| Background and aims:Non-alcoholic fatty liver disease(NAFLD)has been reported to be the most common chronic liver disease throughout the world owing to the increased prevalence of obesity and metabolic syndrome,up to 25%of individuals with NAFLD would develop non-alcoholic steatohepatitis.To date,pharmacotherapy available for the treatment of NASH is limited,therefore it is necessary to clarify the pathogenesis of NASH to prevent further progress of the disease.An obvious feature of NASH is infiltration of inflammatory cells,and macrophages are suggested to be the first barrier against hepatocyte injury.Therefore,it is particularly important to explore the role and function of liver macrophages in the pathogenesis of NASH.However,as far as we know,the systemic analysis of alterations of liver macrophage population in the progression of NASH has not been performed.The aim of the study was to investigate the role of liver macrophages in the development of liver inflammation and injury caused by non-alcoholic steatohepatitis,and explore the effect of free fatty acids on macrophage polarization through Tim-3,These findings are likely to provide an ideal starting point and opportunity for further research on the pathogenesis and clinical intervention of NASH.Methods:1.Evolving alterations of hepatic macrophage population in the disease progression of non-alcoholic steatohepatitis in a mouse model An experimental mouse model of NASH was established by MCD diet feeding,during the 4 weeks of MCD diet,hepatic tissues and serum were collected,the level of steatohepatitis were detected by serum ALT and AST level and by histological analysis of liver tissue determined by H&E staining.Hepatic mononuclear cells were isolated from experimental mouses,the proportions of CD11b+F4/80+ and CD206+F4/80+ were detected weekly by flow cytometry.In adition,qRT-PCR were used to investigate the change of Arg-1,FIZZ and iNOS mRNA?2.Mechanism of fatty acids induced change of macrophage polarization MCD diet was used to establish the NASH model,and detected the change of Tim-3 on F4/80+ hepatic macrophageoFree fatty acids were used to stimulate the THP-1?Raw264.7 and human peripheral blood monocyte,Investigate the change of Tim-3 and HLA-DR by flow cytometry.Detect the change of HLA-DR on Tim-3+THP-1 cell after FFA treating.Observe the change of CD206 on M-CSF induced monocyte after FFA treating.Results:1.Evolving alterations of hepatic macrophage population in the disease progression of non-alcoholic steatohepatitis in a mouse model(1)During NASH progression,the percentage of F4/80+ macrophages in liver MNCs(mononuclear cells)increased on the 1st week and kept on a high level in the following weeks.(2)The increased macrophages were CD11b+ F4/80+,which were thought to be recruited from the peripheral blood.(3)CD206+ F4/80+ M2 macrophage increased and peaked on the 1st week,and gradually fell to a level lower than normal at the 4th week.(4)The level of serum pro-inflammatory cytokines such as TNF-? and IL-6,and anti-inflammatory cytokines such as IL-10,was altered in parallel with macrophage polarization.2.Mechanism of fatty acids induced change of macrophage polarization(1)The level of Tim-3 on F4/80+ hepatic macrophage increased and peaked on the 1st week,and gradually fell to a level lower than normal at the 4th week.(2)FFA can inhibit the expression of Tim-3 on macrophage,and promote macrophage polarizate to M1.(3)The activation role on THP-1 cell of FFA was weaken through overexpression of Tim-3.(4)FFA can make M2 macrophage polarizate to M1Conclusions:1?During NASH progression,there is a increase of recruitment of bone marrow drived macrophage to the liver and a transfer from the early anti-inflammatory M2 type to the pro-inflammatory M1 type coincided with the hepatic inflammation and necrosis2?FFA can promote macrophage polarizate to M1 through depressing the expression of Tim-3... |
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