| [Background and Objective]Hepatic fibrosis, characterized by the overproduction and accumulation of hepatic extracellular matrix (ECM), was the common and important pathological change in the chronic liver diseases, through which the chronic hepatitis developed into cirrhosis. Hepatic stellate cell (HSC) has been widely recognized as the main resource of hepatic ECM and the cellular basis for hepatic fibrosis.Recently, many reports have shown that the molecular mechanism for activation of HSC was involved with the mitogen-activated protein kinase (MAPK) signal cascades, which are the pivot of the cell signal transduction through protein phosphorylation. In mammalian cells, three MAPK families have been characterized: extracellular signal-regulated kinase (ERK), Jun kinase (JNK) and p38 MAPK. Extracellular signals, such as growth factors, peptide hormones and neurotransmitters, activate ERK cascades to execute complex cellular programs, like proliferation, differentiation and apoptosis, which involved in the fibrogenesis of many organs, such as liver, kidney, lung and pancreas.90 kDa ribosomal S6 kinases (RSK; also known as p90rsk), which were among the first substrates of ERK, belong to a family of growth factor-activated serine/threonine kinases. A number of cellular functions of RSK have been proposed, such as the regulation of gene expression, cell cycle and protein synthesis. RSK has been proved to be involved in the pathogenesis of some tumors and some other chronic diseases, but there is no report of the relationship between RSK and hepatic fibrosis up to now.The purpose of the present study was to investigate the expression and location of RSK during the development of hepatic fibrosis and the correlation of RSK with ERK and collagen by Northern blot, immunohistochemistry analysis andimmunofluorescent double labeling laser scanning confoal microscope to identify the role of RSK in the pathogenesis of hepatic fibrosis and the relationship between ERK cascades and the pathogenesis of hepatic fibrosis. [Methods]1. Construction of the model of rat hepatic fibrosis with dimethylnitrosamine (DMN)45 male Sprague-Dawley (SD) rats were randomly divided into model group (n=30) and control group (n=15). Rat hepatic fibrosis was induced by inter-abdomen injection of DMN, 10 mg/kg, three times every week for three weeks. The rat livers were obtained for study at the end of one, two, three weeks' injection respectively. The character of hepatic fibrosis was confirmed with HE and VG staining.2. Investigation of the expression of RSK and collagen in the development of rat hepatic fibrosisThe individual samples of RNA were mixed in the same model stage and the mRNA expression of RSK was measured by Northern blot. The protein expression of RSK and collagen type I , III in the hepatic tissues assessed by immunohistochemistry analysis, and the image data were tested by statistical analysis.3. Identification of the correlation between RSK and ERK in HSCa-smooth muscle actin (a-SMA), that is typical of activated HCS, was selected for this study to determine the localization of RSK in fibrotic liver. The co-localizations of RSK with a-SMA, ERK and type I collagen were visualized by immunofluorescent double labeling laser scanning confoal microscope. Image analysis was performed using the standard system operating software provided with the microscope.The expression vector (pAV-ERKlsiRNA), expressing the small interfering RNA (siRNA) targeting to the ERK1 mRNA had been constructed and transfected into HSC-T6 to establish the stable expression cell line (HSC- pAV-ERKlsiRNA) in our prevenient study. The changes of ERK and RSK expression between HSC-T6 and HSC- pAV-ERKlsiRNA were quantitated with Western blot simultaneously.[Results]1. Establishment of the model of rat hepatic fibrosisPathological analysis show that there are increasing hepatocyte necrosis and fibrogenesis with the time of DMN injection. At the end of DMN treatment there are intact pseudo-lobules formed. Our fibrotic model provides... |
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