| Background and purposesThe farnesoid X Receptor (FXR) is a member of nuclear homone receptor superfamily. It is highly expressed in the enterohepatic system where it functions as intracellular sensor for bile acids. Ligand dependent FXR activation induces transcriptional responses to coordinately regulate bile acid, cholesterol, triglyceride and glucose metabolism. The major function of FXR is to control bile acid homeostasis by regulating the expression of genes involved in bile acid synthesis, transportation, conjugation and detoxification. Liver cancer is one of the most common malignant tumors in the world with an extremely poor prognosis. Recently growing interest is focused on the associations of liver cancer with metabolic diseases such as obesity, nonalcoholic fatty liver and diabetes mellitus. The possible causal link between metabolic diseases and liver cancer suggests that metabolic dysfunction of liver may be an important step during liver carcinogenesis. In the first part of our study, we identified a novel mouse model of liver cancer development due to the deletion of FXR. We used this animal model to investigate the molecular mechanism of liver tumorgenesis in a background of bile acid metabolic deregulation.Liver regeneration is a process consisting of several well-orchestrated phases and multiple pathways. The regulation of growth of liver transplants appears to follow the same principles as those regulate liver regeneration after partial hepatectomy in the laboratory animals. A recent study showed that bile acid signaling is required for mice normal liver regeneration. Cyp7A1 is the rate-limiting enzyme in the classic pathway of bile acid synthesis. Cyp7a1 is regulated mainly at the transcription level by a number of factors including nuclear receptors, protein kinase C activator, cytokine, growth factor, and importantly, bile acid. In the second part of our study, we use 70% hepatectomy as mice model to investigate the molecular mechanism of bile acid induced inhibition of Cyp7a1 gene expression during mice liver regeneration. Understanding the mechanisms of liver regeneration is crucial for the development of new therapies for many sever liver diseases. MethodsPart one1. Wild-type mice 4 weeks old were treated once with liver carcinogen DEN or PBS, followed by feeding a standard diet or a 0.2% cholic acid diet for 30 weeks. Female FXR–/– mice at 11 months old were fed with a 2% cholestyramine diet for 3 months.2. Mice liver morphological analysis: Gross examination of mice liver ; H&E staining, BrdU staining ,TUNEL staining andα-fetoprotein immunohistochemical were performed on mice liver paraffin slices.3.Total RNAs were isolated from mice livers.IFNγ,TNFα,IL-6 NTCP, CYP7a1, CYP8b, SHP, CyclinD1 and CyclinE1 mRNA was quantified by quantitative real-time PCR.4.Mice Serum was isolated and total bile acids were measured using bile acid L3K assay kit. Serum ALT was measured at the City of Hope Helford Research Hospital.Part two1. Male wild-type, FXR–/–and SHP-/- mice between 8 and 10 weeks old were subjected to 70% hepatectomy. For special feeding, mice were fed with 0.2% cholic acid diet or 2% cholestyramine diet for five days before operation. After surgery, they were kept feeding with the same food until being sacrificed.2. Total RNAs were isolated from mice livers. Cyp7a1, SHP, PXR, c-jun mRNA were measured by Northern Blot and /or Real time quantitative PCR.3. Total and Phosphorylated JNK protein , c-jun protein was determined by Western blot.ResultPart one1. Aging FXR–/– mice spontaneously developed liver tumors.2. Pathological changes including hepatomegaly, degeneration, necrosis, apoptosis and inflammation were found in the livers of aging FXR-/- mice.3. Active proliferation was found in the livers of aging FXR-/- mice.4. Pro-inflammatory genes and cyclin genes were upregulated in the liver of aging FXR-/- mice.5. In the aging FXR-/- mice, bile acid level was elevated in serum and livers and genes involved in the bile acid metabolism were deregulated. 6. 0.2% CA diet promoted liver tumors in wilt type mice pre-treated with DEN. 2% cholestyramine diet reduced the malignant lesions in aging FXR-/- mice.Part two1. During the mice liver regeneration after 70% hepatectomy, Cyp7a1 gene expression was regulated by bile acid signaling in a manner of negative feedback.2. In the early stage of liver regeneration, FXR- and SHP- independent pathways involved in the bile acids induced Cyp7a1 gene suppression. In the late stage of liver regeneration, the inhibition of Cyp7a1 transcription might be in FXR- and SHP- dependent fashion.3. In the acute phase of liver regeneration, Cyp7a1 gene expression was suppressed via the coordination of FXR- dependent and FXR–independent pathways.4. In the acute phase of liver regeneration, JNK signaling pathway was activated via FXR–independent pathways.5. In WT and FXR-/- mice , SHP mRNA level was significantly increased at one hour after 70% hepatectomy, but was dramatically decreased at six hour after operation.6. In the acute phase of liver regeneration, PXR mRNA level showed no significant change in both WT and FXR-/- mice.ConclusionPart One1. The bile acid accumulation due to the deletion of FXR gene might be the predominant contribution to the liver tumorigenesis in FXR-/- mice.2. As FXR-/- mice aged, persistent higher level of bile acids would cause the prolonged liver injury and chronic inflammation.3. During the course of liver damage repair, the infiltration of the inflammatory cells, the upregulation of pro-inflammatory genes, the deregulation of cell cycle and the imbalance of cell split and apoptosis might cause the abnormal proliferation of the parenchyma and mesenchyme cells and finally led to the formation of liver tumor in the aging FXR-/- mice.4. FXR-/- mice is a good mice model for the investigation of the molecular mechanism of hepatocarcinogenesis in a background of metabolic deregulation. Part Two1. During the different stages of mice liver regeneration after 70% hepatectomy, bile acid signaling-induced inhibition of Cyp7a1 gene transcription was via different mechanisms. In the early stage, this inhibition might be via the activation of both FXR dependent and FXR–independent pathways. In the late stage, the regulation of Cyp7a1 gene expression might be mainly in a FXR- and SHP–dependent fashion.2. In the acute phase of mice liver regeneration, Bile acids-activated JNK signaling cascade might contribute to the inhibition of Cyp7a1 expression through FXR–independent pathway. This pathway might display the feature of rapid and short-term regulation and help to manage the suddenly occurred overload of bile acid by 70% hepatectomy via prompt suppression of Cyp7a1 gene transcription.3. In the acute phase of liver regeneration, SHP might be involved in the bile acid- induced inhibition of Cyp7a1 expression via both FXR and JNK mediated pathways. SHP gene expression was modulated by a self negative feedback regulation.4. In the acute phase of liver regeneration, PXR signaling pathway might not be involved in the bile acid- induced suppression of Cyp7a1 expression.
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