高脂饮食性非酒精性脂肪性肝病大鼠肝脏PPAR-γ表达增强

目的　探讨过氧化物酶体增值物活化受体γ(PPAR-γ)及其亚型在高脂饮食所致非酒精性脂肪性肝病(NAFLD)大鼠肝脏的表达及其意义。方法　模型组SD大鼠给予高脂肪高胆固醇饮食饲养 ,分批于实验第 8、12、2 6、2 4周处死 ,同期设普通饮食饲养大鼠作对照。免疫组织化学和RT-PCR分别检测大鼠肝脏PPAR-γ的表达。结果　模型组大鼠第 8周呈现单纯性脂肪肝 ,第 12～ 2 4周从脂肪性肝炎进展为脂肪性肝炎伴肝纤维化。免疫组织化学和RT PCR显示 ,随着造模时间延长 ,肝脏PPAR-γ的表达逐渐增强。模型组肝脏PPAR-γ1mRNA表达于第 2 4周达到高峰 (与对照组相比升高 3 .5倍 ,P <0 .0 1) ,PPAR-γ2 mRNA表达于造模第 16周时达高峰 (较对照组升高 5 .8倍 ,P <0 .0 1)。相关分析显示 ,仅PPAR-γ2 mRNA与肝脂变程度之间关系密切 (r =0 .89,P <0 .0 5 )。结论　持续 2 4周的高脂饮食可以成功复制大鼠NAFLD模型 ,模型大鼠肝脏PPAR-γ表达增强 ,NAFLD大鼠肝细胞可能部分具有脂肪细胞的特征 ,即脂肪变的肝细胞发生成脂性改变     

己酮可可碱对非酒精性脂肪肝大鼠肝组织线粒体解耦联蛋白表达的影响

目的 探讨己酮可可碱(PTX)对非酒精性脂肪肝性肝病(NAFLD)大鼠线粒体解耦联蛋白(UCP)-2 mRNA表达的影响. 方法 SD大鼠60只,正常喂养1周后随机分为3组,每组20只.其中对照组20只,以普通饲料喂养,实验组和干预组各20只,以高脂饲料喂养,干预组高脂饮食4周后,在饮水中加用已酮可可碱(PTX)(100 mg·Kg-1·d-1),于第24周处死,采用反转录聚合酶链反应(RT-PCR)技术检测肝脏UCP-2mRNA的表达. 结果 对照组大鼠肝脏UCP2mRNA呈微量表达(1.2±0.1),实验组大鼠肝脏UCP2mRNA呈强表达(4.0±0.3),干预组大鼠肝脏UCP2mRNA呈弱表达(3.0±0.2),3组间UCP-2mRNA表达差异有统计学意义(F=160.67,P＜0.01). 结论 己酮可可碱可下调NAFLD大鼠肝细胞UCP-2mRNA的表达.     

消瘀化痰方对非酒精性脂肪肝大鼠肝脏解偶联蛋白2 mRNA表达的影响

目的 观察消瘀化痰方对非酒精性脂肪肝(NAFLD)大鼠肝脏解偶联蛋白2 mRNA(UCP2 mRNA)表达的影响,探讨其防治NAFLD的部分作用机制.方法采用喂饲高脂饲料的方法复制NAFLD大鼠模型,实验分为正常对照组、模型组、东宝肝泰对照组和消瘀化痰方高、低剂量组.提取肝脏总RNA,运用半定量RT-PCR技术观察各组大鼠肝脏UCP2 mRNA的表达情况,同时测定各组大鼠血清总胆固醇(TC)、甘油三酯(TG)、游离脂肪酸(FFA)和肝组织匀浆TC、TG的含量,并做病理组织切片.结果模型组大鼠UCP2 mRNA的表达增强,血脂和肝脏脂质含量明显升高,肝脏呈明显脂肪变性.经药物治疗后,各治疗组大鼠肝脏UCP2 mRNA的表达减弱,血脂和肝脏脂质含量显著降低,肝脂变程度明显减轻.结论消瘀化痰方通过调控NAFLD大鼠肝脏UCP2 mRNA的适度表达,可能是其治疗NAFLD的分子机制之一.     

