Inflammatory Cytokines Exacerbate Lipid Accumulation And Injury In Primary Hepatocytes Via Up-regulating Srebp-1

PART ONE：THE ISOLATION AND CULTURE METHOD OFMOUSE PRIMARY HEPATOCYTESObjectives: Liver is a very essential organ for lipid metabolism. Theisolation and culture method of mouse primary hepatocyes was estabishedfor subsequent lipid metabolism of liver.Methods: The mouse primary hepatocytes were isolated by a two-stepperfusion with type IV collagenase in situ. The trypan blue staining wasused to detect cells viability. The attachment of primary hepatocytes wasobserved at different time points. Albumin in supernatant which wassecreted by primary hepateocytes had been detected at different time pointsfor a week. Intracellular Albumin and glycogen was examined byimmunocytochemistry staining and PAS staining for the identification ofhepatocytes.Results: trypan blue results show: the highest cells viability was up to90%. The fresh isolated cells are almost alone and stereo. after4hours, most of hepatocytes have been attached. A typical dual-nucleus structure and flatcytoplasm were observed in hepatocytes. After1day, dual-nucleus structurewas more obvious and nucleoluses were clearly visible and cytoplasm wasmore flat. after2days,cell cytoplasm was turn into polygon, and maintainthis form for several days. Almost all of the isolated cells were primaryhepatocytes by the identification of PAS staining andimmunocytochemistry staining for glycogen and albumin. Albumin haddeferent levels in supernatant at different time points in a week.Conclusion: primary hepatocytes from mouse were successful isolatedand cultured and could be used for subsequent research in a week. PART TWO: EFFECTS OF INFLAMMATION CYTOKINES ONLIPID ACCUMULATION AND INJURY IN PRIMARYHEPATOCYITESObjectives: in recent years, chronic systemic inflammation has a veryimportant role in the metabolic disease. In this study we will discuss whetherinflammatory cytokines increase TG and FFA levels and cause damage inmouse primary hepatocytes.Methods: The cells were treated with inflammatory cytokines (TNF-α or IL-6) in the absence or presence of palmitic acid (PA). Lipid accumulationwas observed by Oil red O staining and quantitative detection of intracellularTG and FFA. Oxidative stress status was determined by H2O2and ROS tests.ER stress was detected by IRE1, ATF6, GRP78mRNA levels.Results: Oil Red O staining and quantitative Measurement of TG andFFA demonstrated that inflammatory stress increased lipid accumulation inthe primary hepatic cells with or without palmitic acid loading. Results alsoshowed that inflammatory cytokines can increase H2O2and ROS levels andIRE1, ATF6, GRP78gene expression levels.Conclusion: inflammatory cytokines cause lipid accumulation anddamage in mouse primary hepatocytes. PART THREE: THE EXPRESSION OF SREBP-1AND ITSDOWNSTREAM TARGET GENGE IN PRIMARY HEPATOCYTESUNDER INFLAMMTORY STRESSObjective: SREBP-1, a nuclear transcription factor, which is abundantin liver, regulates the synthesis of fatty acids and triglyceride in the liver viaregulating the expression of its downstream target genes ACC alpha and FAS. Our study is to observe the expression of SREBP-1and ACC alpha andFAS in primary hepatocytes under inflammatory stress.Methods： The mouse primary hepatocytes were treated withinflammatory cytokines (TNF-α or IL-6) in the absence or presence ofpalmitic acid (PA).The mRNA and protein expression of SREBP-1and ACCalpha and FAS in primary hepatocytes were determined by real-time PCRand Western blotting.Results：Our data shows that Inflammatory cytokines can up-regulatethe mRNA and protein expression of SREBP-1and ACC alpha and FAS inmouse primary hepatocytes with or without palmitic acid loading.Conclusion：the expression of SREBP-1and ACC alpha and FAS in mouseprimary hepatocytes can increase under inflammatory stress.