Immunopharmacology and inflammation Effects of palmitoylethanolamide and silymarin combination treatment in an animal model of kidney ischemia and reperfusion Daniela Impellizzeri a , Giuseppe Bruschetta a , Akbar Ahmad b , Rosalia Crupi a , Rosalba Siracusa a , Rosanna Di Paola a , Irene Paterniti a , Marco Prosdocimi c , Emanuela Esposito a , Salvatore Cuzzocrea a,d,n a Department of Biological and Environmental Sciences, Uni versity of Messina, Viale Ferdinando Stagno D’Alcontres, 31-98166 Messina, Italy b Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX, USA c Prismic Pharmaceuticals, Inc., Hayden Corporate Center, Suite 100 8283 North Hayden Road, Scottsdale 85258, Arizona d Manchester Biomedical Research Centre, Manchester Royal Infirmary, School of Medicine, University of Manchester, UK article info Article history: Received 9 January 2015 Received in revised form 21 April 2015 Accepted 13 May 2015 Available online 14 May 2015 Keywords: Renal disease Inflammation Oxidative stress Cytokines Apoptosis abstract The aim of this study was to investigate the efficacy of PEA þsilymarin as a combination treatment in a mouse model of renal I/R and to verify whether PEA þsilymarin could exert more potent effects com- pared to the single substances even if administered at lower doses. Mice were subjected to bilateral renal artery occlusion (30 min) and reperfusion (6 h) and received intraperitoneally silymarin (100, 30 and 10 mg/kg) or PEA (1 mg/kg) or PEA (1 mg/kg) þsilymarin (10 mg/kg) 15 min before release of clamps. Specific indicators of renal dysfunction, tubular injury, myeloperoxidase activity and malondialdehyde levels were measured. The nuclear factor κB pathway and apoptotic mechanisms were also investigated. The treatment with silymarin reduced kidney dysfunction, histological damage, neutrophil infiltration and oxidative stress in a dose dependent manner. Furthermore, PEA þsilymarin showed a significant potentiated effect. Therefore, NF-κB and apoptosis pathways were also significantly inhibited. Our results clearly demonstrate that PEA þsilymarin treatment attenuated the degree of renal inflammation. & 2015 Elsevier B.V. All rights reserved. 1. Introduction Renal Ischemia reperfusion (I/R) injury is characterized by a complex of events, resulting in injury to and the eventual death of renal cells (Patel et al., 2005a). The expression of adhesion mole- cules (Chamoun et al., 2000), the transmigration of polymorpho- nuclear leukocytes (PMNs) into renal tissues and subsequent production of reactive oxygen species (ROS) and nitric oxide (NO) (Rabb et al., 1997) contribute significantly to the development of renal damage. Palmitoylethanolamide (PEA), an endogenous fatty acid amide, is an important analgesic, anti-inflammatory, and neuroprotective mediator, acting at several molecular targets as mast cells (MC) (Di Paola et al., 2012). A previous study showed that PEA at dose of 10 mg/kg attenuated the renal dysfunction and injury associated with I/R (Di Paola et al., 2012). Furthermore, our recent review also discussed how PEA treatment could represent a new therapeutic approach for the treatment of chronic kidney disease (CKD) (Im- pellizzeri et al., 2014) and a recent work also demonstrated the potential beneficial effects of long-term treatment with PEA on conditions associated with kidney disease, modulating key en- zymes in ROS/RNS synthesis and/or scavenging and angiotensin II (AT) receptors homeostasis (Mattace Raso et al., 2013). In particular, during kidney inflammation, PEA could modulate the nuclear factor NF-κB pathway and attenuate NF-κB-induced inflammatory factors such as interleukin-1 (IL-1), or tumor ne- crosis factor-α (TNF-α), inhibit infiltration and activation of MC, reduce masengial matrix proliferation induced by ROS which then resulted in albuminuria (Chen et al., 2009; Di Paola et al., 2012). However, PEA lacks a direct antioxidant capacity to prevent the formation of free radicals, and to counteract the damage of DNA, lipids and proteins, all of which are important events occurring in renal diseases. For this reason, the anti-inflammatory action of PEA combinated with an antioxidant could potentiate its pharmaco- logical effects. Flavonoids are naturally occurring substances that possess Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ejphar European Journal of Pharmacology http://dx.doi.org/10.1016/j.ejphar.2015.05.010 0014-2999/& 2015 Elsevier B.V. All rights reserved. n Corresponding author at: Department of Biological and Environmental Sci- ences, University of Messina, Viale Ferdinando Stagno D’Alcontres, no. 31 98166 Messina, Italy. E-mail addresses: dimpellizzeri@unime.it (D. Impellizzeri), bruschettag@unime.it (G. Bruschetta), aahmad@unime.it (A. Ahmad), rcrupi@unime.it (R. Crupi), rosiracusa@gmail.com (R. Siracusa), dipaolar@unime.it (R. Di Paola), ipaterniti@unime.it (I. Paterniti), prosdocimimarco@gmail.com (M. Prosdocimi), eesposito@unime.it (E. Esposito), salvator@unime.it (S. Cuzzocrea). European Journal of Pharmacology 762 (2015) 136–149