Molekulare Mechanismen der Leberfibrogenese als Grundlage für neue Therapiestrategien der chronischen Hepatitis

Liver fibrosis and its end stage, liver cirrhosis, are the results of repeated chronic liver injury. Regardless of its etiology, it is characterized by a massive accumulation of extracellular matrix proteins and a remodelling of the liver architecture. To date there exists no specific therapy for liver fibrosis and the treatment is restricted on eliminating the causative agent. In this work the effects of Propranolol treatment as a classical drug therapy on the fibrosis progression in primary sclerosing cholangitis (PSC) was evaluated. Therefore the MDR2-/- mouse model was characterized and then chosen as a suitable model for PSC and its treatment. In order to estimate a possible benefit of the Propranolol treatment on PSC, histochemical and immunohistochemical stainings of the livers were prepared. The histological assessment of fibrosis and inflammation showed a significant amelioration in the stage of fibrosis (p<0.05) and grade of inflammation (p<0.05) in the Propranolol treated mice compared to the controls. Transcript level determination of profibrogenic and vasoconstrictive mediators in liver extracts by real-time PCR did not confirm the positive results of the histological assessment. Therefore portal areas and areas in the parenchyma were selectively examined by using laser microdissection technology. The transcript levels of procollagen 1A1 as dominant extracellular matrix protein in fibrotic processes as well as the profibrogenic mediators TGF-ß, TNF-a and CTGF were analyzed. In the portal areas of the treated mice the transcript levels of these mediators were reduced by more than 50% compared to the controls. In addition, the expression of the profibrogenic and vasoconstrictive mediators angiotensinogen, endothelin-1 and their receptors were evaluated. The most pronounced effect was found in changes of the endothelin-1 and endothelin receptor A transcript levels. The expression level of the receptor was reduced by 50% in the Propranolol treated mice in the portal areas, while Endothelin-1 transcripts showed an even higher reduction with 66%. The results of the histological assessment as well as the reduction of profibrogenic mediator transcripts in the portal areas of Propranolol treated MDR2-/- mice showed the positive effect of Propranolol treatment on the progression of liver fibrosis in an experimental PSC model. In a second approach, the role of miRNAs during fibrogenesis was analyzed in order to identify possible new therapeutic targets for treating liver fibrosis. Due to the unknown function of miRNAs in fibrogenesis, the expression of the liver specific miR-122 was tested on an experimental bile duct occlusion model and a hepatitis C patient collective. Both tested collectives showed an increase of fibrosis accompanied by the reduction of miR-122 expression. Moreover, a highly negative correlation between miR-122 expression and hydroxyproline content of the livers(r=-0.786) as well as granulocyte infiltration (r=-0.715) was found. Because myofibroblasts are the crucial cell type of liver fibrogenesis in many chronic liver diseases and in order to identify other important miRNAs during fibrogenesis, in vitro experiments with primary hepatic stellate cells were performed. Using microarray and real-time PCR analysis divergent regulated miRNAs during myofibroblastic differentiation were identified. miR-125b, miR-22, miR-143, miR-221, and miR-222 were the highest upregulated miRNAs during myofibroblastic differentiation, while miR-126 was downregulated. miR-125b was chosen for further examination. A sequence comparison and a reporter assay revealed that miR-125b is the vertebrate homolog of lin-4, a miRNA playing a crucial role in the larval development of the nematode C.elegans. A well known target of lin-4 is the pluripotency factor lin-28. The sequence comparison of the lin-28 3’UTR in different species and the use of a luciferase reporter construct showed that lin-28 is also a target of miR-125b. The expression of lin-28 during myofibroblastic differentiation was opposed to the miR-125b expression and as consequence of the lin-28 downregulation let-7 family members were upregulated. In this study miR-125b was shown to be a miRNA playing an important role in differentiation and setting up an assumption for new therapeutic strategies by its characterisation.