{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T14:54:11Z","timestamp":1773068051122,"version":"3.50.1"},"reference-count":55,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,3,7]],"date-time":"2026-03-07T00:00:00Z","timestamp":1772841600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"JSPS KAKENHI","award":["23K08620"],"award-info":[{"award-number":["23K08620"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Hepatic encephalopathy is a severe complication of liver failure, traditionally investigated through brain\u2013liver interactions; however, the involvement of extrahepatic organs remains poorly understood. This study examined splenic histopathological changes in a mouse model of acute hepatic encephalopathy induced by ammonium acetate administration, focusing on iron metabolism and macrophage activation. Although conventional hematoxylin and eosin staining revealed no overt structural abnormalities, unstained spleen sections demonstrated abundant black deposits, predominantly in the red pulp. Prussian blue staining confirmed a significant increase in hemosiderin-positive cells; however, a subset of black deposits was iron-negative. Immunohistochemical analyses revealed that these iron-negative pigments were localized within F4\/80-positive macrophages and colocalized with heme oxygenase-1 (HO-1), suggesting enhanced heme degradation. Ultrastructural observations further identified electron-dense granules consistent with hematin accumulation in splenic macrophages. Hematological analyses revealed significant reductions in red blood cell count and hemoglobin levels, indicating accelerated erythrocyte destruction. Collectively, these findings demonstrate that acute hepatic encephalopathy induces splenic macrophage activation, accompanied by disordered iron metabolism and hematin accumulation. This study highlights the spleen as a previously underappreciated extrahepatic organ involved in the pathophysiology of hepatic encephalopathy and suggests that splenic heme\u2013iron handling may represent a novel therapeutic target.<\/jats:p>","DOI":"10.3390\/ijms27052463","type":"journal-article","created":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T10:16:12Z","timestamp":1773051372000},"page":"2463","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Splenic Macrophage Activation and Disordered Heme\u2013Iron Metabolism in a Mouse Model of Acute Hepatic Encephalopathy"],"prefix":"10.3390","volume":"27","author":[{"given":"Kanako","family":"Tadokoro","sequence":"first","affiliation":[{"name":"Department of Functional Morphology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose 204-8588, Tokyo, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nozomi","family":"Ito","sequence":"additional","affiliation":[{"name":"Department of Functional Morphology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose 204-8588, Tokyo, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Riku","family":"Terashima","sequence":"additional","affiliation":[{"name":"Department of Functional Morphology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose 204-8588, Tokyo, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kairi","family":"Horigome","sequence":"additional","affiliation":[{"name":"Department of Functional Morphology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose 204-8588, Tokyo, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kiyoharu","family":"Kawakami","sequence":"additional","affiliation":[{"name":"Department of Functional Morphology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose 204-8588, Tokyo, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6717-7167","authenticated-orcid":false,"given":"Kazuhiko","family":"Nakadate","sequence":"additional","affiliation":[{"name":"Department of Functional Morphology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose 204-8588, Tokyo, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1967","DOI":"10.1007\/s00198-016-3512-z","article-title":"Osteoporosis across chronic liver disease","volume":"27","author":"Guarino","year":"2016","journal-title":"Osteoporos. Int."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1097\/MOG.0000000000000527","article-title":"Updates on the pathophysiology and therapeutic targets for hepatic encephalopathy","volume":"35","author":"Alsahhar","year":"2019","journal-title":"Curr. Opin. Gastroenterol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"17","DOI":"10.4254\/wjh.v16.i1.17","article-title":"Role of fecal microbiota transplant in management of hepatic encephalopathy: Current trends and future directions","volume":"16","author":"Shah","year":"2024","journal-title":"World J. Hepatol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Cheon, S.Y., and Song, J. (2021). The Association between Hepatic Encephalopathy and Diabetic Encephalopathy: The Brain-Liver Axis. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22010463"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2017","DOI":"10.14309\/ajg.0000000000000762","article-title":"Incidence and Bedside Predictors of the First Episode of Overt Hepatic Encephalopathy in Patients With Cirrhosis","volume":"115","author":"Tapper","year":"2020","journal-title":"Am. J. Gastroenterol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1007\/s12664-024-01520-6","article-title":"Pediatric acute liver failure: Current perspective in etiology and management","volume":"43","author":"Samanta","year":"2024","journal-title":"Indian. J. Gastroenterol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1689","DOI":"10.1007\/s11011-024-01402-y","article-title":"Infection, inflammation and hepatic encephalopathy from a clinical perspective","volume":"39","author":"Ntuli","year":"2024","journal-title":"Metab. Brain Dis."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Sepehrinezhad, A., Moghaddam, N.G., Shayan, N., and Sahab Negah, S. (2024). Correlation of ammonia and blood laboratory parameters with hepatic encephalopathy: A systematic review and meta-analysis. PLoS ONE, 19.","DOI":"10.1371\/journal.pone.0307899"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Gandolfi, D., Boiani, G.M., Bigiani, A., and Mapelli, J. (2021). Modeling Neurotransmission: Computational Tools to Investigate Neurological Disorders. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22094565"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Nakadate, K., and Kamata, S. (2022). Severe Acute Hepatic Dysfunction Induced by Ammonium Acetate Treatment Results in Choroid Plexus Swelling and Ventricle Enlargement in the Brain. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23042010"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Nakadate, K., Sono, C., Mita, H., Itakura, Y., and Kawakami, K. (2023). Severe Acute Liver Dysfunction Induces Delayed Hepatocyte Swelling and Cytoplasmic Vacuolization, and Delayed Cortical Neuronal Cell Death. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24087351"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1765","DOI":"10.1007\/s00270-018-1992-2","article-title":"Refractory Hepatic Encephalopathy After Elective Transjugular Intrahepatic Portosystemic Shunt: Risk Factors and Outcomes with Revision","volume":"41","author":"Rowley","year":"2018","journal-title":"Cardiovasc. Interv. Radiol."},{"key":"ref_13","first-page":"142","article-title":"Hepatic Encephalopathy: An Update on the Pathophysiology and Therapeutic Options","volume":"5","author":"Elwir","year":"2017","journal-title":"J. Clin. Transl. Hepatol."},{"key":"ref_14","first-page":"177","article-title":"Acute-on-chronic liver failure: The brain","volume":"17","author":"Cordoba","year":"2011","journal-title":"Curr. Opin. Crit. Care"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1001\/archsurg.133.1.56","article-title":"Residual splenic function after laparoscopic splenectomy: A clinical concern","volume":"133","author":"Targarona","year":"1998","journal-title":"Arch. Surg."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1038\/s41577-023-00857-x","article-title":"Splenic stromal niches in homeostasis and immunity","volume":"23","author":"Alexandre","year":"2023","journal-title":"Nat. Rev. Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"F1000 Faculty Rev-533","DOI":"10.12688\/f1000research.13943.1","article-title":"Managing portal hypertension in patients with liver cirrhosis","volume":"7","author":"Sauerbruch","year":"2018","journal-title":"F1000Research"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1007\/s00428-018-2355-8","article-title":"Pathology of idiopathic non-cirrhotic portal hypertension","volume":"473","author":"Guido","year":"2018","journal-title":"Virchows Arch."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"558","DOI":"10.1177\/019262339602400504","article-title":"Histopathological evaluation of liver, pancreas, spleen, and heart from iron-overloaded Sprague-Dawley rats","volume":"24","author":"Whittaker","year":"1996","journal-title":"Toxicol. Pathol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"15515","DOI":"10.1038\/s41598-023-42760-4","article-title":"Oxidative stress induces mitochondrial iron overload and ferroptotic cell death","volume":"13","author":"Chen","year":"2023","journal-title":"Sci. Rep."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"612","DOI":"10.1084\/jem.14.6.612","article-title":"The Relation of Hematin to Pathological Pigment Formation","volume":"14","author":"Brown","year":"1911","journal-title":"J. Exp. Med."