The Role And Mechanism Of Lipopolysaccharide On Rat Hepatic Stellate Cells-mediated Hepatic Fibrosis

Background and Aims: Hepatic fibrosis is a common pathological process from various liver diseases to cirrhosis and even liver cancer. The role of lipopolysaccharide(LPS) in the development of chronic liver disease has attracted more and more attention due to the deep research on the relationship between intestinal endotoxin and hepatic fibrosis. However, the exact mechanism of LPS in the pathogenesis of liver diseases has not been fully clarified. Recently, our study showed that the level of LPS was elevated in the portal vein of rats fed ethanol for ten weeks which played an important role in alcoholic hepatic fibrosis. The aim of this study was to further elucidate the role and mechanism of LPS on hepatic stellate cells(HSC)-mediated hepatic fibrosis in male Sprague-Dawley(SD) rats challenged with LPS alone.Methods: twenty-four male SD rats weighing 120g±20g were randomly divided into normal control group(n=12) and LPS group(n=12) which were fed isocaloric Liber-Decarli liquid diets for 8 weeks. Thereafter, they were fed same diets and challenged with or without LPS(3mg/kg) one time per week for three weeks by tail vein injection.Plasma concentrations of LPS in the portal veins were determined by spectrophotometry. The content of type I collagen A1 and hydroxyproline(Hyp) in liver of rats were measured by ELISA. Immunohistochemistry was used to determine FSP-1 for identification of HSC while F4/80 for recognition of Kupffer cells in liver tissues of rats. Double labeling immunofluorescence stain was performed to detect the expression of TLR4 in FSP-1 positive HSC and the expression of TLR4 or TGF-β1 in F4/80 positive Kupffer cells. IL-1, IL-6, TNF- α and TGF-β1 m RNA from liver samples were detected by Real-Time quantitative PCR(RT-q PCR).The degrees of liver inflammation and fibrosis were evaluated by HE staining and aniline blue staining individually. The HSC of SD rats were isolated by Collagenase-Pronase digestion method to evaluate the effect and mechanism of LPS on TGF-β1-mediated fibrosisin a hepatic stellete cell model.Collagen I, Bambi and p Smad2 protein from HSC were determined by Western blot while Collagen I and Bambi m RNA detected by RT-q PCR.Results: Our results showed that there were obvious edema and inflammatory cell infiltration, enhanced expression of proinflammatory cytokine IL-1, IL-6, TNF-α m RNA and profibrogenic factor TGF-β1 m RNA, extended areas of collagen deposition and increased contents of collagen I A1 and Hyp in liver tissues of rats challenged with LPS compared with the normal control rats. Double immunofluorescence stain showed that FSP-1 positive HSC and F4/80 positive Kupffer cells could express TLR4 and that LPS were able to activate HSC over-expressing TLR4 and Kupffer cells over-expressing TLR4 and TGF-β1. In vitro study showed that LPS could inhibit the expression of Bambi m RNA and protein as a pseudo receptor of TGF-β1 and enhance TGF-β1-mediated Collagen I m RNA and protein synthesis in HSC. However, LPS has no effect on the TGF-β1 /Smad2 signaling pathway in HSC.Conclusions: LPS can activate TLR4 signaling pathway in HSC and Kupffer cells which synthesize proinflammatory cytokines and profibrogenic factor TGF-β1. LPS also enhances TGF-β1-induced Collagen I synthesis in HSC by inhibiting the expression of Bambi.