动态观察非酒精性脂肪肝大鼠肝脏抵抗素的表达

目的观察非酒精性脂肪性肝病(NAFLD)模型大鼠肝脏抵抗素mRNA的动态表达,探讨抵抗素在大鼠NAFLD发病中的作用。方法雄性Wistar大鼠48只随机分为正常对照组(C组)和模型组(M组),C组给予普通饲料,M组给予高脂饮食喂养,分别于9,13,17周末处死各组大鼠。测定大鼠血清肿瘤坏死因子α(TNF-α)、游离脂肪酸(FFA)、甘油三酯(TG)、总胆固醇(TC),以及肝组织TG,测定空腹血糖(FBS)、空腹胰岛素(FINS),并计算胰岛素敏感指数(ISI)。应用半定量RT-PCR检测各组大鼠肝脏组织抵抗素mRNA的表达;HE染色观察肝脏组织病理变化并计算炎症活动度计分。结果第9,13,17周末M组大鼠抵抗素mRNA相对表达量显著高于C组(P<0.01),且随造模时间延长表达量显著增加(P<0.01)。M组大鼠血清FFA、TG、TC、TNF-α及肝组织TG较同期C组均显著升高(P<0.01),ISI显著降低(P<0.01)。相关分析显示,M组大鼠各时点肝脏抵抗素mRNA相对表达量与血清TNF-α水平均呈正相关(r=0.787,0.888,0.873,P<0.05,P<0.01,P<0.01);在第9,13周末与肝脏炎症活动度计分呈正相关(r=0.861,0.892,P<0.01);而与ISI在第9周末呈负相关(r=-0.843,P<0.01)。结论高脂饮食NAFLD模型大鼠肝脏抵抗素基因表达随造模时间的延长而增加,抵抗素可以通过胰岛素抵抗及对炎症因子的调控参与NAFLD的发生发展。     

高脂饮食非酒精性脂肪性肝病大鼠肝脏成脂相关基因表达增强

目的探讨非酒精性脂肪性肝病（NAFLD）大鼠肝脏成脂相关基因mRNA表达的动态变化.方法模型组SD大鼠给予高脂饮食饲养,分批于实验第4、8、12、16、24周处死,同期设普通饮食饲养大鼠作对照.RT-PCR分别检测肝脏固醇调节元件结合蛋白1c（SREBP-1c）及其靶基因脂肪酸合成酶（FAS）、脂联素、抵抗素mRNA表达.结果模型组大鼠4周肝脏可见散在性肝细胞脂肪变性,8周为单纯性脂肪性肝炎,12～24周从脂肪性肝炎进展为脂肪性肝炎伴肝纤维化.从实验第4周起,模型组大鼠肝脏SREBP-1c和FAS mRNA表达逐渐增强,至24周时分别较对照组升高5～6倍和2～2.5倍;模型组大鼠肝脏从第12周起出现脂联素和抵抗素mRNA表达,两者表达量均随造模时间延长而增强.相关分析显示,SREBP-1c、FAS、脂联素、抵抗素mRNA表达量均与肝脂肪变程度呈正相关（r值分别为0.808、0.834、0.592、0.577,P值均＜0.01）.结论高脂饮食NAFLD大鼠肝脏SREBP-1c、FAS、脂联素、抵抗素等成脂基因表达增强,提示脂肪变性的肝细胞可能部分具有脂肪细胞的特征,即发生了成脂性改变.     

