The Study On The Regulatory Mechanism Of Mucins In Hepatolithiasis

Hepatolithiasis(HL) is one of the most common diseases primarily involving biliary tract in China. With a high recurrence rate after operation and repeated episodes of biliary infection, HL is associated with biliary cirrhosis and even cholangiocarcinoma. The radical cure for HL is dependent upon the revealing of its pathogenesis, which is the focus of this study.Clinical research suggested that over-secretion of mucus caused by chronic proliferous inflammation in intrahepatic bile duct was the sources of lithogenesis. Gel-forming mucins, the important component of mucus, have been observed in calculus and considered as the core components. Recent study indicated that overexpression of gel-forming mucins induced by LPS was the principal pathological phenomenon during hepatolithus formation. It indicated that gel-forming mucins were the key factor in the development of hepatolithus, and it is very important to investigate their up-stream signal transduction event and identify the key regulator for suppressing their activity.The EGFR axis was the main up-stream signal transduction pathway in promoting the overexpression of secretory mucins. TACE dissected the ectodomain of TGF-αand promoted its maturation in epithelial tissue. We presumed that activation of TACE and subsequent maturity of TGF-αwould be the key process for phosphorylation of EGFR and the down-stream signal transduction. Based on previous research, we presumed that the ERGF axis play an important role in the pathogenesis of HL.In present study, MUC1, MUC2, MUC3, MUC5AC, and MUC6 in the intrahepatic bile duct tissue of the HL patients and normal persons were detected using immunohistochemistry method and image analysis. The results showed that there was a variable expression rate of these MUCs in normal intrahepatic bile duct, with the overexpression of the secretory mucin MUC5AC in HL patients. A close correlation between the secretion of MUC5AC and the formation of biliary stone was observed. It prompted us to focus on regulation of MUC5AC expression. It is very important to investigate the up-stream signal transduction event and identify key regulator for suppressing its activity.In vitro, simulating the environment for biliary stone formation, we stimulated HIBEC with LPS, the common risk factor of HL. Then, we detected the expressions of the key factors, TGF-α,TACE and EGFR, in the EGFR AXIS, and the relationship between the secretion of MUC5AC and these regulators were analyzed. Results suggested that the protein level of TACE was significantly reduced, while TGF-αand p-EGFR were significantly up-regulated after stimulation of LPS. Subsequently, the expression levels both mRNA and protein of MUC5AC were increased dramatically. These results confirm our preliminary hypothesis that the EGFR AXIS is one of the primary signal pathways in regulation of MUC5AC expression in HIBEC, and that TGF-α,TACE and EGFR were the key factors in this pathway.To further clarify the regulatory mechanism of MUC5AC expression, various interventions targeted at TACE, TGF-αand EGFR in the "EGFR axis", were administrated to reveal the critical regulating points. The results showed that the activity of TACE was reduced obviously in HIBEC transfected with TACE SiRNA or exposed to TACE blocker TAPI-1, and subsequently the expression levels of TGF-αand p-EGFR were significantly decreased, resulting in the not increase of MUC5AC. If given appropriate exogenous TGF-α, the inhibition of MUC5AC can be reversed. The expression of MUC5AC was significantly reduced by AG1478, the inhibitor of p-EGFR, which can stimulate the expression of MUC5AC without influencing TGF-αlever. These results suggest that LPS secreted from enterogenic gram-negative bacillus could activate TACE, which promotes the phosphorylation of EGFR through the dissection of pre-TGF-α, resulting in the over-expression of MUC5AC in the earlier stages of HL. Among these checkpoints, TGF-α-dependant phosphorylation of EGFR may play a pivotal role in regulation of MUC5AC and dissection of pre-TGF-αby TACE is the rate-determining step in the activation of EGFR AXIS.Our study showed that the activation of EGFR axis and its down-stream signal transduction would be the principal pathway for regulation of mucin expression. It would be effective for inhibiting the formation of calculus to suppress and reverse the overexpression of secretory mucins by preventing EGFR's phosphorylation in biliary epithelial cell. Our findings provide new evidences for the elucidation of the pathogenesis of HL and offer us informative instruction for prevention and cure for HL.