OBJECTIVETo determine whether Toll-like receptor 4 (TLR4) is involved in development of gut leakiness in alcoholic steatohepatitis using an in vivo animal model and an in vitro cell culture system.

METHODSMice were fed an alcohol (ethanol group, EtOH) or isocaloric liquid diet (control group, Ctrl). Successful establishment of the alcoholic steatohepatitis model was assessed at week 6 by measuring serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and evaluating the liver pathology using hematoxylin and eosin (HandE) staining of liver tissues. Gut permeability was assessed by measuring serum endotoxin and urine lactulose/mannitol (L/M) levels and evaluating HandE-stained colon tissues. Intestinal and colon tissue expression levels of TLR4 were assessed by immunohistochemistry. Cultured Caco-2 cells were exposed to 25 - 400 mmol/L EtOH and changes in TLR4 were assessed by enzyme-linked immunoassay and in permeability were assessed by intracellular uptake of FD4.

RESULTSThe mice in the EtOH group had significantly higher levels of serum ALT (46.5 +/- 6.9 U/L vs. Ctrl: 30.9 +/- 4.4 U/L, P less than 0.01), serum AST (53.3 +/- 7.9 U/L vs. Ctrl: 29.3 +/- 3.8 U/L, P less than 0.01), serum endotoxin (0.33 +/- 0.05 Eu/L vs. Ctrl: 0.27 +/- 0.04 Eu/L, P less than 0.01), and urine L/M (2.59 +/- 0.44% vs. Ctrl: 2.17 +/- 0.31%, P less than 0.05). The mice in the EtOH group also had significantly higher expression levels of TLR4 in intestinal tissues (13.1 +/- 2.0 ng/ml vs. Ctrl: 7.4 +/- 1.2 ng/L, P less than 0.01) and in colonic tissues (18.5 +/- 2.7 ng/ml vs. Ctrl: 9.1 +/- 1.6 ng/ml, P less than 0.01). The intestinal histopathology of the two groups was not different. Immunohistochemical staining of colonic tissues showed brown particles distributed in the endochylema and membrane of the EtOH group, which was almost completely absent in the Ctrl group. EtOH treatment of Caco-2 cells led to a dose-dependent increase in TLR4 expression and in cellular permeability.

CONCLUSIONChronic alcohol exposure induced TLR4 expression and cellular permeability in gut tissues. Activation of TLR4 may be involved in development of gut leakiness in alcoholic liver disease.