【作者】 杨玲；

【导师】 刘学忠；

【作者基本信息】 扬州大学 ， 临床兽医学， 2020， 硕士

【摘要】 非酒精性脂肪肝是以肝实质细胞脂肪变性和脂质过度沉积为主要特征的脂质代谢综合征,棕榈酸是游离脂肪酸中含量最高的饱和脂肪酸,大量游离脂肪酸不及时代谢积聚肝脏可诱导肝细胞发生脂肪变性,但棕榈酸诱导肝细胞脂肪变性的发生机制尚未明确。烟酸在哺乳动物体内转化为有生物活性的烟酰胺,参与机体碳水化合物、脂类合成和分解代谢过程,有研究表明烟酸可减少甘油三酯合成,减轻脂肪变性程度,但其作用机制尚不清楚。本研究以棕榈酸诱导BRL 3A细胞脂肪变性建立非酒精性脂肪肝体外模型,揭示棕榈酸致BRL 3A细胞脂肪变性的分子作用机制,并探究烟酸对BRL 3A细胞脂肪变性是否具有保护作用并且揭示其保护机制;为临床非酒精性脂肪肝病及畜禽肝脏脂质代谢紊乱性疾病的防治提供新的理论依据。1.棕榈酸致BRL 3A细胞毒性损伤作用为研究棕榈酸致BRL 3A细胞毒性损伤作用,本试验采用CCK-8法、实时无标记细胞分析技术(RTCA)检测棕榈酸对BRL3A细胞增殖毒性的影响;免疫荧光(IF)染色法观察细胞核及骨架形态的变化;油红O染色观察脂滴生成情况并测定TG含量;透射电镜(TEM)观察细胞超微结构变化及脂滴生成情况;建立BRL3A细胞脂肪变性模型。结果显示:与对照组相比,0.2、0.4 mM棕榈酸对细胞增殖无影响,0.6 mM棕榈酸抑制细胞增殖;随棕榈酸浓度升高,细胞核发生皱缩、变形、碎裂,细胞骨架被破坏,微丝断裂;0.4 mM棕榈酸处理细胞24 h,脂滴大量蓄积,发生明显脂肪变性;TG含量随棕榈酸浓度升高呈极显著性增加(P<0.01);0.4mM棕榈酸处理后细胞核膜受损,核皱缩变形,线粒体脊断裂,细胞质中大量脂滴生成。结果表明:棕榈酸可致BRL 3A细胞形态损伤并诱导脂质沉积,发生脂肪变性。2.棕榈酸致BRL3A细胞脂肪变性机制的研究为探究棕榈酸诱导BRL3A细胞脂肪变性机制,用不同浓度(0.2、0.4、0.6mM)棕榈酸处理细胞24 h,采用流式细胞术(FCM)检测ROS含量;0.4 mM棕榈酸处理细胞24 h,IF染色后用高内涵细胞成像技术检测ChREBP胞核转位情况;qRT-PCR检测ChREBP、Acaca、Fasn、Dgat1、Dgat2 转录水平;Western blot 检测 ChREBP、ACC、FAS、DGAT1、DGAT2表达水平;并用ChREBP-siRNA敲减ChREBP表达后检测ACC、FAS、DGAT1、DGAT2蛋白表达水平。结果显示:与对照组相比,随棕榈酸浓度升高,细胞内ROS水平显著性(P<0.05)或极显著性(P<0.01)增加;0.4 mM棕榈酸处理细胞24 h后ChREBP核/胞浆荧光值极显著性增加(P<0.01);ChREBP转录水平极显著性升高(P<0.01),Fasn、Acaca、Dgat1、Dgat2转录水平显著升高(P<0.05);总ChREBP蛋白表达显著升高(P<0.05),核内 ChREBP 及 FAS/AC/DGAT1/DGAT2 蛋白表达均极显著性升高(P<0.01);si-ChREBP(50 nM)转染36h时,ChREBP mRNA转录水平极显著性降低(P<0.01),蛋白表达显著性降低(P<0.05),ACC、FAS、DGAT1、DGAT2蛋白表达显著降低(P<0.05);结果表明:棕榈酸可引起BRL3A细胞氧化应激;并通过提高ChREBP表达及核转位效率,上调ACC/FAS/DGAT1/DGAT2表达水平,促进TG合成,诱导脂肪变性。3.烟酸在棕榈酸致BRL 3A细胞损伤及脂肪变性中的作用及其机制为探究烟酸在棕榈酸致BRL 3A细胞损伤及脂肪变性中的作用及其机制,采用CCK-8及RTCA筛选烟酸处理浓度;IF观察棕榈酸和烟酸共处理对细胞核及骨架的影响;油红O染色观察脂滴生成情况及测定TG含量;TEM观察细胞超微结构变化情况;FCM检测ROS含量;IF 检测 ChREBP 胞核转位情况;qRT-PCR 及 Westrern Blot 检测 ChREBP、Acaca/ACC、Fasn/FAS、Dgat1/DGAT1、Dgat2/DGAT2mRNA转录水平及蛋白表达水平。结果显示:与对照组相比,0.6mM烟酸促进细胞增殖,1.0 mM烟酸作用24h对细胞增殖活性无影响;与棕榈酸组相比,共处理组细胞核损伤程度减轻,微丝断裂程度减轻,骨架完整;脂滴生成量减少,TG含量显著性降低(P<0.05);与对照组相比,棕榈酸组细胞内ROS水平显著性(P<0.05)增加;与棕榈酸组相比,共处理组细胞内ROS水平极显著性(P<0.01)降低;与对照组相比,棕榈酸组ChREBP核/胞浆荧光值极显著性增加(P<0.01);与棕榈酸组相比,共处理组ChREBP荧光比值极显著性降低(P<0.01);与棕榈酸组相比,共处理组ChREBP、Acaca、Dgat1转录水平极显著性降低(P<0.01),Fasn、Dgat2转录水平显著降低(P<0.05);与棕榈酸组相比,共处理组ChREBP表达显著降低(P<0.05),核内ChREBP蛋白表达极显著降低(P<0.01),FAS、ACC、DGAT2表达极显著性降低(P<0.01),DGAT2表达显著降低(P<0.05)。结果表明:1.0 mM烟酸对BRL 3A细胞无损伤作用,可缓解由棕榈酸所致BRL 3A细胞损伤;降低氧化应激水平;减少棕榈酸诱导BRL 3A细胞脂滴生成量,减少TG含量;并通过下调ChREBP蛋白表达水平及抑制ChREBP胞核转位,下调脂肪合成下游通路ACC、FAS、DGAT2蛋白表达,缓解由棕榈酸所致BRL3A细胞脂肪变性程度。更多还原