高脂饮食对实验性大鼠非酒精性脂肪肝UCP2表达的影响

目的:通过高脂饮食制作非酒精性脂肪肝(NAFLD)动物模型;观察高脂饮食对实验性大鼠NAFLD解偶联蛋白2(UCP2)的影响。方法:通过高脂饮食建立大鼠非酒精性脂肪性肝病模型。观察肝脏病理改变并检测肝脏UCP2表达情况。结果:与正常对照组相比,高脂饮食大鼠肝脏UCP2表达显著上升,肝组织广泛脂肪变性,形成单纯性脂肪肝。继续给予高脂饮食使肝脏UCP2表达水平进一步增加,肝脏发生进一步病理改变而形成脂肪性肝炎。结论:高脂饮食成功地复制了NAFLD动物模型;NAFLD时肝脏UCP2表达上调,可能是机体的一种适应性反应;但是UCP2过度表达,可能诱导或加剧肝脏病理改变。     

非酒精性脂肪性肝病大鼠血浆前列环素与血栓素的变化及其与肝脏损伤的关系

目的 探讨非酒精性脂肪性肝病(NAFLD)大鼠血浆前列环素(PG12)和血栓索(TX)A2的动念变化及其与肝组织学改变之间的关系。 方法 48只模型组SD大鼠给予高脂肪高胆固醇饮食饲养,分批于第8、12、16、24周处死,24只正常饮食大鼠作对照。放射免疫法检测血浆PGI 2和TXA 2的稳定代谢产物6酮-前列环素1α(PGF1 α)和TXB2含量,光镜观察肝组织切片病理学改变。 结果 模型组大鼠8周呈现单纯性脂肪肝,12～24周从脂肪性肝炎进展至脂肪性肝纤维化。模型组大鼠血浆TXB 2在造模第8、24周分别为(52.4±3.15)ng/L和(117.7±7.47)ng/L,对照组为(41.1±1.45)ng/L,t值为9.12和31.34,P<0.01和P<0.001。 血浆PGF1 α水平在造模8、24周分别为(31.1±1.6)ng/L和(3.4±2.4)ng/L,对照组为(36.5±1.7)ng/L,t值为6.27和34.62,P<0.01和,P<0.001。模型组大鼠血浆TXB2和PGF1 α水平分别与其肝组织损伤程度呈显著正相关(r=0.537,P<0.001)及负相关(r=-0.452,P<0.01)。 结论 持续24周的高脂饮食可以成功复制大鼠NAFLD模型,模型大鼠血浆TXA 2与PGI 2平衡失调,可能参与NAFLD的发病。     

非酒精性脂肪性肝病大鼠肝脏纤溶酶原激活物及其抑制物mRNA表达

目的　探讨非酒精性脂肪性肝病 (NAFLD)大鼠肝脏组织型纤溶酶原激活物 (t PA )及纤溶酶原激活物抑制物 1(PAI 1)基因表达及其意义。方法　高脂饮食建立SD大鼠NAFLD模型 ,分批于造模第 8、12、16、2 4周处死 ,同期设普通饮食喂养大鼠作对照。通过H E染色和苦味酸VG染色观察肝组织学改变 ,应用RT PCR对肝脏t PA和PAI 1mRNA的表达进行相对定量分析。结果　模型组大鼠于实验 8、12、2 4周分别形成单纯性脂肪肝、脂肪性肝炎以及脂肪性肝炎并肝纤维化。与对照组相比 ,模型组大鼠肝脏PAI 1mRNA表达随造模时间延长而增强 ,于实验 2 4周达高峰( 1.0 2± 0 .11比 0 .5 1± 0 .0 9,P <0 .0 1) ,并与其肝脂肪变及肝组织学损伤程度呈正相关 (r分别为 0 .492和 0 .3 72 ,P分别 <0 .0 1和 <0 .0 5 )。肝脏t PAmRNA表达随造模时间延长而逐渐减少 ,于实验 2 4周降至最低 ( 0 .89± 0 .11比 1.62± 0 .10 ,P <0 .0 1) ,但其仅与肝组织学损伤程度总积分呈负相关 (r =-0 .3 68,P <0 .0 5 )。结论　高脂饮食大鼠肝脏PAI 1及t PA基因表达改变可能参与NAFLD的发病     

