UNCORRECTED PROOF 1 Proteasome inhibitors protect the steatotic and non-steatotic liver graft against cold 2 ischemia reperfusion injury ☆ 3 Mohamed Amine Q1 Zaouali a , Fawzia Bardag-Gorce b , Teresa Carbonell c , Joan Oliva b , Eirini Pantazi a , 4 Mohamed Bejaoui a , Hassen Ben Abdennebi d , Antoni Rimola e , Joan Roselló-Catafau a, e, ⁎ 5 a Experimental Hepatic Ischemia-Reperfusion Unit, Institut d´Investigacions Biomèdiques de Barcelona-Consejo Superior de Investigaciones Científicas, Barcelona, Spain 6 b Harbor-UCLA Medical Center, Torrance, CA, USA 7 c Department de Fisiologia, Facultat de Biología, Universitat de Barcelona, Barcelona, Spain 8 d Laboratory of Human Physiology, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia 9 e Ciberehd, Hospital Clínic, Barcelona, Spain 10 11 abstract article info 12 Article history: 13 Received 18 September 2012 14 and in revised form 20 December 2012 15 Available online xxxx 16 17 18 19 Keywords: 20 Ischemia reperfusion injury 21 Steatotic liver 22 UW preservation solution 23 Bortezomib 24 MG132 25 UPS inhibitor 26 Background: The dramatic shortage of organs leads to consider the steatotic livers for transplantation 27 although their poor tolerance against ischemia reperfusion injury (IRI). Ubiquitin proteasome system 28 (UPS) inhibition during hypothermia prolongs myocardial graft preservation. The role of UPS in the liver 29 IRI is not fully understood. Bortezomib (BRZ) treatment at non-toxic doses of rats fed alcohol chronically 30 has shown protective effects by increasing liver antioxidant enzymes. We evaluated and compared both 31 proteasome inhibitors BRZ and MG132 in addition to University of Wisconsin preservation solution (UW) 32 at low and non-toxic dose for fatty liver graft protection against cold IRI. 33 Experimental: Steatotic and non-steatotic livers have been stored in UW enriched with BRZ (100 nM) or 34 MG132 (25 μM), for 24 h at 4 °C and then subjected to 2-h normothermic reperfusion (37 °C). Liver injury 35 (AST/ALT), hepatic function (bile output; vascular resistance), mitochondrial damage (GLDH), oxidative 36 stress (MDA), nitric oxide (NO) (e-NOS activity; nitrates/nitrites), proteasome chymotrypsin-like activity 37 (ChT), and UPS (19S and 20S5 beta) protein levels have been measured. 38 Results: ChT was inhibited when BRZ and MG132 were added to UW. Both inhibitors prevented liver injury 39 (AST/ALT), when compared to UW alone. BRZ increased bile production more efficiently than MG132. Only 40 BRZ decreased vascular resistance in fatty livers, which correlated with an increase in NO generation 41 (through e-NOS activation) and AMPK phosphorylation. GLDH and MDA were also prevented by BRZ. In 42 addition, BRZ inhibited adiponectin, IL-1, and TNF alpha, only in steatotic livers. 43 Conclusion: MG132 and BRZ, administrated at low and non toxic doses, are very efficient to protect fatty liver 44 grafts against cold IRI. The benefits of BRZ are more effective than those of MG132. This evidenced for the first 45 time the potential use of UPS inhibitors for the preservation of marginal liver grafts and for future applications 46 in the prevention of IRI. 47 © 2013 Published by Elsevier Inc. 48 49 50 51 52 Introduction 53 Organ shortage is a main cause of the patients' death in the 54 waiting list for transplantations and marginal livers are needed to 55 expand the number of available donors for liver transplantation 56 (Imber et al., 2002; Selzner and Clavien, 2001). The increased propor- 57 tion of fatty liver grafts in bank donors in Western countries is a direct 58 consequence of unhealthy lifestyles associated with the consumption 59 of alcohol and inappropriate diets (Zaouali et al., 2011). Consequently, 60 the steatotic liver grafts use for transplant purposes increases the risk of 61 postoperative morbidity and mortality. In fact, they are generally 62 discarded for transplantation when liver steatosis is severe (upper 63 than 60%) (McCormack et al., 2011; Peralta and Rosello-Catafau, 2004; 64 Todo et al., 1989a). 65 Ischemia–reperfusion injury (IRI) is a consequence from the 66 hypothermic liver graft conservation in preservation solution and 67 the subsequent normothermic reperfusion after transplantation 68 (Zaouali et al., 2010a). Steatotic liver grafts are more vulnerable to 69 cold IRI than non-steatotic ones. Moreover, the accumulation of fat in 70 sinusoids originates severe alterations in liver microcirculation and 71 exacerbates mitochondrial damage and oxidative stress during reperfu- 72 sion, compromising thus the suitable liver graft revascularization after Experimental and Molecular Pathology xxx (2013) xxx–xxx ☆ This study was kindly supported by the Ministerio de Sanidad y Consumo (project grant PIO81988) (Madrid, Spain). Mohammed Amine Zaouali wishes to thank the Societat Catalana de Trasplantament, for their fellowships. ⁎ Corresponding author at: Experimental Hepatic Ischemia-Reperfusion Unit, IIBB-CSIC, C/Rosselló 161, 7th floor, E-08036, Barcelona, Spain. Fax: +34 933638301. E-mail address: joan.rosello@iibb.csic.es (J. Roselló-Catafau). YEXMP-03440; No of Pages 8 0014-4800/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.yexmp.2012.12.005 Contents lists available at SciVerse ScienceDirect Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp Please cite this article as: Zaouali, M.A., et al., Proteasome inhibitors protect the steatotic and non-steatotic liver graft against cold ischemia reperfusion injury, Experimental and Molecular Pathology (2013), http://dx.doi.org/10.1016/j.yexmp.2012.12.005