【Abstract】 Nonalcoholic fatty liver is a lipid disorder metabolic syndrome characterized by steatosis and excessive lipid deposition in liver parenchyma cells.Palmitic acid is the highest content of saturated fatty acids in free fatty acids.A large number of free fatty acids can not be metabolized and accumulated in time to induce steatosis in hepatocytes,but the mechanism of steatosis induced by palmitic acid in hepatocytes is not clear.Niacin is transformed into bioactive nicotinamide in mammals and participates in carbohydrate,lipid synthesis and catabolism.Some studies have shown that niacin can reduce triglyceride synthesis and the degree of steatosis,but its mechanism is not clear.In our study,palmitic acid was used to induce steatosis in BRL 3A cells to establish a non-alcoholic fatty liver model in vitro.To reveal the molecular mechanism of steatosis in BRL 3A cells induced by palmitic acid,to explore the protective effect of niacin on steatosis and its protective mechanism,and to provide a new theoretical basis for the prevention and treatment of nonalcoholic fatty liver disease and liver lipid metabolism disorder in livestock and poultry.1.Effect of palmitic acid on toxic damage of BRL 3A cellsTo explore the toxic effect of palmitic acid on BRL 3A cells,CCK-8 method and real-time label free cell analysis technique were used to determined the proliferative toxicity,immunofluorescence staining was used to observe the morphological changes of nucleus and skeleton,and oil red O staining was used to observe the formation of lipid droplets and the content of TG,transmission electron microscope was used to observe the ultrastructural changes.The results showed that compared with the control group,0.2,0.4 mM palmitic acid had no effect on cell proliferation;0.6 mM palmitic acid inhibited cell proliferation.With the increase of palmitic acid concentration,the nucleus shrank,deformed and fragmented,the cytoskeleton was destroyed and the microfilament was broken.0.4 mM palmitic acid treatment for 24 hours,lipid droplets accumulated and obvious steatosis occurred.With the increase of palmitic acid concentration,the nucleus shrank,deformed and fragmented,the cytoskeleton was destroyed and the microfilament was broken.And the TG content increased significantly with the increase of palmitic acid concentration;the nuclear membrane was damaged,the nucleus wrinkled and deformed,the mitochondrial ridge was broken,and a large number of lipid droplets were formed in the cytoplasm.The results showed that palmitic acid could damage the morphology and induce lipid droplet accumulation.2.Study on the mechanism of steatosis induced by palmitic acidTo investigate the mechanism of BRL 3 A cells steatosis induced by palmitic acid;the cells were treated with different concentrations of palmitic acid(0.2,0.4,0.6 mM)for 24 h,and the ROS content was determined by flow cytometry.The cells were treated with 0.4 mM palmitic acid for 24 h,evaluated the nuclear translocation of ChREBP;and the