肝脏表达Niemann-Pick C1-Like 1调节肝X受体诱导的小鼠胆固醇分泌

目的:探讨小鼠肝脏过表达人NPC1L1对LXR诱导的小鼠胆固醇分泌的影响。方法:给予野生型小鼠(WT)和肝脏过表达人NPC1L1的转基因小鼠(L1Tg)胃饲LXR激动剂(T0901317)7 d后,抽提小鼠粪便总脂质并检测核心甾醇的含量,检测小鼠的血浆脂质水平,分析肝脏ABCG5和ABCG8的mRNA表达水平。结果:L1Tg小鼠与WT小鼠相比,除血浆游离胆固醇含量显著升高外,粪便核心甾醇含量及肝脏ABCG5和G8的mRNA水平差异无统计学意义;在胃饲T0901317 1 w后,WT小鼠的粪便核心甾醇含量由[(3.22±0.44)升高到(28.68±1.05)μmol.d-1.100 g-1],血浆总胆固醇、游离胆固醇、胆固醇酯和磷脂的含量均显著升高,同时肝脏的ABCG5和G8的mRNA水平也分别上调了5倍和2倍;然而,L1Tg小鼠经T0901317处理后,与T0901317处理的WT小鼠相比,粪便核心甾醇的分泌减少了56%,血浆游离胆固醇含量升高了40%,肝脏的ABCG5和G8的mRNA水平分别降低了52.4%和40.6%。结论:肝脏特异表达人NPC1L1降低了LXR诱导的小鼠粪便核心甾醇的分泌,并升高了血浆游离胆固醇水平,可能与下调LXR诱导的肝脏ABCG5和G8 mRNA表述水平有一定关系。     

肝脏表达的Niemann-Pick C1-Like 1对小鼠胆汁胆固醇浓度的调节作用以及依折麦布对其功能的影响

目的探讨苦瓜蛋白MD28对Caco-2细胞胆固醇转运相关基因表达的影响,阐明苦瓜蛋白MD28抗动脉粥样硬化的作用机制。方法Caco-2细胞经培养传代分为四组:①Caco-2细胞对照组;②Caco-2胆固醇转运细胞模型组;③Caco-2胆固醇转运细胞模型+苦瓜蛋白MD28处理组;④Caco-2细胞+苦瓜蛋白MD28处理组。加相应的处理因素处理后,高效液相色谱法测量细胞内胆固醇含量,油红O染色观察细胞内的脂滴,逆转录聚合酶链反应法和WesternBlot分别检测细胞中?...     

Niemann-Pick C1-Like 1表达缺陷缓解肝X受体激动剂诱导的小鼠肝脏脂肪性变

目的探讨Niemann-Pick C1-Like 1在肝X受体激动剂诱导的肝脏脂肪性变中的作用。方法给C57BL/6小鼠和Niemann-Pick C1-Like 1基因敲除小鼠喂食0.015%胆固醇饮食21天,胃饲溶剂或肝X受体激动剂T0901317一周后,收集小鼠肝脏,称重;抽提肝脏脂质并用酶法检测脂质含量;用实时定量聚合酶链反应法检测肝脏表达与脂肪合成相关基因固醇调节元件结合蛋白1c、脂肪酸合成酶和硬脂酰CoA去饱和酶1的mRNA水平。结果在胃饲T0901317一周后,C57BL/6小鼠肝脏由1.1±0.1g增大到2.8±0.3g,肝脏甘油三酯含量由34.2±18.1mg/g增至232.2±67.9mg/g,固醇调节元件结合蛋白1c、脂肪酸合成酶和硬脂酰CoA去饱和酶1mRNA水平在肝脏的表达也显著增高;而Niemann-Pick C1-Like 1基因敲除小鼠肝脏由0.9±0.1g增大到1.5±0.1g,肝脏甘油三酯含量由43.7±26.5mg/g增至104.9±62.1mg/g,脂肪酸合成酶和硬脂酰CoA去饱和酶1mRNA水平在肝脏的表达也显著增高。但在T0901317诱导下,Niemann-Pick C1-Like 1基因敲除小鼠肝脏脂肪酸合成酶mRNA水平仍比C57BL/6小鼠低63%。结论Niemann-Pick C1-Like 1表达缺陷降低肝X受体激动剂诱导的肝脏固醇调节元件结合蛋白1c和脂肪酸合成酶的表达,缓解小鼠肝脏脂肪性变。     

