Immunogenomics reveal molecular circuits of diclofenac induced liver injury in mice
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Eun-Hee Lee1, Jung-Hwa Oh1,2, Saravanakumar Selvaraj3, Se-Myo Park1, Mi-Sun Choi1, Reinhard Spanel3,4, Seokjoo Yoon1,2,*, Jürgen Borlak3,*
1Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon, 305-343, Republic of Korea
2Department of Human and Environmental Toxicology, School of Engineering, Korea University of Science and Technology, Daejeon, 305-343, Republic of Korea
3Centre for Pharmacology and Toxicology, Hannover Medical School, 30625 Hannover, Germany
4Institute for Clinical Pathology, 41747 Viersen, Germany
*These authors contributed equally to this work
Jürgen Borlak, e-mail: firstname.lastname@example.org
Keywords: diclofenac, NSAID, gene expression profiling, inflammation, immune response
Received: September 25, 2015 Accepted: January 12, 2016 Published: February 25, 2016
Diclofenac is a non-steroidal anti-inflammatory drug and its use can be associated with severe adverse reactions, notably myocardial infarction, stroke and drug-induced liver injury (DILI). In pursue of immune-mediated DILI mechanisms an immunogenomic study was carried out. Diclofenac treatment of mice at 30 mg/kg for 3 days caused significant serum ALT and AST elevations, hepatomegaly and degenerative changes including hepatic glycogen depletion, hydropic swelling, cholesterolosis and eosinophilic hepatocytes with one animal presenting subsegmental infarction due to portal vein thrombosis. Furthermore, portal/periportal induction of the rate limiting enzyme in ammonia detoxification, i.e. carbamoyl phosphate synthetase 1 was observed. The performed microarray studies informed on > 600 differential expressed genes of which 35, 37 and 50 coded for inflammation, 51, 44 and 61 for immune and 116, 129 and 169 for stress response, respectively after single and repeated dosing for 3 and 14 days. Bioinformatic analysis defined molecular circuits of hepatic inflammation with the growth hormone (Ghr)− and leptin receptor, the protein-tyrosine-phosphatase, selectin and the suppressor-of-cytokine-signaling (Socs) to function as key nodes in gene regulatory networks. Western blotting confirmed induction of fibronectin and M-CSF to hallmark tissue repair and differentiation of monocytes and macrophages. Transcript expression of the macrophage receptor with collagenous structure increased > 7-fold and immunohistochemistry of CD68 evidenced activation of tissue-resident macrophages. Importantly, diclofenac treatment prompted strong expression of phosphorylated Stat3 amongst individual animals and the associated 8- and 4-fold Soc3 and Il-6 induction reinforced Ghr degradation as evidenced by immunoblotting. Moreover, immunohistochemistry confirmed regulation of master regulatory proteins of diclofenac treated mice to suggest complex pro-and anti-inflammatory reactions in immune-mediated hepatic injury. The findings encourage translational research.
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