17 This system includes the receptor activator of NF-κB (RANK),18, 19 its ligand, RANKL,18 and the decoy receptor for RANKL, osteoprotegerin (OPG).20 Although the study focused on the roles of the RANKL/OPG system in osteoporosis caused by liver transplantation, the data suggest that hepatic I/R may affect RANKL and OPG expression.17 Moreover, another study has shown that the RANKL/OPG system is involved in chronic liver diseases, such as primary biliary cirrhosis and chronic hepatitis C, and suggested that liver inflammation may induce RANKL and OPG expression.21 Because NF-κB activation is known to play pivotal roles in hepatic I/R injury and the interaction of RANK and RANKL appears to have

a direct relationship with hepatic inflammation, we sought to determine PLX4032 the role of the RANK/RANKL/OPG system in the hepatic pathophysiological response to I/R. ALT, alanine amino transferase; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; I/R, ischemia/reperfusion; PD-0332991 datasheet KC, keratinocyte chemokine; MIP-2, macrophage

inflammatory protein-2; MPO, myeloperoxidase; NF-κB, nuclear factor kappaB; OPG, osteoprotegerin; RANK, receptor activator of NF-κB; RANKL, receptor activator of NF-κB ligand; TNF-α, tumor necrosis factor-α. Male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) weighing 20-26 g were used in all experiments. This project was approved by the University of Cincinnati Animal Care and Use Committee and was in compliance with the National Institutes of Health guidelines. The animals underwent either sham surgery or I/R. Partial hepatic ischemia was induced as described.7 Briefly, mice were Mirabegron anesthetized with sodium pentobarbital (60

mg/kg, intraperitoneally). A midline laparotomy was performed and an atraumatic clip was used to interrupt blood supply to the left lateral and median lobes of the liver. After 60 or 90 minutes of partial hepatic ischemia, the clip was removed to initiate hepatic reperfusion. Sham control mice underwent the same protocol without vascular occlusion. Some mice were injected intraperitoneally with 400 μg/mouse of anti-mouse CD254 (RANKL) antibody (BioLegend, San Diego, CA) or rat IgG2a (Sigma-Aldrich, St. Louis, MO) at the time of clip removal. Some mice were injected intraperitoneally with phosphate-buffered saline (PBS) or recombinant mouse RANKL (R&D Systems, Minneapolis, MN), dissolved in PBS at 1 hour prior to ischemia or at the time of clip removal (i.e., after ischemic period). Mice were sacrificed after the indicated periods of reperfusion and blood and samples of the left lateral lobe were taken for analysis. Blood was obtained by cardiac puncture for analysis of serum alanine amino transferase (ALT) as an index of hepatocellular injury. Measurements of serum ALT were made using a diagnosis kit by bioassay (Wiener Laboratories, Rosario, Argentina).