解耦联蛋白2在急性肝功能衰竭大鼠肝脏中的动态表达和意义

目的 探讨急性肝功能衰竭(ALF)大鼠肝脏线粒体解耦联蛋白(UCP)2的表达趋势及意义.方法 健康雄性SD大鼠36只,分为对照组和模型组,模型组冉分为6、12、24、36和48 h 5个亚组,每组6只.模型组腹腔内注射D-氨基半乳糖(D-Gal)和脂多糖(LPS)诱导大鼠ALF模型.采用HE染色,光学显微镜下观察肝组织损伤情况,采用RT-PCR和免疫组织化学检测不同时间点肝脏UCP2 mRNA转录及其蛋白表达,同时测定各时间点血清ALT、AST和肝组织丙二醛(MDA)的变化.各实验组问数值比较采用SNK检验.结果 模型组肝组织呈炎性细胞浸润和明显坏死的ALF特征;模型组ALT、AST、MDA值均明显高于对照组[(24.0±2.0)U/L,(82.3士16.9)U/L,(2.55±0.22)μmol/g],且造模24 h达高峰[(8346.7±1363.1)U/L,(9766.7±1274.1)U/L,(8.34±1.13)μmol/g;均P<0.05];UCP2蛋白和UCP2 mRNA在正常肝组织中几乎不表达,D-Gal和LPS处理后6 h表达均硅著增加(P<0.05),24 h表达最强,且模型组相邻时间点之间差异有统计学意义(P<0.05).结论 成功构建大鼠ALF模型,大鼠ALF时UCP2蛋白和UCP2mRNA的表达水平与肝损伤程度及氧化应激水平有关.     

非酒精性脂肪性肝病大鼠肝脏脂联素受体的表达

目的 探讨非酒精性脂肪性肝病(NAFLD)大鼠肝脏脂联素受体(AdipoR)mRNA的表达.方法 喂养高脂饲料建立NAFLD大鼠模型,分别于2、4、8、12周检测血清生化指标,并取肝组织测肝指数(肝湿重/体重),以RT-PCR法检测肝脏AdipoR1和AdipoR2 mRNA的表达,肝组织冰冻切片苏丹Ⅲ脂肪染色、石蜡切片苏木素-伊红常规染色和Masson三色纤维染色,镜下观察.结果 模型组大鼠2、4、8、12周肝脏AdipoR1 mRNA表达逐步上升,AdipoR2 mRNA表达逐步下降,两者分别于4周、2周开始显著差异于对照组(P均<0.01).模型组肝脏AdipoR2表达与肝指数(r=-0.431,P=0.006)、纤维化评分(r=-0.353,P=0.025)均呈显著负相关.结论 NAFLD大鼠肝脏AdipoR1 mRNA表达增加,AdipoR2 mRNA表达减少,提示肝脏AdipoR表达异常可能参与NAFLD的发病机制.     

Obesity-related fatty liver is unchanged in mice deficient for mitochondrial uncoupling protein 2

Nonalcoholic fatty liver disease (NAFLD), a prevalent condition associated with obesity, has the potential of evolving into end-stage liver disease. The biochemical mechanisms that define the progression of NAFLD are not well known, but reactive oxygen species (ROS) have been implicated in this process. Uncoupling protein (UCP) 2 is a mitochondrial inner-membrane protein that mediates proton leak, uncouples adenosine triphosphate (ATP) synthesis, and negatively regulates ROS production. UCP2 expression is increased in various animal models of NAFLD. Up-regulation of UCP2 may compromise cellular ATP levels and worsen liver damage, or it may be protective by ROS reduction in NAFLD. This study aimed to obtain a definitive answer as to whether increased UCP2 expression contributes to NAFLD. UCP2-/- mice were exposed to obesity by crossbreeding with ob/ob mice and by long-term high-fat feeding to study the effect of UCP2 deficiency on the outcome of NAFLD. Steatohepatitis score of crossbred mice (ob/ob/ko) was similar to that of ob/ob mice at 25 weeks. No compensatory increase was observed in the expression of UCP5 in ob/ob/ko livers. To unmask the effects of absent leptin and its potential proinflammatory actions, steatosis was also induced in UCP2-/- mice by a high-fat diet continued for 6 months. Serum alanine aminotransferase (ALT) levels remained normal, and the steatohepatitis score in UCP2-/- mice was the same as in wild-type controls. We conclude that increased expression of UCP2 in the livers of mice with genetically or diet-induced obesity exerts neither protective nor deleterious effects on the severity of fatty liver disease.     