高胆固醇血症小鼠肝脏的差异表达蛋白

运用以双向电泳为基础的蛋白质组学研究手段,比较分析高胆固醇血症小鼠与正常小鼠肝脏差异表达蛋白。用高脂饲料复制小鼠高胆固醇血症模型,饲喂14周后高脂组小鼠血浆中总胆固醇为9.5±3.9 mmol/L,肝脏中总胆固醇为35.3±3.7 mg/g,均明显高于对照组(P<0.001)。提取两组小鼠肝脏总蛋白,双向凝胶电泳分离蛋白质,所获得的凝胶图谱经软件分析后确定差异点及其等电点与分子质量,利用质谱技术对差异蛋白进行肽质量指纹谱分析鉴定,再与蛋白质数据库中的小鼠肝脏标准图谱匹配验证。双向凝胶电泳分离蛋白质样品的分辨率好、重复性较高,两组间至少有16个点有稳定的差异表达(2倍以上),鉴定其中4个点个为主要尿蛋白系列、2个点为碳酸酐酶Ⅲ、2个点为谷胱甘肽S-转移酶P2。实验结果提示:主要尿蛋白、碳酸酐酶Ⅲ及谷胱甘肽S-转移酶等受雄激素调控的蛋白质表达下调,可能与饮食引起的高胆固醇血症相关。     

高胆固醇饮食对小鼠肝脏内皮脂酶mRNA表达的影响

目的内皮脂酶（endothelial lipase，EL）是新近发现的一种脂肪酶，它与肝脂肪酶（HL）、脂蛋白脂肪酶同属于甘油三酯脂肪酶基因家族，参与高密度脂蛋白（HDL）的代谢。本研究旨在观察高胆固醇饮食对小鼠肝脏EL mRNA表达的影响，并同时检测小鼠血脂水平的改变，研究EL mRNA表达水平与血脂的关系。     

养肝利胆颗粒对胆固醇结石小鼠肝脏基因表达的影响

[目的]探讨养肝利胆颗粒对胆固醇结石C57小鼠肝脏基因表达的影响。[方法]38只C57BL／6雌性小鼠随机分为正常对照组（n=10）、模型组（n=15）和养肝利胆颗粒组（n=13）。其中后2组小鼠采用高脂饮食诱发法建立胆固醇结石模型。造模同时,养肝利胆颗粒组小鼠予养肝利胆颗粒2．1g·kg^-1·d^-1灌胃治疗。8周后观察各组小鼠的成石率,并用OligO GE Array芯片检测肝脏基因表达的改变。[结果]正常对照组成石率为0;模型组成石率为73．33％;养肝利胆颗粒组成石率为30．77％,明显低于模型组（P〈0．01）。模型组较正常对照组上调基因34条,养肝利胆颗粒组较模型组下调基因12条。[结论]养肝利胆颗粒可以通过调控胆固醇脂质代谢、炎症应答等相关基因表达来发挥防治胆石症的作用。     

胆囊胆固醇结石患者肝脏受体类似物1基因表达差异的研究

目的研究胆囊胆固醇结石患者肝脏受体类似物1(LRH-1)表达,探讨其与胆固醇结石病发病的关系。方法27例胆囊胆固醇结石患者,男6例,女21例,平均年龄52．44岁。14例无胆石症者为对照(肝脏肿瘤2例,胆囊息肉患者12例),男9例,女5例,平均年龄47．50岁。测定胆汁脂类成分和计算胆汁胆固醇饱和指数。实时定量PCR法测定肝脏LRH-1 mRNA的表达量。结果胆石组LRH-1表达14．18±9．37,高于对照组7．86±6．19,(P＜0．05),胆石组胆汁呈胆固醇过饱和(1．17±0．27)。结论本研究提示肝脏LRH-1表达增高与胆囊胆固醇结石病有关。     

肝脏调节胆固醇代谢稳态的研究进展

胆固醇代谢稳态是机体发挥生理功能的重要保障。胆固醇代谢紊乱引起的体内胆固醇异常积聚可导致肝脏脂肪病变以及高胆固醇血症,与动脉粥样硬化等心血管疾病的发生发展密切相关。肝脏在维持机体胆固醇代谢稳态过程中发挥着关键性作用,其胆固醇代谢涉及摄取、合成、生物转化以及外排等多个环节。文章就肝脏调节胆固醇代谢稳态的最新进展作一综述。     