},{"key":"ref_22","first-page":"436","article-title":"Formalin pigment (acid hematin) and related pigments","volume":"42","author":"Pizzolato","year":"1976","journal-title":"Am. J. Med. Technol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Bozza, M.T., and Jeney, V. (2020). Pro-inflammatory Actions of Heme and Other Hemoglobin-Derived DAMPs. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.01323"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Recalcati, S., Gammella, E., and Cairo, G. (2019). Ironing out Macrophage Immunometabolism. Pharmaceuticals, 12.","DOI":"10.3390\/ph12020094"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2303","DOI":"10.1007\/s12011-022-03362-9","article-title":"Accumulation of Intracellular Ferrous Iron in Inflammatory-Activated Macrophages","volume":"201","author":"Ma","year":"2023","journal-title":"Biol. Trace Elem. Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"895","DOI":"10.1016\/j.jhep.2024.06.016","article-title":"Inflammasomes in chronic liver disease: Hepatic injury, fibrosis progression and systemic inflammation","volume":"81","author":"Taru","year":"2024","journal-title":"J. Hepatol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1861","DOI":"10.3748\/wjg.v26.i16.1861","article-title":"Macrophages in metabolic associated fatty liver disease","volume":"26","author":"Alharthi","year":"2020","journal-title":"World J. Gastroenterol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1038\/s41418-024-01266-w","article-title":"SLC15A3 plays a crucial role in pulmonary fibrosis by regulating macrophage oxidative stress","volume":"31","author":"Luo","year":"2024","journal-title":"Cell Death Differ."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1080\/08958370701496103","article-title":"A comparison of the production of reactive oxygen species by suspended particulate matter and diesel exhaust particles with macrophages","volume":"19","author":"Ohyama","year":"2007","journal-title":"Inhal. Toxicol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"453","DOI":"10.3904\/kjim.2017.317","article-title":"Role of the renin-angiotensin system in hepatic fibrosis and portal hypertension","volume":"33","author":"Shim","year":"2018","journal-title":"Korean J. Intern. Med."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Liu, H., Nguyen, H.H., Hwang, S.Y., and Lee, S.S. (2023). Oxidative Mechanisms and Cardiovascular Abnormalities of Cirrhosis and Portal Hypertension. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms242316805"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Mikhailova, D.M., Skverchinskaya, E., Sudnitsyna, J., Butov, K.R., Koltsova, E.M., Mindukshev, I.V., and Gambaryan, S. (2024). Hematin- and Hemin-Induced Spherization and Hemolysis of Human Erythrocytes Are Independent of Extracellular Calcium Concentration. Cells, 13.","DOI":"10.3390\/cells13060554"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Haines, D.D., and Tosaki, A. (2020). Heme Degradation in Pathophysiology of and Countermeasures to Inflammation-Associated Disease. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21249698"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Lu, K. (2023). Cellular Pathogenesis of Hepatic Encephalopathy: An Update. Biomolecules, 13.","DOI":"10.3390\/biom13020396"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Somnay, K., Wadgaonkar, P., Sridhar, N., Roshni, P., Rao, N., and Wadgaonkar, R. (2024). Liver Fibrosis Leading to Cirrhosis: Basic Mechanisms and Clinical Perspectives. Biomedicines, 12.","DOI":"10.3390\/biomedicines12102229"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"22476","DOI":"10.1038\/srep22476","article-title":"Involvement of splenic iron accumulation in the development of nonalcoholic steatohepatitis in Tsumura Suzuki Obese Diabetes mice","volume":"6","author":"Murotomi","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"848","DOI":"10.1177\/15353702221077218","article-title":"Iron-accumulating splenocytes may exacerbate non-alcoholic steatohepatitis through the production of proinflammatory cytokines and reactive oxygen species","volume":"247","author":"Murotomi","year":"2022","journal-title":"Exp. Biol. Med."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2144","DOI":"10.12659\/MSM.896494","article-title":"The Role of Iron and Iron Overload in Chronic Liver Disease","volume":"22","author":"Milic","year":"2016","journal-title":"Med. Sci. Monit."