Peroxisome proliferator activated receptor-gamma in pathogenesis of experimental fatty liver disease

AIM: To study the expression of peroxisome proliferator activated receptor-y (PPARy) in the liver of rats with fatty liver disease (FLD) and to explore the role of PPARy in the pathogenesis of FLD to provide the basis for using PPARy ligand to treat patients with FLD.METHODS: Forty Wistar rats were divided into 4 groups of ten rats each randomly: normal group (group A), alcohol group (group B), fat-rich diet group (group C), alcohol and fat-rich diet group (group D). The rats were sacrificed at the end of the 16th week from the feeding day. Alanine aminotransferase (ALT), tumor necrosis factor-alfa (TNFa)in serum and malondialdehyde (MDA) in liver homogenate were determined; livers were collected for observing pathologic changes by HE, Sudan IV, Masson stain under microscope. The morphologic results were analyzed by picture quantitative analysis technique. The changes of ultrastructure were also examined under electron microscope.The expression of PPARy in liver was detected by immunoh-istochemistry and RT-PCR. The correlations between the expression of PPARy and biochemical indexes, and liver histology were analyzed.RESULTS: The steatosis, inflammation, necrosis and fibrosis were present in livers of different experimental groups,especially in livers of alcohol and fat-rich diet group. The content of immunodetectable PPARy was decreased remarkably in the livers of model rats (group B-D); the level in alcohol and fat-rich diet group (3.43+ 1.48) was significantly lower than that in normal group (18.34+3.73), alcohol group(8.82+2.52) and fat-rich diet group (11.73+2.51) (all P&lt;0.01).The level of PPARy mRNA was also lower in the livers of model rats (group B-D) than in‘livers of controls. The expression of PPARy in rat liver correlated negatively with the degree of its inflammation, necrosis and fibrosis, as well as the level of serum TNFα and the content of MDA in liver homogenates, but not with steatosis or serum ALT.CONCLUSION: Decreased expression of PPARy may play an important role in the development of hepatocellular inflammation, necrosis and fibrosis of rats with FLD. Thus,activating PPARy by its ligand can be anticipated to provide a therapy target for FLD.     