易成石和抗成石小鼠肝脏胆固醇代谢调节酶的基因表达特征

目的通过研究胆囊胆固醇结石易成石小鼠(C57L鼠)和抗成石小鼠(AKR鼠)肝脏胆固醇代谢调节酶的基因表达差异来探讨胆囊胆固醇结石形成的分子机制.方法用致石饲料(含15%脂肪、1.25%胆固醇和0.5%胆酸)喂养C57L和AKR鼠4周,分别在喂养前、后检查其肝脏重量、肝脏胆固醇含量和胆囊内结石形成情况,并用RT-PCR法分析参与肝脏胆固醇代谢调节的肝脏3羟基3甲基-戊二酰辅酶A还原酶(HMG-CoAR)、胆固醇7α羟化酶(C7H)、乙酰基辅酶A-胆固醇酰基转移酶(ACAT)和低密度脂蛋白受体(LDLR)的mRNA含量.结果致石饲料喂养前、后AKR鼠肝脏胆固醇含量均明显高于C57L鼠,致石饲料诱导4周后C57L鼠胆囊内有胆固醇结石形成,而AKR鼠则无.致石饲料诱导前AKR鼠肝脏HMG-CoAR的mRNA表达水平明显高于C57L鼠,而ACAT、LDLR、C7H的mRNA水平在两组小鼠间差异无统计学意义(P＞0.05).致石饲料诱导后,AKR鼠肝脏HMG-CoAR和C7H的mRNA水平分别下降31%和30%,但C57L鼠无明显改变,其中AKR和C57L鼠肝脏LDLR的mRNA表达水平则分别提高14%和41%.结论致石饲料作为一种环境因素能揭示C57L和AKR鼠肝脏胆固醇代谢调节酶的基因表达特征.致石饲料诱导后C57L鼠肝脏HMGCoAR基因表达水平的下调不足和LDLR基因表达水平的显著上升可能在其胆囊胆固醇结石形成过程中发挥着一定作用.     

ABCA1基因转录调节与胆固醇代谢及动脉粥样硬化的关系

ABCA1（ATP-binding cassette transporter A1）可利用ATP参与胆固醇和磷脂从细胞内转运至贫脂或无脂的apoA1，促进高密度脂蛋白（HDL）生成，并在胆固醇逆转运（RCT）中起重要作用。ABCA1受多种因素调节，其中以核受体超家族的转录调节最为重要。上调ABCA1表达增加RCT、提高HDL血浆浓度是防治动脉粥样硬化（AS）的一个新方向，特异性肝X受体（LXR）激动剂可能是抗AS新药的希望所在。     

Cyclodextrin overcomes deficient lysosome-to-endoplasmic reticulum transport of cholesterol in Niemann-Pick type C cells

A handoff model has been proposed to explain the egress from lysosomes of cholesterol derived from receptor-mediated endocytosis of LDL. Cholesterol is first bound by soluble Niemann-Pick C2 (NPC2) protein, which hands off the cholesterol to the N-terminal domain of membrane-bound NPC1. Cells lacking NPC1 or NPC2 accumulate LDL-derived cholesterol in lysosomes and fail to deliver LDL cholesterol to the endoplasmic reticulum (ER) for esterification by acyl-CoA acyltransferase (ACAT) and for inhibition of sterol regulatory element-binding protein cleavage. Here, we support this model by showing that the cholesterol transport defect in NPC1 mutant cells is restricted to lysosomal export. Other cholesterol transport pathways appear normal, including the movement of cholesterol from the plasma membrane to the ER after treatment of cells with 25-hydroxycholesterol or sphingomyelinase. The NPC1 or NPC2 block in cholesterol delivery to the ER can be overcome by 2-hydroxypropyl-β-cyclodextrin, which leads to a marked increase in ACAT-mediated cholesterol esterification. The buildup of cholesteryl esters in the cytosol is expected to be much less toxic than the buildup of free cholesterol in the lysosomes of patients with mutations in NPC1 or NPC2.     

Niemann-Pick C1 (NPC1)/NPC1-like1 Chimeras Define Sequences Critical for NPC1’s Function as a Filovirus Entry Receptor

We recently demonstrated that Niemann-Pick C1 (NPC1), a ubiquitous 13-pass cellular membrane protein involved in lysosomal cholesterol transport, is a critical entry receptor for filoviruses. Here we show that Niemann-Pick C1-like1 (NPC1L1), an NPC1 paralog and hepatitis C virus entry factor, lacks filovirus receptor activity. We exploited the structural similarity between NPC1 and NPC1L1 to construct and analyze a panel of chimeras in which NPC1L1 sequences were replaced with cognate sequences from NPC1. Only one chimera, NPC1L1 containing the second luminal domain (C) of NPC1 in place of its own, bound to the viral glycoprotein, GP. This engineered protein mediated authentic filovirus infection nearly as well as wild-type NPC1, and more efficiently than did a minimal NPC1 domain C-based receptor recently described by us. A reciprocal chimera, NPC1 containing NPC1L1’s domain C, was completely inactive. Remarkably, an intra-domain NPC1L1-NPC1 chimera bearing only a ~130-amino acid N–terminal region of NPC1 domain C could confer substantial viral receptor activity on NPC1L1. Taken together, these findings account for the failure of NPC1L1 to serve as a filovirus receptor, highlight the central role of the luminal domain C of NPC1 in filovirus entry, and reveal the direct involvement of N–terminal domain C sequences in NPC1’s function as a filovirus receptor.     

