ABSTRACT
1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG), a chemically synthesized monoacetyldiaglyceride, is one of the constituents in Sika deer antlers and has been known traditionally as having immunomodulatory effects. However, the mechanism by which PLAG controls neutrophil migration, which evokes liver injury in the hepatitis animal model, remains largely unknown. This study was designed to evaluate the immunomodulatory effects of PLAG on cytokine secretion and neutrophil migration in vivo and in vitro. Concanavalin A (Con A) induced leukocyte infiltration in the liver and increased plasma cytokine levels. Pretreatment with PLAG reduced the levels of interleukin (IL)-4, IL-6, IL-10, and CXCL2, but maintained interferon (IFN)-γ levels and modulated neutrophil recruitment toward the liver. Furthermore, the mRNA and protein levels of IL-4 and CXCL2 in liver tissue were also decreased in the Con A-treated mice. Liver histology analyses showed that PLAG reduced Con A-induced hepatic necrosis, which was accompanied by leukocyte infiltration. The in vitro studies revealed that PLAG reduced IL-4 secretion in Con A stimulated T cell and blocked signal transducer and activator of transcription 6 (STAT6) Con A induced hepatocyte. PLAG attenuated IL-4 induced activation of atypical protein kinase C (PKC)/STAT6 in hepatocytes and inhibited neutrophil migration toward the liver tissue through suppression of IL-8/vascular cell adhesion molecule (VCAM) expression. These results suggest that PLAG could mitigate excess neutrophil migration into liver tissue and potentially have a therapeutic effect on immune-mediated liver injury.
Subject(s)
Chemical and Drug Induced Liver Injury/drug therapy , Concanavalin A/adverse effects , Cytokines/genetics , Cytokines/metabolism , Diglycerides/administration & dosage , Animals , Cell Line, Tumor , Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/metabolism , Chemokine CXCL2/genetics , Chemokine CXCL2/metabolism , Diglycerides/pharmacology , Disease Models, Animal , Gene Expression Regulation/drug effects , HL-60 Cells , Hep G2 Cells , Humans , Interleukin-10/genetics , Interleukin-10/metabolism , Interleukin-4/genetics , Interleukin-4/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Male , MiceABSTRACT
NDRG (N-Myc downstream-regulated gene)-2 is a member of the NDRG family. Although it has been suggested that NDRG2 is involved in cellular differentiation and tumor suppression, its intracellular signal and regulatory mechanism are not well known. Here, we show the differential expression of NDRG2 in human colon carcinoma cell lines and tissues by reverse transcription-polymerase chain reaction and immunohistochemical analyses with monoclonal antibody against NDRG2. NDRG2 was strongly expressed in normal colonic mucosa and colonic adenomatous tissues (25 of 25) but not in all invasive cancer tissues [44 of 99 (44%)]. Most distinctive results indicated that the high expression level of NDRG2 has a positive correlation with tumor differentiation and inverse correlation with tumor invasion depth and Dukes' stage of colon adenocarcinoma. To investigate the roles of NDRG2 in tumorigenesis, we used in vitro cell culture system. SW620 colon cancer cell line with a low level of intrinsic NDRG2 protein was transfected with NDRG2-expressing plasmid. TOPflash luciferase reporter assay showed that the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) was reduced by NDRG2 introduction, but not by the introduction of mutant NDRG2 generated by deletion or site-directed mutagenesis. Intracellular beta-catenin levels were slightly reduced in the NDRG2-transfected SW620 cells and this regulation of beta-catenin stability and TCF/LEF activity were mediated through the modulation of glycogen synthase kinase-3beta activity by NDRG2 function. Our results suggest that NDRG2 might play a pivotal role as a potent tumor suppressor by the attenuation of TCF/beta-catenin signaling for the maintenance of healthy colon tissues.