":"52bba266093248419a0bec35efbb5fb31structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8370586/v1/aef1af73b3f0a973d68f1e1a.xml"},{"id":100599780,"identity":"51adcb8a-003a-41bf-bd53-1007d44dc942","added_by":"auto","created_at":"2026-01-19 14:44:59","extension":"html","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":73355,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8370586/v1/bb4018b297d79dece17090b5.html"},{"id":100599823,"identity":"10bc15ab-d3aa-4c09-87fd-9bdda352d295","added_by":"auto","created_at":"2026-01-19 14:45:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3468584,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin-Eosin (HE) Stained Sections of Patients with Chronic Constrictive Pericarditis. Panels A and B: Microscopic findings of pericardial tissue sections from patients with nonspecific pericarditis (Scale bars = 500 μm for both); Panels C and D: Microscopic findings of pericardial tissue sections from patients with tuberculous pericarditis (Scale bars = 1 mm and 250 μm, respectively).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8370586/v1/d25bb676a10a60b0ec49ee03.png"},{"id":100599832,"identity":"45d4b860-82b5-4c76-a44f-01a099706a24","added_by":"auto","created_at":"2026-01-19 14:45:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3227784,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of α-SMA in Pericardial Tissue from Different Patients (Scale bars = 100 μm for all panels).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8370586/v1/efffdddaee6ff11fa9654d72.png"},{"id":100599781,"identity":"04d3f46c-1594-47a1-a4f5-a995488d7836","added_by":"auto","created_at":"2026-01-19 14:44:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3602033,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of Col-III in Pericardial Tissue from Different Patients (Scale bars = 100 μm for all panels).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8370586/v1/cffd595b7e69f25322f78d6c.png"},{"id":100600134,"identity":"8ea94f30-a8d1-4a31-89ed-d05bac2bef8e","added_by":"auto","created_at":"2026-01-19 14:46:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3411132,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of TLR4 in Pericardial Tissue from Different Patients (Scale bars = 100 μm for all panels).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8370586/v1/0bdf4f9c29a26cb74c6e2127.png"},{"id":100600050,"identity":"87d745d1-1c4f-4ae2-84fb-3219eebce4d6","added_by":"auto","created_at":"2026-01-19 14:46:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3468650,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of HMGB1 in Pericardial Tissue from Different Patients (Scale bars = 100 μm for all panels).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8370586/v1/ca7c7892551a739274ee7fb6.png"},{"id":100599773,"identity":"66baa044-44d4-43f2-8fab-1461c637c78f","added_by":"auto","created_at":"2026-01-19 14:44:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":165453,"visible":true,"origin":"","legend":"\u003cp\u003eAssociation Between TLR4, HMGB1 and α-SMA.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8370586/v1/72b6d695006d712bc3ac1f61.png"},{"id":100599783,"identity":"04f7594c-4f25-49b8-8fef-ce54719f1832","added_by":"auto","created_at":"2026-01-19 14:45:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":145242,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of TLR4 and HMGB1 in Patients with and without Postoperative LCOS.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8370586/v1/ccfefb6a21aa47ca2b355af2.png"},{"id":100599926,"identity":"af179c93-26ab-4410-bb1d-ceea5f2c0ef6","added_by":"auto","created_at":"2026-01-19 14:45:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":752880,"visible":true,"origin":"","legend":"\u003cp\u003eROC Curve of TLR4 (A) and HMGB1 (B) for Predicting Postoperative LCOS.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8370586/v1/c6adbd516430d709e8ef8738.png"},{"id":105576456,"identity":"70a01f4e-5a7c-4543-97c9-6b66e4f3b0ae","added_by":"auto","created_at":"2026-03-27 13:44:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17241796,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8370586/v1/4ff342f8-765c-48a5-bb76-59cd370ee6ae.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The role of HMGB1/TLR4 in pericardial fibrosis and postoperative low cardiac output syndrome in constrictive pericarditis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eConstrictive pericarditis is a rare but life-threatening disease, and the most common cause is tuberculosis worldwide\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Fibrosis is the core pathological feature of constrictive pericarditis which ultimately results in diastolic heart failure\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Pericardiectomy is the only definitive treatment option but is accompanied with high perioperative risk\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Low cardiac output syndrome (LCOS) is the main postoperative complication and a major cause of postoperative mortality\u003csup\u003e[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. At present, it has remained unclear about the factors that are involved in the pericardial fibrotic process and the occurrence of postoperative LCOS.