Unraveling the Role of Toll-Like Receptors (TLR) and Regulators of TLR Signaling in Cholestatic Liver Injury

Biliary atresia (BA) is a complex disorder of unknown origin. Our recent study shows that the oxidative stress marker 8-hydroxydeoxyguanosine (8-OHdG) is highly elevated in the early stage of BA. The elevation of 8-OHdG is positively correlated with the degree of liver inflammation. Our group also reveals a role of toll-like receptors (TLRs) in the pathogenesis of BA. BA is one of the most important cholestatic liver disorders in human being, in which inflammatory cytokines play important roles. Since TLR signaling initiates inflammatory cytokine production, we hypothesize that TLR signaling is implicated in cholestatic liver injury. In order to test the hypothesis, we propose a three-year project that is designed to accomplish the following specific aims: First year We will characterize TLR signaling in cholestatic liver injury and correlate it with the expression of inflammatory cytokines and interferons by 1. Screening the all the members of TLR family and important signaling molecules, including MyD88, TRIF, IRAK, TRAF and IRF with the use of real-time quantitative RT-PCR (QRT-PCR),Western blotting and immunohistochemistry to identify the those molecules of significant difference between the jaundiced rats and the sham rats and correlate with inflammatory cytokine and interferon production. 2. Studying how potential ligands for TLR signaling cholestatic liver, including lipopolysaccharides (LPS) and hydrophobic bile acids, exert its function in hepatic stellate cells (HSC). 3. To characterize the effects of agonist and antagonist of the TLR (for example TLR7 agonist R837 & antagonist IRS661) with significant change during cholestasis on HSC under bile acid treatment. Second year In vivo verification of regulators of TLR signaling and in vitro study of their modulation by 1. Using QRT-PCR, Western blotting and immunohistochemistry to identify negative regulators for adaptor molecules in the signaling pathway, including SOCS1, MyD88s, Triad3A, IRAKM, IRAK2c and IRAK2d in cholestatic rat liver. 2. Employing siRNA of these regulators in HSCs to explore their roles in TLR signaling under the specific condition of LPS or bile acid treatment. Third year Elucidation the effect of corticosteroids on modulation of TLR signaling in cholestatic liver injury by 1. Administration of corticosteroids in cholestatic rats with or without endotoxin cholangitis and study the expression of TLRs and their signaling molecules, as well as regulators. 2. Correlate the above findings with the histopathological changes of the rat liver as well as survival of the jaundiced rats after endotoxin cholangitis. Through the proposed 3-year study, we may provide substantial novel information regarding TLR signaling in the pathogenesis of cholestatic liver injury, which is in turn help us to understand, at least in part, the pathogenesis of BA.

Project ID:PC9709-0928
External Project ID:NSC97-2314-B182-010-MY3