Relation between hepatic expression of ATP-binding cassette transporters G5 and G8 and biliary cholesterol secretion in mice

BACKGROUND/AIM: Mutations in genes encoding the ATP-binding cassette (ABC)-transporters ABCG5 and ABCG8 underlie sitosterolemia, which is characterized by elevated plasma levels of phytosterols due to increased intestinal absorption and impaired biliary secretion of sterols. The aim of our study was to correlate the expression levels of Abcg5 and Abcg8 to biliary cholesterol secretion in various (genetically-modified) mouse models. METHODS: Bile was collected from genetically-modified mice fed a chow diet, or from mice fed either a chow diet, or chow supplemented with either 1% diosgenin, 0.1% simvastatin, or a synthetic liver X receptor agonist, for determination of biliary lipids. Livers and small intestines were harvested and expression levels of Abcg5, Abcg8 and Abcb4 were determined by real-time polymerase chain reaction. RESULTS: Intestinal expression of Abcg5 and Abcg8 did not show much variation between the various models. In contrast, a linear correlation between hepatic expression levels of Abcg5 and Abcg8 and biliary cholesterol secretion rates was found. This relation was independent of Abcb4-mediated phospholipid secretion. However, in diosgenin-fed mice showing cholesterol hypersecretion, hepatic Abcg5 and Abcg8 expression levels remained unchanged. CONCLUSIONS: Our results strongly support a role for Abcg5 and Abcg8 in regulation of biliary cholesterol secretion, but also indicate the existence of a largely independent route of cholesterol secretion.     

Apolipoprotein E regulates dietary cholesterol absorption and biliary cholesterol excretion: studies in C57BL/6 apolipoprotein E knockout mice

The present study examined the role of apolipoprotein E (apoE) in the regulation of dietary cholesterol absorption and biliary cholesterol excretion. Increasing dietary cholesterol from 0.02% to 0.5% in C57BL/6 wild-type mice decreased the percentage of dietary cholesterol that is absorbed by 25%, and this decrease was associated with a 2-fold increase in gallbladder biliary cholesterol concentration. In contrast, increasing dietary cholesterol from 0. 02% to 0.5% in C57BL/6 apoE knockout mice produced no significant suppression of the percentage dietary cholesterol absorption and increased gallbladder biliary cholesterol concentration only 16%. Whereas in wild-type mice, the increase in dietary cholesterol increased the hepatic excretion of biliary cholesterol 4-fold, there was only a 2-fold increase in apoE knockout mice. On both the low- and the high-cholesterol diets, whole liver and isolated hepatocyte cholesterol content was higher in the apoE knockout mice. These results suggest that, in response to dietary cholesterol, apoE may play a critical role in decreasing the percentage absorption of dietary cholesterol and increasing biliary cholesterol excretion. These observations suggest a mechanism whereby the absence of apoE contributes to the propensity for tissue cholesterol deposition and accelerated atherogenesis in apoE knockout mice.     

Melatonin reduces cholesterol accumulation and prooxidant state induced by high cholesterol diet in the plasma, the liver and probably in the aorta of C57BL/6J mice

We examined the hypolipidemic and antioxidative effects of melatonin in plasma, liver and aorta of C57BL/6J mice fed on a high cholesterol (HC) diet. Mice were fed normal mice chow containing 1.5% cholesterol and 0.5% cholic acid for 4 months with or without melatonin (10 mg/L in drinking water) treatment. HC diet was observed to increase cholesterol, triglyceride and diene conjugate (DC) levels in plasma and liver. There was a tendency towards an increase in cholesterol level in the aorta following HC diet. In addition, aortic DC levels were higher than those of control group. No fatty streaks or plaques developed in the aorta of mice following HC diet, but in some sections, derangement of the endothelial layer was detected. Melatonin treatment was found to reduce plasma, liver cholesterol and DC levels as well as liver triglyceride levels in hypercholesterolemic mice. Aortic cholesterol and DC levels were also reduced in hypercholesterolemic mice when given melatonin, although not statistically significant. There were no differences in aortic histopathological findings of mice fed on a HC diet with and without melatonin treatment. In conclusion, our results indicate that melatonin reduces HC diet-induced cholesterol accumulation and prooxidant state in the plasma, liver and probably in the aorta.