\u003c/p\u003e \u003cp\u003eToll-like receptors (TLRs) are a class of transmembrane structural proteins that play a crucial role in the innate immune system\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that they may participate in the activation of fibroblasts, and among them, TLR4 has been proven to exert a significant pro-fibrotic effect\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. In rat constrictive pericarditis model, activation of the TLR4 signaling pathway stimulates the expression of α-smooth muscle actin (α-SMA) and promotes the process of myocardial fibrosis\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. High-Mobility Group Box 1 (HMGB1) is a highly conserved nuclear protein that is widely expressed in nearly all cell types, and a growing body of research has shown that HMGB1 is closely associated with various fibrotic diseases\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. TLR4 is one of the most extensively studied receptors for HMGB1, and inhibiting the expression of HMGB1 could inactivate the HMGB1/TLR4 pathway to impede the development of fibrosis\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, the expression patterns and functional roles of TLR4 and HMGB1 in the pericardial tissue of patients with constrictive pericarditis have remained unclear. We aimed to analyze the expression of TLR4 and HMGB1 in pericardial tissue, the associations between their expression and that of key fibrotic proteins, α-SMA and collagen III (Col-III), as well as their impact on the development of postoperative LCOS in patients with constrictive pericarditis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy population\u003c/h2\u003e \u003cp\u003eA retrospective analysis was conducted on the clinical records of patients diagnosed with constrictive pericarditis who were treated in our department between May 2023 and April 2025. Pericardial tissues surgically resected from 24 eligible patients were selected for immunohistochemical staining. No patient had histories of autoimmune or established connective tissue diseases. The clinical and pathological data of included patients were collected from the hospital electronic medical records system. This study\u0026rsquo;s protocol received approval from the Institutional Review Board of Hangzhou Red Cross Hospital (No. 2025149-001), with the exemption of written informed consent provided by patients.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSurgical procedure\u003c/h3\u003e\n\u003cp\u003eAll enrolled patients underwent general anesthesia, followed by pericardiectomy conducted via median sternotomy with no cardiopulmonary bypass employed during the procedure. The surgical resection extent covered at least the anterolateral pericardium located between the two phrenic nerves, the basal pericardium on the diaphragmatic surface, the pericardium on the great arteries, and the pericardium extending from the superior vena cava-right atrium junction to the inferior vena cava-right atrium junction.\u003c/p\u003e\n\u003ch3\u003ePostoperative Outcomes\u003c/h3\u003e\n\u003cp\u003ePostoperative outcomes of all enrolled patients were documented, with postoperative LCOS designated as the primary outcome event. LCOS presented with myocardial dysfunction alongside a cardiac index (CI)\u0026thinsp;\u0026lt;\u0026thinsp;2.0 L/min/m\u0026sup2;, systolic blood pressure\u0026thinsp;\u0026lt;\u0026thinsp;90 mmHg, and confirmed tissue hypoperfusion in patients without hypovolemia\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003ePericardial tissue sections were stained with Hematoxylin-Eosin (HE). The paraffin-embedded tissue sections were firstly deparaffinized, followed by antigen retrieval and blocking. The sections were incubated with primary antibody against α-SMA (dilution ratio: 1:1500; #14395-1-AP, ProteinTech), Col-III (dilution ratio: 1:500; #22734-1-AP, ProteinTech), TLR4 (dilution ratio: 1:400; #19811-1-AP, ProteinTech) and HMGB1 (dilution ratio: 1:200; #10829-1-AP, ProteinTech), followed by incubation with a secondary antibody, color development and counterstaining. The procedures were conducted via the Roche Automated Multifunctional Histopathological Detection System (BenchMark XT). The stained sections were examined by two pathologists. All tissue sections after immunohistochemical staining were subjected to image acquisition under the same microscope and camera settings. For each sample, 3 non-overlapping fields of view were randomly selected for analysis.