The novel Janus kinase inhibitor ruxolitinib confers protection against carbon tetrachloride-induced hepatotoxicity via multiple mechanisms Sara H. Hazem a , Mohamed E. Shaker a,⇑ , Sylvia A. Ashamallah b , Tarek M. Ibrahim a a Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt b Department of Pathology, Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt article info Article history: Received 15 April 2014 Received in revised form 3 June 2014 Accepted 16 June 2014 Available online 25 June 2014 Keywords: Hepatic inflammation Ruxolitinib Janus kinase Carbon tetrachloride Oxidative stress JAK/STAT abstract Therapeutic targeting of the JAK/STAT pathway, the principal signaling mechanism for numerous cyto- kines, might be an effective approach for limiting inflammation in different organs, including the liver. Therefore, we investigated whether targeting this pathway by the novel JAK inhibitor ruxolitinib could mitigate hepatic damage provoked by carbon tetrachloride (CCl 4 ). Male mice received ruxolitinib treat- ments (75 and 150 mg/kg, oral) 2 h prior to intoxication with CCl 4 (10 ml/kg of 0.3% v/v CCl 4 solution in olive oil, intraperitoneal) for 24 h. Our results showed that ruxolitinib treatments dose-dependently alleviated CCl 4 -induced hepatic injury and necroinflammation, as indicated by biochemical markers of injury and histopathology. We unraveled also the mechanisms involved in these hepatoprotective effects. These comprise (i) reducing infiltration of neutrophils and macrophages, as demonstrated by reducing myeloperoxidase activity and F4/80 positive macrophages; (ii) abating apoptosis of hepatocytes, as denoted by decreasing hepatocytes positive for Bax protein; (iii) inhibiting elevation of TNF-a, IL-1b and IL-10; (iv) inhibiting NF-jB activation and translocation to the nucleus, as visualized immunohisto- chemically; (v) attenuating activation of the IL-23/IL-17 pathway via targeting IL-17, but not IL-23; (vi) antagonizing hepatic oxidative stress by increasing the antioxidant levels (reduced glutathione, glutathione-S-transferase and superoxide dismutase) and decreasing products of lipid peroxidation (malondialdehyde and 4-hydroxynonenal) and total nitrate/nitrite; and (vii) more interestingly, modulat- ing hepatocyte regeneration according to the extent of damage, as quantified by PCNA-immunohisto- chemistry. In conclusion, our study sheds light on the therapeutic usefulness and the potential underlying mechanisms of the novel JAK inhibitor ruxolitinib in hepatic inflammatory disorders. Ó 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Hepatic inflammation is a key process in the pathogenesis of several disorders, such as drug-induced liver injury, ischemia/ reperfusion, nonalcoholic and alcoholic steatohepatitis, as well as viral and bacterial infections [1,2]. Moreover, hepatic inflammation is a major determinant for the progression of hepatic fibrosis to cirrhosis and carcinogenesis [3,4]. Among the various pathways implicated in hepatic inflammation, the Janus kinase/signal trans- ducers and activators of transcription (JAK/STAT) pathway appears to be the most intimately related to signal transduction of proin- flammatory cytokines. JAKs are a family of intracellular non-receptor tyrosine kinases that transmit signals arising from the interaction of various cytokines with extracellular receptor to the nucleus via phosphor- ylating STAT3 [5]. Generally, JAK/STAT signaling regulates many cellular processes including development, cell proliferation, differentiation and apoptosis [6]. In the liver, JAKs are activated by several proinflammatory cytokines and growth factors, such as interferon-c, interleukin (IL)-4, IL-6, IL-12, IL-13 and growth hormones [7], as well as hepatitis viral proteins [8]. Recently, acti- vation of the JAK/STAT pathway has been shown to regulate fibro- genic cytokines like transforming growth factor-b1 and connective tissue growth factor [9], as well as enhancement of liver fibrosis and cancer [10,11]. Hence, drugs that target this pathway will indeed dampen the signaling of these proinflammatory and fibro- genic cytokines involved in hepatic inflammation and fibrosis in humans. JAK inhibitors are a novel class of medications that inhibit the activity of one or more of JAK enzymes, including JAK1, JAK2, JAK3, TYK2 [12]. Of these inhibitors, ruxolitinib is considered the most important predecessor. Ruxolitinib, a novel oral JAK1/JAK2 inhibitor, is the first approved therapy for the treatment of myelofibrosis by FDA on November 16, 2011 [13]. Mechanistically, http://dx.doi.org/10.1016/j.cbi.2014.06.017 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +20 502246253; fax: +20 502247496. E-mail address: mshaker2222@yahoo.com (M.E. Shaker). Chemico-Biological Interactions 220 (2014) 116–127 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint