高脂饮食对大鼠非酒精性脂肪肝中解偶联蛋白2的诱导作用

背景:至今非酒精性脂肪肝的发病机制不十分清楚,而线粒体膜上的解偶联蛋白可使线粒体氧化磷酸化解偶联,导致线粒体合成ATP效率降低,通过能量代谢的改变,参与脂肪肝的发生。目的:观察解偶联蛋白2在大鼠非酒精性脂肪肝动物模型各时相点的表达规律。设计:随机对照动物实验。单位:解放军第三军医大学大平医院野战外科研究所消化科。材料:实验于2004-11/2005-12在解放军第三军医大学大坪医院野战外科研究所国家重点实验室进行。取清洁级Wistar成年雄性大鼠64只,普通饲料正常喂养1周后,随机分为模型组和正常对照组2组,每组32只,每组又分饲养2,4,8,12周4个时间点,每个时间点8只。方法:模型组大鼠喂高脂饮食(基础饲料88%、猪油10%、胆固醇2%)饲养12周,正常对照组普通饲料喂养,分笼(每笼6只)饲养。主要观察指标:两组于相应时间点麻醉后采血,处死动物,迅速取出肝脏。①免疫组织化学和Westernblot技术检测肝组织中解偶联蛋白2表达变化。②生化检测大鼠血清三酰甘油、游离脂肪酸和丙氨酸氨基转移酶活性。③检测肝组织丙二醛含量。结果:64只大鼠全部进入结果分析。①血清生化指标:模型组饲养4,8,12周时血清三酰甘油、游离脂肪酸和丙氨酸氨基转移酶水平明显高于正常对照组,以8,12周最显著(P<0.01)。②模型组解偶联蛋白2蛋白表达随脂肪肝程度的加重,其表达逐渐增强,而正常对照组几乎阴性表达。③肝组织丙二醛含量:模型组饲养4,8,12周明显高于正常对照组,以8,12周最显著[(4.07±0.15),(4.93±0.14),(5.20±0.20)μmol/g;(3.14±0.20),(3.12±0.18),(3.13±0.16)μmol/g,P<0.01]。结论:随着非酒精性脂肪的形成和程度加重,血清三酰甘油、游离脂肪酸和丙氨酸氨基转移酶及肝组织丙二醛含量增加,解偶联蛋白2表达也逐渐增强,提示高脂饮食可诱导肝细胞表达解偶联蛋白2,使细胞内ATP生成减少,促进脂肪肝的形成和发展。

Inductive effect of high fat diet on uncoupling protein 2 in rats with nonalcoholic fatty liver

BACKGROUND: Up to now the pathogenesy of nonalcoholic fatty liver(NAFL) is unclear. Uncoupling protein (UCP) of mitochondrial membrane can make oxidative phosphorylation uncoupling in mitochondrium, and then result in decreasing the efficiency of ATP synthesis by mitochondrium and participate the progression of fatty liver by changing energy metabolism.OBJECTIVE: To observe the expressive rule of UCP-2 in NAFL rat models at different time phases.DESIGN: Randomized control animal trial.SETTING: Department of Gastroenterology, Research Institute of Surgery,Daping Hospital, Third Military Medical University of Chinese PLA.MATERIALS: The experiment was performed at National Key Laboratory,Research Institute of Surgery, Daping Hospital, Third Military Medical University of Chinese PLA from November 2004 to December 2005. A total of 64 clean-grade Wistar adult male rats were selected. After being fed with common feed for 1 week, the rats were randomly assigned into model group and normal control group with 32 rats in each group. In each group there were 4 time points: feeding for 2, 4, 8 and 12 weeks, 8 ones at each time point.METHODS: The rats in model group received high fat diet (88% basic feed, 10% pork fat and 2% cholesterol) for 12 weeks. Those in normal control group received common feed in different cages (6 rats in each cage).MAIN OUTCOME MEASURES: Blood was collected from anesthetic rats of the two groups at corresponding time point. Animals were killed,and then liver was extracted rapidly. ①Change of expression of UCP-2in liver was determined with immunohistochemical method and Western blot technique. ②Activity of triacylglycerol, free fatty acid (FFA) and alanine aminotransferase (ALT) in serum was measured with biochemical method. ③Content of malondialdehyde (MDA) in liver tissues was measured.RESULTS: Totally 64 rats were involved in the result analysis. ①Biochemical indicators in serum: Levels of triacylglycerol, FFA and ALT in serum were significantly higher in the model group than in the normal control group at weeks 4, 8 and 12, especially at weeks 8 and 12 (P ＜ 0.01). ②The expression of UCP-2 enhanced gradually with the exacerbation of fatt yliver in the model group, while it was negative in the normal control group.③Content of MDA in liver tissues: It was significantly higher in the model group than in the normal control group at weeks 4, 8 and 12, especially at weeks 8 and 12 [(4.07±0.15), (4.93±0.14), (5.20±0.20) μmol/g; (3.14±0.20), (3.12±0.18), (3.13±0.16) μmol/g,P ＜ 0.01].CONCLUSION: With the formation and aggravation of nonalcoholic fatty liver, levels of triacylglycerol, FFA and ALT in serum and content of MDA in liver tissues increase, and the expression of UCP-2 also increase. It is indicated that high fat diet can induce expression of hepatocyte UCP-2,decrease of ATP progression in cells and accelerate the formation and development of fatty liver.