{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T05:55:25Z","timestamp":1790920525952,"version":"4.1.0"},"reference-count":34,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T00:00:00Z","timestamp":1790899200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Nutr."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Obstructive cholestasis is characterized by disturbances in bile acid homeostasis, amino acid metabolism, inflammatory responses, and hepatocellular injury. Although RIP3\/MLKL-pathway proteins have been implicated in cholestatic liver damage, their relationship with metabolic alterations remains incompletely understood. This study aimed to identify necroptosis-related genes and associated metabolic-pathway alterations in early bile duct ligation (BDL)-induced cholestatic liver injury through integrated bioinformatics analyses and experimental validation.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>Gene expression datasets GSE29776 and GSE166867 were retrieved from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and necroptosis-related differentially expressed genes (NRDEGs) were identified. Functional enrichment analyses, protein\u2013protein interaction network construction, least absolute shrinkage and selection operator (LASSO) regression, and random forest analysis were performed to prioritize candidate genes. A murine BDL model was established for validation using biochemical assays, histological analyses, RT-qPCR, and western blotting. Phosphorylated RIP3 and MLKL were evaluated both relative to GAPDH and as phosphorylation-to-total-protein ratios.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Eight NRDEGs were identified, including Tnfaip3, Sqstm1, Chmp2b, Ifngr2, Slc25a4, Ifngr1, Camk2d, and Glul. Functional enrichment analyses revealed significant enrichment in pathways related to metabolic regulation, transmembrane transporter activity, inflammatory signaling, and necroptosis. Machine-learning analyses prioritized Sqstm1, Ifngr1, and Glul as candidates for exploratory characterization. Glul, which encodes glutamine synthetase (GLUL), was markedly downregulated in cholestatic liver tissues. Experimental validation confirmed bile acid accumulation, reduced Glul mRNA expression and GLUL protein abundance, and increased GAPDH-normalized RIP3, p-RIP3, MLKL, and p-MLKL abundance in BDL mice. Both the p-RIP3\/RIP3 and p-MLKL\/MLKL ratios were significantly increased, supporting enhanced phosphorylation of RIP3 and MLKL relative to the corresponding total-protein levels.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>In early BDL-induced cholestatic liver injury, reduced hepatic Glul mRNA expression and GLUL protein abundance occurred concurrently with increased RIP3 and MLKL phosphorylation relative to the corresponding total-protein levels. These findings support enhanced RIP3\/MLKL-associated signaling but do not independently establish pathway-dependent necroptotic flux. Further mechanistic validation is required.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fnut.2026.1912372","type":"journal-article","created":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T05:35:55Z","timestamp":1790919355000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Reduced hepatic GLUL expression accompanies altered RIP3\/MLKL-pathway protein abundance in early cholestatic liver injury following bile duct ligation"],"prefix":"10.3389","volume":"13","author":[{"given":"Baiyu","family":"Yao","sequence":"first","affiliation":[{"name":"Department of General Surgery, Shengjing Hospital of China Medical University","place":["Shenyang, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fanhe","family":"Xiao","sequence":"additional","affiliation":[{"name":"Department of General Surgery, Shengjing Hospital of China Medical University","place":["Shenyang, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhong","family":"Tian","sequence":"additional","affiliation":[{"name":"Department of General Surgery, Shengjing Hospital of China Medical University","place":["Shenyang, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Kong","sequence":"additional","affiliation":[{"name":"Department of Clinical Nutrition, Shengjing Hospital of China Medical University","place":["Shenyang, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,10,2]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"791","DOI":"10.1016\/j.mayocp.2015.03.017","article-title":"The cholangiopathies","volume":"90","author":"Lazaridis","year":"2015","journal-title":"Mayo Clin Proc"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"1756284818787400","DOI":"10.1177\/1756284818787400","article-title":"Cholestatic liver diseases: new targets, new therapies","volume":"11","author":"Santiago","year":"2018","journal-title":"Ther Adv Gastroenterol"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"1393","DOI":"10.1002\/hep.28991","article-title":"New therapeutic concepts in bile acid transport and signaling for management of cholestasis","volume":"65","author":"Trauner","year":"2017","journal-title":"Hepatology"},{"key":"ref4","doi-asserted-by":"publisher","first-page":"619","DOI":"10.1016\/j.jhep.2017.04.026","article-title":"Biliary bile acids in hepatobiliary injury\u2014what is the link?","volume":"67","author":"Fickert","year":"2017","journal-title":"J Hepatol"},{"key":"ref5","doi-asserted-by":"publisher","first-page":"1221","DOI":"10.1124\/pharmrev.124.000978","article-title":"Bile acid signaling in metabolic and inflammatory diseases and drug development","volume":"76","author":"Li","year":"2024","journal-title":"Pharmacol Rev"},{"key":"ref6","doi-asserted-by":"publisher","first-page":"G554","DOI":"10.1152\/ajpgi.00223.2019","article-title":"Bile acid receptors FXR and TGR5 signaling in fatty liver diseases and therapy","volume":"318","author":"Chiang","year":"2020","journal-title":"Am J Physiol Gastrointest Liver Physiol"},{"key":"ref7","doi-asserted-by":"publisher","first-page":"1243","DOI":"10.1111\/apt.16602","article-title":"Review article: therapeutic aspects of bile acid signalling in the gut-liver axis","volume":"54","author":"Simbrunner","year":"2021","journal-title":"Aliment Pharmacol Ther"},{"key":"ref8","doi-asserted-by":"publisher","first-page":"719","DOI":"10.1038\/s41579-023-00904-3","article-title":"The gut-liver axis and gut microbiota in health and liver disease","volume":"21","author":"Hsu","year":"2023","journal-title":"Nat Rev. 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