The Activation Pattern Of Kupffer Cells In Nonalcoholic Fatty Liver Disease

Background and Objective:Recently, with the development of economy and the modification of life style, nonalcoholic fatty liver disease(NAFLD) has been one of the most important causes of chronic liver disease in our country. NAFLD is a spectrum of progressively linking conditions from simple hepatic steatosis (NAFL) to nonalcoholic steatohepatitis (NASH) with extensive fibrosis leading to cirrhosis and eventually in some cases, hepatocellular carcinoma (HCC).The pathogenesis of NAFLD is still unclear. As our knowledge is expanding on the role of macrophages in danger recognition, immune tolerance, and lipid homeostasis, the significance of liver macrophages in NAFLD is increasingly appreciated. Kupffer cells represent are strategically located in the liver sinusoids, which provide the anatomical structure for capillary-level confluence of portal vein and hepatic artery tributaries. Activated Kupffer cells are enabled to induce liver injury by releasing a variety of biologically active mediators including cytokines, chemokines, proteolytic enzymes, ROS, and nitric oxide.Two main macrophage phenotypes have been suggested, mirroring the Thl/Th2polarization scheme. These include the classically activated (or M1) macrophages or the alternatively activated (or M2) macrophages. Thl-related cytokines like IFNyas well as microbicidal stimuli such as lipopolysaccharides (LPS) polarize macrophages to an M1phenotype. M1macrophages are engaged in inflammatory, microbicidal and tumoricidal activities. In contrast, Th2cytokines like IL-4and IL-13polarize macrophages to an alternatively activated (or M2) phenotype. These macrophages dampen inflammation, promote tissue remodeling and repair, and help in angiogenesis and fibrosis. KCs transiting phenotypes between the two states may affect the mechanism of NFALD.This research aims to explore the role of KCs in the pathogenesis of NAFLD in vivo.Methods:(1) Animal and Diet:Male C57BL/6J mice (6-8weeks) were individually housed and fed either a standard chow diet(SCD) or a high-fat liquid diet(HFD). All the mice were fed the diets ad libitum and maintained under a12-h light/dark cycle for the entire duration of the feeding protocol. In order to explore exact times that needed, the mice were studied4,8or12weeks after being placed on the diet, at which times steatosis is evident. There were at least4mice in each experimental group. The general situation of the mice, serum parameters, liver biopsy and liver TG content were evaluated.(2) Isolation of hepatic nonparenchymal cells by flow cytometry sorting:Mice were anesthetized after overnight fast, livers were first digested in situ and then digested in vitro. The cell suspension underwent differential centrifugation to remove HC, non-parenchymal cells were obtained by density gradient centrifugation. KCs were sorted by flow cytometry, labling CD45-APC and F4/80-PE antibody. The cells that present CD45+F4/80+were KCs.(3) Oil red O staining:Cell monolayers were washed twice with PBS and fixed for15minutes with12%neutral formalin. Then staining in Oil Red O solution for 15minutes and washing with60%isopropanol. Subsequently, counterstaining with hematoxylin for5minutes and washing thoroughly in running tap water.(4) RNAs were extracted using the Ultrapure RNA kit (Kangwei, CM0581) and performed following the manufacturer’s instructions.RT-PCR amplification mixtures (10μl) containedl0.5μl template cDNA, SYBER Green master mix buffer (Quanti-Tect, Qiagen, Hilden, Germany) and400nM (10pmol/reaction) sense and antisense primer.Reactions were run on a EcoTM Real-Time PCR detector. The results were analysed by EcoTM version3.1. The expression of genes of interest was normalised to the housekeeper gene (GAPDH) and calculated using AACt method.Results:(1) After4weeks of HFD treatment, the hepatic steatosis was mild. After8,12weeks of HFD treatment.approximately30%-60%of all hepatic parenchymal cells were filled with multivesicular fat(mainly microvesicular), but no necroinflammatory lesion was observed. The HFD mouse liver exhibited clear signs of steatosis and had TG levels that were higher than the liver TG levels of SCD mice(2) The purity of KCs can reach to more than95%using flow cytometry.(3) A number of enlarged lipid droplets accumulated in the cytoplasm of KCs from HFD-fed mice.(4)Hat fat-laden KCs produced higher level of pro-inflammatory cytokines, biasing to M1phenotypeConclusions:(1) The percentage of Kupffer cells were not changed during hepatic steatosis.(2) Kupffer cells experienced morphological changes in hepatic steatosis, that was became foam cells. (3) The activation pattern of Kupffer cells in fatty liver was trending to classical activation, which may promote the progress of NAFLD.