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Zhang, L., Dai, X., Wang, L., Cai, J., Shen, J., Shen, Y., Li, X., and Zhao, Y. (2022). Iron overload accelerated lipid metabolism disorder and liver injury in rats with non-alcoholic fatty liver disease. Front. Nutr., 9.","DOI":"10.3389\/fnut.2022.961892"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1433","DOI":"10.3324\/haematol.2010.024992","article-title":"Changes in parameters of oxidative stress and free iron biomarkers during treatment with deferasirox in iron-overloaded patients with myelodysplastic syndromes","volume":"95","author":"Ghoti","year":"2010","journal-title":"Haematologica"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1186\/s12974-019-1430-7","article-title":"Iron accentuated reactive oxygen species release by NADPH oxidase in activated microglia contributes to oxidative stress in vitro","volume":"16","author":"Yauger","year":"2019","journal-title":"J. Neuroinflamm."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1097\/PAP.0000000000000112","article-title":"The Pathology of Acute Liver Failure","volume":"23","author":"Lefkowitch","year":"2016","journal-title":"Adv. Anat. Pathol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1093\/oxfordjournals.bmb.a011632","article-title":"Apoptosis and the immune system","volume":"53","author":"Ekert","year":"1997","journal-title":"Br. Med. Bull."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"348","DOI":"10.1055\/s-2006-951603","article-title":"Measurement of portal pressure and its role in the management of chronic liver disease","volume":"26","author":"Bosch","year":"2006","journal-title":"Semin. Liver Dis."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"27","DOI":"10.5009\/gnl230072","article-title":"Recent Advances in the Pathogenesis and Clinical Evaluation of Portal Hypertension in Chronic Liver Disease","volume":"18","author":"Kotani","year":"2024","journal-title":"Gut Liver"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"484","DOI":"10.1089\/ars.2017.7089","article-title":"Scavenging Reactive Oxygen Species Production Normalizes Ferroportin Expression and Ameliorates Cellular and Systemic Iron Disbalances in Hemolytic Mouse Model","volume":"29","author":"Tangudu","year":"2018","journal-title":"Antioxid. Redox Signal"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1111\/j.1600-0609.2008.01143.x","article-title":"Iron homeostasis: New players, newer insights","volume":"81","author":"Edison","year":"2008","journal-title":"Eur. J. Haematol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"3458","DOI":"10.1039\/D1LC00598G","article-title":"In vitro assay for single-cell characterization of impaired deformability in red blood cells under recurrent episodes of hypoxia","volume":"21","author":"Qiang","year":"2021","journal-title":"Lab. Chip"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"520","DOI":"10.1001\/jama.1976.03260310034018","article-title":"Hematin treatment of acute porphyria. Early remission of an almost fatal relapse","volume":"235","author":"Peterson","year":"1976","journal-title":"JAMA"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"80","DOI":"10.7326\/0003-4819-79-1-80","article-title":"Effect of hematin in acute porphyric relapse","volume":"79","author":"Watson","year":"1973","journal-title":"Ann. Intern. Med."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1515\/hsz-2013-0241","article-title":"The role of oxidative stress during inflammatory processes","volume":"395","author":"Lugrin","year":"2014","journal-title":"Biol. Chem."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1007\/s11906-010-0100-z","article-title":"Oxidative stress and hypertension: Current concepts","volume":"12","author":"Briones","year":"2010","journal-title":"Curr. Hypertens. Rep."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"12","DOI":"10.4110\/in.2009.9.1.12","article-title":"Heme oxygenase-1: Its therapeutic roles in inflammatory diseases","volume":"9","author":"Pae","year":"2009","journal-title":"Immune Netw."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"1541","DOI":"10.2174\/1389450111009011541","article-title":"Heme oxygenase-1 and iron in liver inflammation: A complex alliance","volume":"11","author":"Immenschuh","year":"2010","journal-title":"Curr. Drug Targets"},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Ryter, S.W. (2022). Heme Oxygenase-1: An Anti-Inflammatory Effector in Cardiovascular, Lung, and Related Metabolic Disorders. Antioxidants, 11.","DOI":"10.3390\/antiox11030555"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/5\/2463\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T10:18:27Z","timestamp":1773051507000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/5\/2463"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,7]]},"references-count":55,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["ijms27052463"],"URL":"https:\/\/doi.org\/10.3390\/ijms27052463","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,7]]}}}