transcription and expression of ChREBP,Acaca/ACC,Fasn/FAS,Dgatl/DGAT1 and Dgat2/DGAT2 were determined by qRT-PCR and Western blot.The results showed that the intracellular ROS level increased significantly(P<0.05 or P<0.01)with the increase of palmitic acid concentration;and the ratio of ChREBP fluorescence in intracellular/nuclear increased significantly after treatment with 0.4 mM palmitic acid for 24 h.And the transcription and expression of ChREBP,Acaca/ACC,Fasn/FAS,Dgatl/DGAT1 and Dgat2/DGAT2 were increased significantly(P<0.05 or P<0.01).At36 after si-ChREBP transfection,the transcription level of ChREBP and the protein expression of ACC,FAS,DGAT1 and DGAT2 decreased significantly(P<0.05 or P<0.01).The results showed that palmitic acid could increase the expression of ACC/FAS/DGAT1/DGAT2,promote TG synthesis and induce steatosis by up-regulating the expression of ChREBP and improving the efficiency of nuclear translocation.3.The role of niacin in palmitic acid-induced BRL3A cells injury and steatosis and its mechanismTo investigate the role and mechanism of niacin in toxic damage and steatosis of BRL 3 A cells induced by palmitic acid.CCK-8 and RTCA were used to screen niacin concentration;IF was used to observe the effect of palmitic acid and niacin co-treatment on nucleus and skeleton;and determined nuclear translocation of ChREBP.And observed lipid droplet production and cell ultrastructural changes;determined the TG content;the transcription and expression of ChREBP,Acaca/ACC,Fasn/FAS,Dgatl/DGAT1 and Dgat2/DGAT2 were determined by qRT-PCR and Western Blot.The results showed that compared with the control group,1.0 mM niacin had no effect on cell proliferation for 24 h.The intracellular ROS level in co-treatment group was significantly lower than palmitic acid group.The degree of nuclear damage,microfilament breakage and cytoskeleton integrity,lipid droplet production and TG content in co-treatment group were significantly lower than palmitic acid group.The ratio of ChREBP fluorescence in co-treatment group was significantly lower than palmitic acid group,the transcription and expression levels of ChREBP,Acaca/ACC,Fasn/FAS,Dgatl/DGAT1,Dgat2/DGAT2 in co-treatment group were significantly lower than palmitic acid group.The results showed that 1.0 mM niacin could alleviate the damage of BRL 3 A cells induced by palmitic acid,reduce the lipid droplet production of BRL 3A cells induced by palmitic acid,reduce the content of TG,and down-regulate the expression of ChREBP protein and nuclear translocation of ChREBP,thus down-regulate the expression of ACC,FAS and DGAT2 proteins and alleviate the degree of steatosis of BRL 3 A cells induced by palmitic acid.更多还原