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe Mean Optical Density (MOD) was used for quantitative analysis of the immunohistochemically stained images. Image-pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was employed, with the same brown-yellow color set as the uniform criterion for determining positivity in all images. For each image, the Integrated Optical Density (IOD) of positive areas and the pixel area (AREA) of the tissue were measured. The MOD value was then calculated using the formula: MOD\u0026thinsp;=\u0026thinsp;IOD/AREA. Categorical data were compared using the chi-square test, the corrected chi-square test or the Fisher exact test, and continuous data using the t test or Mann\u0026ndash;Whitney U-test depending on the actual condition. Pearson correlation analysis was performed to evaluate the correlations between TLR4, HMGB1 and key fibrosis-related proteins, respectively. Receiver Operating Characteristic (ROC) curve analysis and the Area Under the Curve (AUC) were used to assess the potential diagnostic value of TLR4 and HMGB1 for postoperative LCOS. Statistical analyses were conducted using SPSS 27.0 software (IBM SPSS Inc., Chicago, IL, USA) and R 4.4.0 software (The R Project for Statistical Computing). All statistical tests were two-tailed, and a P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eClinical characteristics\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA total of 24 patients receiving isolated pericardiectomy were included in this study, and 7 (29.2%) cases had postoperative LCOS. The clinical characteristics were presented in the Table 1. Of the 24 patients, 22 (91.7%) had tuberculosis as the etiological factor, while the remaining 2 (8.3%) were considered to have idiopathic constrictive pericarditis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePathological and Immunohistochemical Features\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll patients exhibited fibrotic changes in their pericardial tissue. Among them, abundant collagen fibrous tissue was observed in the tissue sections of patients with idiopathic constrictive pericarditis, whereas massive inflammatory cell infiltration, granulomatous components and coagulative necrosis were noted in those of patients with tuberculous constrictive pericarditis (Figure 1).\u003c/p\u003e\n\u003cp\u003eImmunohistochemical staining showed that α-SMA and Col-III were expressed in the pericardial tissue of all patients. Among these patients, significant differences were observed in the expression levels of α-SMA in pericardial tissue (Figure 2). In contrast, Col-III was abundantly expressed in the pericardial tissue of all patients, with no significant differences noted (Figure 3).\u003c/p\u003e\n\u003cp\u003eAdditionally, TLR4 was expressed in 21 (87.5%) specimens, and 2 specimens from patients with idiopathic constrictive pericarditis showed negative expression. Figure 4 showed the expression of TLR4 in different specimens. HMGB1 expression was observed in the pericardial tissue specimens of all patients, and Figure 5 presented the expression of HMGB1 in different specimens.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAssociation Between TLR4, HMGB1 and α-SMA\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the potential association between the expression of TLR4, HMGB1 and fibrosis, a correlation analysis was further performed to examine the relationship between the expression of TLR4, HMGB1 and key fibrosis-related proteins. Since Col-III was significantly expressed in the pericardial specimens of all patients, the association between TLR4, HMGB1 and α-SMA was analyzed.\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 6, the correlation coefficient between TLR4 and α-SMA was 0.529 (P=0.008), while the correlation coefficient between HMGB1 and α-SMA was 0.516 (P=0.010). Additionally, a significant correlation was observed between TLR4 and HMGB1, with a correlation coefficient of 0.844 (P\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAssociation Between TLR4, HMGB1 and Postoperative LCOS\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe MOD values of TLR4 and HMGB1 in pericardial tissue were significantly higher in patients who developed postoperative LCOS than in those who did not, with P-values of 0.028 and \u0026lt;0.001, respectively (Figure 7). Further analysis using ROC curves showed that the expression of TLR4 and HMGB1 had potential predictive value for the development of postoperative LCOS. The AUC of TLR4 for predicting postoperative LCOS was 0.790, with 95%CI of 0.516-1.000 (Figure 8A); the AUC of HMGB1 for predicting postoperative LCOS was 0.941, with 95%CI of 0.853-1.000 (Figure 8B).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study explored the associations of TLR4 and HMGB1 with pericardial fibrosis and the occurrence of postoperative LCOS through immunohistochemical analysis of pericardial tissues and clinical data analysis in 24 patients with constrictive pericarditis. It provided new biological evidence for understanding the disease progression of constrictive pericarditis and the mechanisms underlying postoperative adverse outcomes.