| Alcoholic liver disease(Alcoholic Hepatitis) is due to the long-term heavy drinking.Fatty liver usually presents at the early stage, then it can develop into alcoholic hepatitis, liver fibrosis and cirrhosis. Alcoholic liver disease is one of the common liver disease, which is a seriously damage to people’s health. In recent years, proportion of alcoholic liver disease accounts for the hospitalized patients is on the rise at the same period, and thus the study of alcoholic liver disease is especially important. As a settlement macrophages in the liver, Kupffer cells(KCs) play a very important role in the pathogenesis of alcoholic liver disease. And it can phagocytosis also has secretory function, so it can cause damage to the liver through a variety of ways. With its unique plasticity and heterogeneity, macrophages may change the function and phenotype according to the external environment. As an "adaptive response" genes, ATF-3(ATF/cyclic adenosine monophosphate(cAMP) response element binding protein(ATF/CREB) family of transcription factor 3) is used by cells in order to adapt to external and/or intracellular changes. It can still regulate macrophage function and inflammatory response associated with signaling pathways.1.Establish the model of alcoholic fatty liver in mice and isolate the biological activity kupffer cells(KCs)in order to study its role in the alcoholic fatty liver disease.C57BL/6 mice were fed with the Lieber-DeCarli diet for 16 days plus one time acute alcohol lavage to establish the model of alcoholic fatty liver. Tissue specimens were collected on the sixteenth day after gastric lavage 9 h later. To verify whether the model was established successfully by detecting the level of serum alanine aminotransferase/glutamicoxalacetic transaminase(ALT/AST)and total cholesterol/ triglycerides(TG/TC), the level of TG/TC of liver tissue homogenate and liver pathological section HE and oil red staining. KCs were isolated by in situ perfusion, and the cell yield and cell changes were detected by flow cytometry. Cytokine levels of organization and the primary cell were detected by q-PCR. The model response to liver injury index of ALT/AST was higher than that of control group,meanwhile HE and oil red staining results are consistent with that. So the alcoholic fatty liver model was successfully established in mice. Cell yield of each mice was about 1.5×106~2.0×106, KCs were double-labeled by murine macrophage cell surface marker F4/80 molecule and white blood cells surface antigen molecules CD45, F4/80 and CD45 double positive cells were selected to flow analysis. Data showed that natural CD68+ was significantly lower in the model, a large number of the infiltration of mononuclear cells were elevated. Florescent real-time quantitative-PCR(q-PCR) results showed that cytokine,tumor necrosis factor(TNF-α), interleukin-6(IL-6),monocyte chemoattractant protein(MCP-1) level increased significantly in liver tissue and primary cell. The model of alcoholic fatty liver was successfully established and there was a high yield of cells in situ perfusion. The incidence of alcoholic fatty liver may relative with hepatic macrophages constitute and phenotypic changes, associated with elevated cytokine mediated infiltration of peripheral mononuclear cells.2. RAW264.7 cells were treated by IFN-γ(10 ng/ml) + LPS(10 ng/ml) for 6 h and IL-4(15 ng/ml) for 48 h to establish the cell model of M1 and M2 polarization.Using Western blot and q-PCR to detect the expression of iNOS and Arg1 of RAW264.7 cells. The cytokine TNF-α,TGF-β,IL-6,IL-10 mRNA expression was detected by q-PCR, and cytokineIL-12,IL-10 expression levels of cell culture supernatant of each group was detected by ELISA. q-PCR and Western blot showed that, after stimulated by IFN-γ(10 ng/ml) + LPS(10 ng/ml) for 6h, M1 marker iNOS protein levels were significantly elevated, TNF-α, IL-6 mRNA levels significantly increased. Meanwhile after IL-4(15 ng/ml) stimulated for 48 h, the significantly elevated levels of Arg1, TGF-β and IL-10 mRNA expression levels were also showed. At the same time, ELISA showed that IL-12 levels were significantly increased in the in the supernatant of M1-type cells, while the IL-10 levels were also significantly increased in M2 type.3. The influence of ATF3 for macrophage polarization induced by IFN-γ(10 ng/ml) + LPS(10 ng/ml) and IL-4(15 ng/ml). Detecting the level of ATF3 of liver tissues in alcoholic fatty liver mouse model by Western blot.Western blot and q-PCR were used to detect the expression of ATF3, STAT3 and phosphorylation STAT3 of RAW264.7 cells. By using q-PCR to detect the cytokine TGF-β,IL-10 mRNA expression and ELISA to detect the expression of cytokine IL-12 and IL-10 in cell culture supernatant. ATF3 siRNA were transfected into RAW264.7, by using the specific use LipofectamineTM 2000.ATF3 was downregulated and its activity was reducd. It was found that, in mice alcoholic fatty liver model,liver tissue ATF3 expression levels were significantly decreased. and the expression of ATF3 had significantly higher expression level and the STAT3 phosphorylation also increased in M2 type. After transfection ATF3 siRNA, the M2 type of polarization was significantly inhibited, the expression levels of Arg1 and phosphorylation of STAT3 were both downregulated. |
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