维生素E对糖尿病大鼠肾脏的保护作用

目的探讨维生素E对糖尿病大鼠肾脏保护作用及其可能机制。方法实验动物分为正常对照组、链脲佐菌素诱导的糖尿病未治疗组、糖尿病给予维生素E（20mg.kg-1.d-1）治疗组,共观察8周。测定尿白蛋白排泄量(UAE),内生肌酐清除率（Ccr）、血浆及肾脏组织一氧化氮（NO）、一氧化氮合成酶（NOS）、内皮素（ET）和肾小球蛋白激酶C（PKC）。结果2周时糖尿病未治疗组Ccr［(6.47±1.51)ml·min-1·kg-1］、尿白蛋白排泄量［(15.60±1.64)μg/24h］、NO［(37.30±3.77)μmol/L］、NOS［(34.89±3.83)U/L］及肾小球细胞膜PKC［(86.85±11.37)pmol·min-1·mgprotein-1］明显高于对照组,ET低于对照组。8周时糖尿病大鼠肾小球细胞膜PKC［(84.18±12.14)pmol·min-1·mgprotein-1］仍明显高于对照组,但NO［(22.75±2.89)μmol/L］及NOS［(21.34±1.92)U/L］低于对照组,ET高于对照组。给予维生素E治疗组8周时,Ccr［(4.46±0.49)ml·min-1·kg-1］及尿白蛋白量［(16.31±1.12)μg/24h］显著低于未治疗组,8周时肾小球细胞膜PKC［(65.19±8.83)pmol·min-1·mgprotein-1］,2周时NO［(33.13±3.77)μmol/L］及NOS［(30.16±2.89)U/L］明显低于未治疗组,维生素E治疗组2周时与8周时的NO及NOS下降幅度明显小于未治疗组。结论维生素E通过抑制蛋白激酶C可以纠正糖尿病早期的肾脏高滤过、高灌注,并与抑制肾脏NO合成有关,抑制蛋白激酶C活性对糖尿病肾病防治尤为重要。     

大鼠肝组织核因子-κB活性变化与过氧化物酶体增殖物激活受体γ表达的关系

目的 观察不同病因所致的脂肪性肝病大鼠肝组织核因子-κB(NF-κB)的活性变化,过氧化物酶体增殖物激活受体γ(PPAR γ)的表达,及其相互关系在脂肪性肝病炎症反应中的作用。方法40只Wistar 大鼠随机分为正常对照组、酒精组、高脂组和酒精加高脂4组,每组10只大鼠,16周断头处死,用HE、苏丹Ⅳ、Masson三色染色观察肝组织光镜下的病理改变和超微结构的变化。用电泳迁移率分析、逆转录聚合酶链反应观察各组大鼠肝组织NF-κB的活性变化与PPAR γ mRNA的表达、各生物化学指标间的相互关系。 结果 模型组大鼠肝组织均表现有不同程度的脂肪变性、炎症、坏死和纤维化,以酒精加高脂组病理损害最重。酒精组和酒精加高脂组的NF-κB活性(142±16.32,238±19.14)明显高于正常对照组(73±9.24,F值分别为6.36、17.93,P值均<0.01)和单纯高脂组(84±10.38,F值分别为5.96、16.20,P值均<0.01),酒精加高脂组的NF-κB活性显著高于酒精组(F=6.23,P<0.01),而高脂组和正常对照组比较差异无统计学意义。酒精组、高脂组和酒精加高脂组大鼠肝组织PPAR γmRNA的表达(0.2530±0.069,0.3647±0.082,0.1226±0.054)均较正常对照组(0.8097±0.094)有不同程度的减弱(F值分别为15.43、7.24、21.45,P值均<0.01)。相关分析显示:酒精组和酒精加高     