\u003c/p\u003e \u003cp\u003eMost current studies on constrictive pericarditis have focused on clinical diagnosis and treatment, while basic research on the pathogenesis and progression of the disease remains significantly scarce. Our study confirmed via immunohistochemistry that all patients exhibited marked expression of Col-III and α-SMA in pericardial tissues, with particularly prominent Col-III expression. This indicated that the fibrotic process was widespread in the pericardial tissues of these patients. We found that both TLR4 and HMGB1 were positively correlated with α-SMA expression which was a marker reflecting the activity of fibrosis, suggesting that TLR4 and HMGB1 may be involved in the pericardial fibrotic process in patients with constrictive pericarditis. However, there was no literature reporting which proteins expressed in the pericardial tissue affected cardiac function and postoperative outcomes in constrictive pericarditis. Based on this, we analyzed the associations between TLR4, HMGB1, and relevant clinical events. The expression levels of TLR4 and HMGB1 in pericardial tissues were significantly higher in patients who developed postoperative LCOS than in those who did not, and they showed potential predictive value for the occurrence of postoperative LCOS.\u003c/p\u003e \u003cp\u003eStudies have shown that the pericardium plays a unique role in cardiac development: some cells in the pericardium may detach, invade the myocardium, and differentiate into cardiac vascular smooth muscle cells and fibroblasts, thereby playing a crucial role in maintaining myocardial integrity\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. As the heart matures, the function of the pericardium stabilizes. However, animal model studies have suggested that myocardial injury could reactivate the pericardium, enabling it to participate in myocardium repair and fibrotic process\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. During acute cardiac ischemic injury, activation of the Wnt/β-catenin signaling pathway is observed in the pericardium, and inhibition of this pathway further suppresses myocardial infarction induced fibrosis\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Therefore, the pericardium plays a potentially important role in cardiac development, repair and disease progression, and the crosstalk between the pericardium and myocardium also affects cardiac function to a certain extent\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTLR4 is a core regulator of innate immunity, and a prospective cohort study has found that TLR4 is independently associated with active tuberculosis infection and may be involved in the pathogenesis and progression of tuberculosis\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. In our study, TLR4 expression was detected in almost all specimens of tuberculous pericarditis. There was a single-cell RNA sequencing study revealing that activation of TLR4 in fibroblasts was a major driver of cardiac fibrosis, confirming the direct role of TLR4 in regulating fibroblasts\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Additionally, as a hub molecule for inflammatory signaling, TLR4 plays an important role in pericardial-myocardial crosstalk. A study has shown that specific activation of pericardial TLR4 significantly upregulated the expression and secretion of pro-inflammatory cytokines via the NF-κB pathway, and these cytokines not only accumulate in the pericardial cavity but also diffuse to adjacent myocardium, forming a local inflammatory microenvironment\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Induction of myocardial inflammation could promote fibrosis, leading to deterioration of contractile function and other functional impairments\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. In our study, the association between TLR4 expression in pericardial tissue and postoperative LCOS might also be attributed to TLR4 mediating pericardial-myocardial crosstalk under inflammatory stimulation, which induced myocardial fibrosis and thereby affected the occurrence of postoperative LCOS.\u003c/p\u003e \u003cp\u003eFurthermore, HMGB1 is an important endogenous ligand of TLR4. The signaling pathway initiated by their binding could drive the activation of cardiac fibroblasts and the fibrotic process, and the inhibition of HMGB1 could reduce TLR4 expression and block pro-fibrotic effects\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. However, most studies on HMGB1 are limited to cell and animal models. We found that HMGB1 was widely expressed in pericardial specimens from constrictive pericarditis patients, with positive staining observed in the nucleus, cytoplasm and extracellular space. Moreover, HMGB1 expression was significantly positively correlated with TLR4 expression, suggesting that HMGB1 and TLR4 might synergistically participate in pericardial fibrosis and pericardial-myocardial crosstalk, thereby influencing the occurrence of postoperative LCOS.\u003c/p\u003e \u003cp\u003eAlthough this study innovatively analyzed the expression of TLR4 and HMGB1 in the pericardium and their associations with postoperative LCOS, it had several limitations. First, the sample size was small and the study was single-centered, leading to unavoidable biases. Second, while immunohistochemistry could reflect protein expression level, it could not clarify functional mechanisms. Future studies are needed to further verify the specific mechanisms of these proteins in pericardial fibrosis through cell and animal experiments. Additionally, this study did not obtain myocardial specimens, so it was unable to directly confirm the pericardial-myocardial crosstalk in constrictive pericarditis and the specific mechanisms by which TLR4 and HMGB1 expression in the pericardium affected postoperative LCOS.