Inhibition of Sphingosine Kinase-2 Suppresses Inflammation and Attenuates Graft Injury after Liver Transplantation in Rats

Abstract

Inflammation mediates/promotes graft injury after liver transplantation (LT). This study investigated the roles of sphingosine kinase-2 (SK2) in inflammation after LT. Liver grafts were stored in UW solution with and without ABC294640 (100 µM), a selective inhibitor of SK2, before implantation. Hepatic sphingosine-1-phosphate (S1P) levels increased ∼4-fold after LT, which was blunted by 40% by ABC294640. Hepatic toll-like receptor-4 (TLR4) expression and nuclear factor-κB (NF-κB) p65 subunit phosphorylation elevated substantially after transplantation. The pro-inflammatory cytokines/chemokines tumor necrosis factor-α, interleukin-1β and C-X-C motif chemokine 10 mRNAs increased 5.9-fold, 6.1-fold and 16-fold, respectively following transplantation, while intrahepatic adhesion molecule-1 increased 5.7-fold and monocytes/macrophage and neutrophil infiltration and expansion of residential macrophage population increased 7.8–13.4 fold, indicating enhanced inflammation. CD4+ T cell infiltration and interferon-γ production also increased. ABC294640 blunted TLR4 expression by 60%, NF-κB activation by 84%, proinflammatory cytokine/chemokine production by 45–72%, adhesion molecule expression by 54% and infiltration of monocytes/macrophages and neutrophils by 62–67%. ABC294640 also largely blocked CD4+ T cell infiltration and interferon-γ production. Focal necrosis and apoptosis occurred after transplantation with serum alanine aminotransferase (ALT) reaching ∼6000 U/L and serum total bilirubin elevating to ∼1.5 mg/dL. Inhibition of SK2 by ABC294640 blunted necrosis by 57%, apoptosis by 74%, ALT release by ∼68%, and hyperbilirubinemia by 74%. Most importantly, ABC294640 also increased survival from ∼25% to ∼85%. In conclusion, SK2 plays an important role in hepatic inflammation responses and graft injury after cold storage/transplantation and represents a new therapeutic target for liver graft failure.

The role of sphingosine 1-phosphate in inflammation and cancer

“…and CXCL-10 mRNA formation, ICAM1 expression and infiltration of monocytes/macrophages and neutrophils (Liu et al, 2012). ABC294640 also reduces CD4+ lymphocyte infiltration and IFN- production (Liu et al, 2012).…”

Suramin Decreases Injury and Improves Regeneration of Ethanol-Induced Steatotic Partial Liver Grafts

“…), and frozen sections were prepared and stained with Oil-Red-O 16 hours after ethanol treatment or 48 hours after implantation. At 48 hours after sham or implantation surgery, livers were perfusion fixed and harvested for histology, as described elsewhere (Zhong et al, 2005;Liu et al, 2012c). Tissue blocks were imbedded in paraffin, and histology was examined after hematoxylin and eosin (H&E) staining (Zhong et al, 2006).…”

“…For quantification of necrosis, 10 images were captured randomly per slide using a Universal Imaging Image-1/AT image acquisition and analysis system (West Chester, PA) with an Axioskop 50 microscope (Carl Zeiss, Inc., Thornwood, NY) and a 10Â objective lens. Necrotic areas were quantified by image analysis using an IPlab 3.7v software (BD Biosciences, Rockville, MD), as previously described elsewhere (Liu et al, 2012c). Mitotic cells were counted in 10 randomly selected fields under the light microscope, and the mitotic index was calculated.…”

“…Quantitative polymerase chain reaction (qPCR) was performed as described elsewhere (Liu et al, 2012c). After total RNA isolation from liver tissue with Trizol (Invitrogen, Grand Island, NY), single-stranded cDNAs were synthesized from RNA (2 mg) using a Bio-Rad iScript cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA).…”

“…Proteins were detected by immunoblot analysis as previously described elsewhere (Liu et al, 2012c) with primary antibodies specific for cleaved caspase-3 (Cell Signaling Technology, Danvers, MA), cleaved caspase-8, TGF-b (Abcam, Cambridge, MA), epidermal growth factor receptor (EGFR), phospho-EGFR (p-EGFR; GenWay Biotech, San Diego, CA), c-Jun N-terminal kinase-1/2 (JNK1/2), phospho-JNK (p-JNK), extracellular signal-regulated kinases-1/2 (ERK1/2), phospho-ERK (p-ERK), Smad2/3, phosphoSmad2/3, p21Cip1 (Santa Cruz Biotechnology, Santa Cruz, CA), and 4-hydroxynonenal adducts (4-HNE; Alpha Diagnostic, San Antonio, TX) at concentrations of 1:100 to 1000, and actin (ICN, Costa Mesa, CA) at a concentration of 1:3000 at 4°C overnight, respectively. Horseradish peroxidase-conjugated secondary antibodies were applied, and detection was by chemiluminescence (Pierce Biotechnology, Rockford, IL).…”