Effect of manganese on heat stress protein synthesis of new—born rats

AIM: To study the effect of manganese (Mn) on heat stressprotein 70 (HSP70) synthesis in the brain and liver of new-bom rats whose mother-rats were exposed to Mn.METHODS: 32 female rats were randomly divided into fourgroups. One group was administrated with physiologicalsaline only as control group, the other three groups wereadministrated with 7.5, 15 and 30 mg@ kg-1 manganesechloride (MnCl2) by intraperitioneal injection every two daysfor two weeks. After delivery, the mother-rats receivedMnCl2 unceasingly for a week with the same method. Thenthe contents of Mn、 Zn、 Cu and Fe in the livers of the new-bom rats were determined by atomic absorptionspectroscopy; The level of HSP70 in the brains and thelivers of the new-born rats as detected by Westsrn-dot-blotting, and the SOD activities were measuredsimultaneously.RESULTS: The contents of Mn in the livers of new-bom ratsof the experimental groups(respective 1.38 ± 0.18, 2.73 ±0.65, 3.44 ± 0.89μg @ g-1) were significantly increasedcompared with the control group(0.88 ± 0.18μg@ g-1; p ＜0.01); The contents of Fe in the livers of new-bom rats of 15and 30 mg@ kg-1 experimental groups (426 ± 125,572 ± 175μg@g-1, respectively) were significantly increased comparedwith the control group(286±42μg@g-1; P＜0.05); the levelsof Zn in the livers of the new-bom rats of three experimentalgroups( 254 ± 49, 263 ± 47, 213 ± 28μg@ g-1, respectively)were lower than those of the control group(335 ± 50μg@g-1;respective P＜0.05, P＜0.01); and the levels of Cu showedno significant difference among the four groups (threeexperimental groups: 75 ± 21, 68 ± 241 and 78 ± 18μg@g-1;control group: 83 ± 9μg@ g-1; p ＞ 0.05). There was asignificant increase in the levels of HSP70 in the brains ofnew-bom rats of the 30 mg@kg-1 group (19.5 × 103 ± 1.3 ×103A; control group: 14.3 × 103 ± 1.4 × 103A; P＜ 0.01),andthe levels of HSP70 in the livers of new-bom rats of threeexperinental groups(respective 19.6 × 103 ± 3.9 × 103A, 18.5× 103 ± 3.8 × 103A, 22.4 × 103 ± 1.9 × 103A ) also increasedthan control group(13.3 × 103 ± 1.0 × 103A; P ＜ 0.01), butthe SOD activities showed no significant difference amongbrains of the four groups (experimental groups: 5.04 ± 0.43,4.83±0.48, 4.60±0.84 ku@g-1; control group: 4.91 ± 0.37ku@g-1; P＞ 0.05). The SOD activities in the livers of 15mg@kg-1 group(5.41 ± 0.44 ku@g-1) was lower than the controlgroup(5.95±0.36 ku@g-1; P＜0.05).CONCLUSION: While mother-rats were exposed tomanganese, the metabolisms of Mn、Zn and Fe of new- bornrats in the livers were influenced and were situated in astress status, thus HSP70 syntheses is induced in the brainsand livers of new-bom rats, but the mechanism of this effectin the developmental toxicity of Mn remains to he furtherstudied.     

Opposing effects of chronic alcohol consumption on hepatic gluconeogenesis for female versus male rats

BACKGROUND: The impact of chronic alcohol consumption on hepatic gluconeogenesis (HGN) between males and females is unknown. To determine the effects of chronic alcohol consumption (8 weeks) on HGN, the isolated liver perfusion technique was used on 24-hr-fasted male and female Wistar rats. METHODS: After surgical isolation, livers were perfused (single pass) for 30 min with Krebs-Henseleit bicarbonate buffer and fresh bovine erythrocytes with no added substrate (washout period). After the washout period, livers were perfused with lactate (10 mM) and [U-14C]lactate (15,000 dpm/ml) using the recirculation method. RESULTS: There was no significant difference in HGN between males and females fed the control diet. In contrast, the females chronically fed the ethanol diet (FE) had significantly lower HGN rates (2.73 +/- 0.37 micromol/min x g liver protein(-1)), whereas males fed the ethanol diet (ME) had significantly higher HGN rates (4.99 +/- 0.45 micromol/min x g liver protein(-1)) than controls (3.83 +/- 0.34 micromol/min x g liver protein(-1)). Concomitant decreases were also observed for both 14C-lactate incorporation into 14C-glucose and rates of lactate uptake for FE, while corresponding increases were observed for 14C-lactate incorporation into 14C-glucose for ME. The livers from ME were able to convert a greater percentage of the lactate into glucose, resulting in the elevation in gluconeogenic capacity. CONCLUSION: Chronic alcohol consumption lowers the hepatic gluconeogenic capacity from lactate in females and elevates HGN in males.     