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study suggested that TLR4 and HMGB1 might be involved in the process of pericardial fibrosis, and the expressions of TLR4 and HMGB1 were significantly associated with the occurrence of postoperative LCOS and exhibited certain predictive value. These findings offered new insights into understanding the pathological mechanisms of constrictive pericarditis, and also provided potential biomarkers for postoperative risk assessment.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLCOS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLow cardiac output syndrome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTLRs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eToll-like receptors\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eα-SMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eα-smooth muscle actin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHMGB1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh-Mobility Group Box 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCol-III\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCollagen III\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecardiac index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematoxylin-Eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMean Optical Density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIntegrated Optical Density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the first or the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Likui Fang, Tianxiang Wang, Guocan Yu, Fangming Zhong and Weihua Li. The first draft of the manuscript was written by Likui Fang and Weihua Li. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the Institutional Review Board of Hangzhou Red Cross Hospital (No. 2025149-001). Because of the retrospective nature of the study and without any specific intervention, the informed consent has been agreed to be waived. The data were maintained with confidentiality. The present study complied with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGillombardo CB, Hoit BD. Constrictive pericarditis in the new millennium. J Cardiol. 2023;S0914\u0026ndash;5087(23):00225\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoit BD. Pathophysiology of the Pericardium. 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Nature. 2008;454(7200):109\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao J, Poss KD. The epicardium as a hub for heart regeneration. Nat reviews Cardiol. 2018;15(10):631\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuan J, Gherghe C, Liu D, et al. Wnt1/betacatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair. EMBO J. 2012;31(2):429\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang M, Xiang FL, Braitsch CM, et al. Epicardium-derived fibroblasts in heart development and disease. J Mol Cell Cardiol. 2016;91:23\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCubillos-Angulo JM, Arriaga MB, Silva EC, et al. Polymorphisms in TLR4 and TNFA and Risk of Mycobacterium tuberculosis Infection and Development of Active Disease in Contacts of Tuberculosis Cases in Brazil: A Prospective Cohort Study. Clin Infect Dis. 2019;69(6):1027\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Thai PN, Shivnaraine RV, et al. Multiscale drug screening for cardiac fibrosis identifies MD2 as a therapeutic target. Cell. 2024;187(25):7143\u0026ndash;e6322.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin FJ, Li SJ, Lu YY, et al. Toll-like receptor 4 activation modulates pericardium-myocardium interactions in lipopolysaccharide-induced atrial arrhythmogenesis. Europace. 2021;23(11):1837\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaya Z, Goser S, Buss SJ, et al. Identification of cardiac troponin I sequence motifs leading to heart failure by induction of myocardial inflammation and fibrosis. Circulation. 2008;118(20):2063\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNi SY, Zhong XL, Li ZH, et al. Puerarin Alleviates Lipopolysaccharide-Induced Myocardial Fibrosis by Inhibiting PARP-1 to Prevent HMGB1-Mediated TLR4-NF-kappaB Signaling Pathway. Cardiovasc Toxicol. 2020;20(5):482\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1. Clinical characteristics of the study population\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"391\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 239px;\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003eN=24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 239px;\"\u003e\n \u003cp\u003eAge，years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e65.5 (18.0-83.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 239px;\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e18 (75.0%)\u003c/p\u003e\n \u003cp\u003e6 (25.