The Role of LPS-TLR4 Pathway on Hepatic Fibrogenesis in Chronic Ethanol-Fed Rats

Abstract

Introduction: Gut-derived lipopolysaccharide (LPS) has been proposed as a key player in the pathogenesis of alcoholic liver disease (ALD). The aims of this study were to investigate the role of LPS on hepatic fibrogenesis in a rat model of chronic alcohol feeding. Methods: Forty-eight Sprague-Dawley male rats were randomly divided as 4 groups and fed isocaloric Liber-DeCarli liquid diets ± alcohol (36% of total caloric value) for 8 weeks or 10 weeks. Plasma concentrations of LPS in portal veins from these rats were determined by spectrophotometric method. Rat liver tissue samples were examined for TLR4 and TGF-β1 mRNA by in situ hybridization. Immunohistochemistry was used to determine FSP-1 and vimentin for identification of activated HSC while F4/80 for recognition of Kuffer cells in serial sections of liver tissues. Computer image analysis was performed to measure the integrated optimal density (IOD) of FSP-1-positive HSC and TLR4 mRNA-positive cells. Hepatic collagen-I and α-SMA protein were analyzed by Western blot while IL-1, IL-6, TNF-α, and TGFβ1 mRNA were accessed by RT-PCR. Histological characteristics were evaluated by H and E staining and Masson’s Trichrome stain. Results: Masson’s Trichrome stain showed that there was a perisinusoidal fibrosis in rats fed ethanol at the end of 10 weeks, which was consistent with increased expression of collagen I by Western blot. Immunohistochemical stain showed there were abundant activated HSCs and some Kuffer cells in livers of rats fed ethanol at the end of 10 weeks, but a few HSC were seen in control-diet fed rats. Plasma LPS levels in portal veins were significantly higher in 10-week alcohol-fed rats than in 10-week controldiet-fed rats, but LPS levels were not increased in 8-week alcohol-fed rats. In situ hybridization analysis showed that a significant increase of TLR4 mRNA could be detected in abundant hepatocytes and some activated HSC and Kuffer cells in 10-week alcohol-fed rat livers, but in fewer endothelial cells of central vein in control-diet-fed rats. Bivariate correlation analysis showed that there was a positive correlation between the expression intensity of TLR4 mRNA in livers and the levels of plasma LPS in the portal vein of 10-week alcohol-fed rats (r=0.856; p<0.05); furthermore, the expressions of proinflammatory cytokine mRNA, including IL-1, IL-6, and TNF-α, as well as profibrogenic factor TGF-β1 mRNA, were increased in livers of 10-week alcohol-fed rats compared with control diet-fed rats. Conclusion: This study highlights the role of gut-derived LPS on hepatic fibrogenesis in a rat model of chronic alcohol feeding.