罗格列酮对大鼠非酒精性脂肪性肝炎逆转机制的研究

目的 探讨过氧化物酶体增殖物激活受体γ（PPAR-γ）激动剂罗格列酮对库普弗细胞中LPS诱导的IκB激酶-β（IKK-β）表达以及对NASH大鼠肝组织核因子-κB（NF-κB）活性和环氧合酶-2（COX-2）表达水平的影响及意义。方法 采用Ⅳ型胶原酶消化和密度梯度离心的方法分离纯化正常Wistar大鼠库普弗细胞,并应用LPS（1μg／ml）及不同浓度的罗格列酮（10 nmol／L和50 nmol／L）干预培养,收集细胞及培养上清液。体内实验：健康雄性Wistar大鼠随机分为正常组和模型组,后者以高脂饲料（2％胆固醇＋10%猪油＋5％玉米油）喂养复制NASH大鼠动物模型,造模成功后,分别以1 mg·kg^-1·d^-1、4mg·kg^-1·d^-1剂量的罗格列酮和等容积等渗盐水灌胃,实验结束后采集大鼠血清和肝组织标本。RT-PCR检测库普弗中细胞IKK-β、肝组织中COX-2 mRNA的表达,Western blot和电泳迁移率改变分析（EMSA）分别检测库普弗细胞中IKK-β蛋白表达和肝组织NF-κB活性变化,ELISA检测细胞培养上清液及血清中TNFα水平。结果 LPS可显著增高体外培养库普弗细胞中IKK-βmRNA、蛋白的表达及上清液中TNFα水平。模型组大鼠肝组织COX-2 mRNA和蛋白的表达均较正常对照组增强,NF-κB活性与COX-2的表达和血清TNFα水平呈正相关。罗格列酮体外可抑制LPS诱导库普弗细胞中IKK-βmRNA和蛋白的表达,体内通过抑制肝组织NF-κB活化,减少COX-2表达,均表明罗格列酮可通过抑制炎症细胞活化,下调炎症介质基因表达,从而减少了NFα等炎症介质合成、释放,抑制肝脏炎症反应。结论 PPAR-γ特异性激动剂罗格列酮通过干预IKK-β／IκB／NF-κβB／TNFα信号通路而发挥抗炎作用,从细胞分子水平逆转NASH大鼠肝组织的炎症反应,这为临床上有效治疗NASH提供了新思路。     

U230A芯片动态观察非酒精性脂肪性肝病大鼠肝脏基因表达

目的探讨大鼠非酒精性脂肪性肝病（NAFLD）发生过程中肝脏基因表达谱的改变。方法通过持续24周高脂饮食诱导大鼠NAFLD模型，应用U230A芯片检测不同造模时期肝脏基因表达，并设普通饮食饲养大鼠作对照。结果与对照大鼠相比，造模4周和8周时差异表达基因数分别为426条和540条，上调基因主要为细胞内磷酸化酶基因、代谢酶基因、脂肪酸结合蛋白基因，细胞色素P450基因以及细胞转录和分化基因等，下调基因主要为离子通道基因、激素受体基因、细胞黏附基因以及细胞骨架基因等；12周时差异表达基因有501条，其中表达上调352条，除上述基因外，还包括白细胞介素，Toll样受体4等炎症和凋亡相关基因；16周时差异表达的基因有665条，其中上调基因430条，炎症和凋亡相关基因表达进一步增加，且Ⅰ型胶原等纤维化相关基因出现表达上调，而细胞再生相关基因表达下调；24周时差异表达的基因有663条，其中上调基因512条，除上述基因表达差异外，主要包括成纤维细胞生长因子，转化生长因子和胰岛素样生长因子等纤维化相关基因。在所有表达差异的基因中，随着时间进展表达持续上调的基因共128条，其中成脂相关基因10条，代谢酶基因46条，炎症相关基因15条、凋亡相关基因10条，纤维化相关基因16条；持续下调的基因有52条，包括激素受体相关基因6条，细胞再生相关基因5条，电子转运基因11条等。结论高脂饮食大鼠肝脏基因谱呈动态改变，并与NAFLD的组织学进展一致。