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 239px;\"\u003e\n \u003cp\u003eEtiology\u003c/p\u003e\n \u003cp\u003eTuberculosis\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eIdiopathic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e22 (91.7%)\u003c/p\u003e\n \u003cp\u003e2 (8.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 239px;\"\u003e\n \u003cp\u003eNYHA functional class\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003cp\u003eII\u003c/p\u003e\n \u003cp\u003eIII\u003c/p\u003e\n \u003cp\u003eIV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003cp\u003e10 (41.7%)\u003c/p\u003e\n \u003cp\u003e11 (45.8%)\u003c/p\u003e\n \u003cp\u003e3 (12.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 239px;\"\u003e\n \u003cp\u003eBMI, kg/m2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e21.7 (15.6-31.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 239px;\"\u003e\n \u003cp\u003eCVP, cmH2O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e24.0 (16.0-36.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 239px;\"\u003e\n \u003cp\u003ePericardial thickness, mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e8.6 (3.5-18.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 239px;\"\u003e\n \u003cp\u003eLVEF, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e58.3 (42.0-68.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 239px;\"\u003e\n \u003cp\u003ePostoperative LCOS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e7 (29.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNotes: Values presented as N (percentage) for categorical variables and median (range) for continuous variables.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAbbreviations: NYHA, New York Heart Association; BMI, body mass index; CVP, central venous pressure; LVEF, left ventricular ejection fraction (measured on echocardiogram).\u003c/p\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":"constrictive pericarditis, low cardiac output syndrome, TLR4, HMGB1","lastPublishedDoi":"10.21203/rs.3.rs-8370586/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8370586/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIt has remained unclear about the factors that are involved in the pericardial fibrotic process and the occurrence of postoperative low cardiac output syndrome (LCOS) in constrictive pericarditis. This study aimed to analyze the role of TLR4 and HMGB1 in pericardial fibrosis, and their impact on the development of postoperative LCOS in patients with constrictive pericarditis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis retrospective study enrolled 24 constrictive pericarditis patients who underwent isolated pericardiectomy at our department from May 2023 to April 2025. Pericardial tissues were subjected to immunohistochemistry to detect the expression of TLR4, HMGB1, α-SMA and collagen III. Mean Optical Density (MOD) was used for quantitative analysis of immunohistochemical staining. Pearson correlation analysis was performed to assess associations between TLR4, HMGB1 and fibrotic markers, while Receiver Operating Characteristic (ROC) curves were used to evaluate their predictive value for postoperative LCOS.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOf the 24 patients, 7 (29.2%) developed postoperative LCOS. TLR4 was expressed in 21 (87.5%) specimens and HMGB1 was expressed in all specimens. Pearson correlation analysis showed positive correlations between TLR4 and α-SMA (r\u0026thinsp;=\u0026thinsp;0.529, P\u0026thinsp;=\u0026thinsp;0.008), HMGB1 and α-SMA (r\u0026thinsp;=\u0026thinsp;0.516, P\u0026thinsp;=\u0026thinsp;0.010), and TLR4 and HMGB1 (r\u0026thinsp;=\u0026thinsp;0.844, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). MOD values of TLR4 and HMGB1 were significantly higher in patients with postoperative LCOS (P\u0026thinsp;=\u0026thinsp;0.028 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). ROC curve suggested that TLR4 (AUC\u0026thinsp;=\u0026thinsp;0.790, 95%CI: 0.516\u0026ndash;1.000) and HMGB1 (AUC\u0026thinsp;=\u0026thinsp;0.941, 95%CI: 0.853\u0026ndash;1.000) had potential predictive value for postoperative LCOS.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eTLR4 and HMGB1 were involved in the pericardial fibrosis and were significantly associated with the occurrence of postoperative LCOS in constrictive pericarditis.\u003c/p\u003e","manuscriptTitle":"The role of HMGB1/TLR4 in pericardial fibrosis and postoperative low cardiac output syndrome in constrictive pericarditis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 13:35:46","doi":"10.21203/rs.3.rs-8370586/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":"31572d50-416f-40fe-8fc8-2ae6e566e372","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-27T13:39:25+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-19 13:35:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8370586","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8370586","identity":"rs-8370586","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}