丙型肝炎病毒核心蛋白下调沉默信息调节因子1导致肝星状细胞活化

目的研究丙型肝炎病毒(HCV)核心蛋白对肝星状细胞沉默信息调节因子1(SIRT1)表达及肝星状细胞活化的影响。方法 HepG2细胞或表达HCV核心蛋白的HepG2细胞与肝星状细胞(LX-2细胞)共培养。应用液体闪烁计数仪、实时荧光定量-PCR、Western印迹检测LX-2细胞SIRT1活性、mRNA及蛋白的表达。Western印迹检测LX-2细胞磷酸化AMP激活的蛋白激酶(p-AMPK)、脂联素受体2(AdipoR2)、转化生长因子-β1(TGF-β1)蛋白的表达。ELISA法检测共培养上清液中人Ⅳ胶原(ColⅣ)、Ⅲ型前胶原肽(PⅢNP)、透明质酸(HA)和人层黏连蛋白(LN)的水平。计量资料采用t检验。结果与LX-2细胞和HepG2细胞共培养组相比,LX-2细胞和表达HCV核心蛋白HepG2细胞共培养组SIRT1活性(0.4±0.1比1.0±0.2,t=6.573,P0.01)、mRNA(0.3±0.1比1.0±0.3,t=5.422,P0.01)和蛋白(0.4±0.1比0.8±0.2,t=4.382,P0.01)水平下降;p-AMPK(0.3±0.1比0.8±0.2,t=5.477,P0.01)和AdipoR2(0.4±0.1比0.8±0.2,t=4.382,P0.01)表达下降;TGF-β1(2.3±0.5比0.8±0.2,t=6.823,P0.01)表达增加;共培养上清液中ColⅣ、PⅢNP、HA和LN的水平增加。SIRT1激动剂白藜芦醇降低TGF-β1的表达。结论 HCV核心蛋白下调肝星状细胞SIRT1活性及表达,下调AdipoR2表达,上调TGF-βl表达,活化肝星状细胞。     

脂滴自噬在丙型肝炎病毒核心蛋白下调沉默信息调节因子1诱导小鼠肝脂肪变性中的作用

目的研究脂滴自噬在HCV核心蛋白下调沉默信息调节因子1(SIRT1)诱导小鼠肝脂肪变性中的作用。方法小鼠随机分为两组,每组10只。实验组小鼠尾静脉注射HCV core重组表达载体。对照组小鼠尾静脉注射磷酸盐缓冲溶液。1个月后处死小鼠。检测肝功能、脂联素、血清和肝内甘油三酰(TG)、肝脏组织病理学检查肝脂肪变性程度。蛋白质免疫法检测肝脏SIRT1蛋白、脂联素受体(AdipoR)蛋白以及脂滴自噬相关蛋白微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、脂肪分化相关蛋白(ADRP)、尾部作用蛋白(TIP-47)和P62蛋白的表达。计量资料采用t检验。结果与对照组相比,HCV组小鼠出现肝脂肪变性;肝脏TG含量明显增加[(80.9±20.1)比(45.8±10.5)μg/mg,t=4.964,P0.01];血清脂联素[(1.05±0.25)比(1.41±0.45)ng/mL,t=2.211,P0.05]水平下降;SIRT1蛋白水平(0.4±0.1比0.9±0.2,t=7.071,P0.01)和AdipoR2蛋白水平(0.4±0.1比0.8±0.2,t=5.656,P0.01)下降;LC3-Ⅱ蛋白(0.8±0.2比0.4±0.1,t=5.656,P0.01)、TIP-47蛋白(0.9±0.3比0.4±0.1,t=5.000,P0.01)和ADRP蛋白(0.8±0.3比0.4±0.1,t=4.000,P0.01)表达增加;而p62蛋白(0.7±0.2比0.8±0.3,t=0.877,P0.05)表达水平差异无统计学意义。结论 HCV核心蛋白下调SIRT1表达,下调脂联素及受体表达,引起不完全脂滴自噬导致肝脂肪变性。     

丙型肝炎病毒核心蛋白下调沉默信息调节因子2相关酶1-AMP激活蛋白激酶信号通路活性致肝细胞能量代谢紊乱

目的 探讨沉默信息调节因子2相关酶1 (SIRT1)-AMP激活的蛋白激酶(AMPK)信号通路在HCV核心蛋白所致肝细胞能量代谢紊乱中的作用.方法 pcDNA3.1-core重组质粒转染HepG2细胞,流式细胞仪测定表达HCV核心蛋白的HepG2细胞的活性氧(ROS),液体闪烁计数仪测定ATP/ADP比例和AMPK α2酶活性,比色法测定烟酰胺腺嘌呤二核苷酸氧化态与还原态之比(NAD+/NADH),荧光定量检测SIRT1酶活性,RT-PCR和Western印迹检测SIRT1、AMPK α2的表达.计量资料比较采用t检验.结果 Western印迹检测证实,HepG2细胞表达相对分子质量为22 000的HCV核心蛋白.与HepG2细胞相比,表达HCV核心蛋白的HepG2细胞ROS水平升高(1.0±0.1比4.0±0.5,t=14.411,P＜0.01);ATP/ADP比例无明显变化(8.2±2.2比9.3±2.8,t=0.757,P＞0.05);AMPK α2酶活性(0.8±0.2比0.2±0,t=7.345,P＜0.01)明显降低;NAD+/NADH比例明显降低(0.08±0.02比0.02±0,t=7.348,P＜0.01);SIRT1酶活性[(0.30±0.05) pmol·μg 1·min-1比(0.15±0.04) pmol·μtg-1·min-1,t=5.738,P＜0.01)]明显降低;SIRT1 mRNA(0.8±0.2比0.4±0.1,t=4.382,P＜0.01)和AMPK α2 mRNA(0.9±0.3比0.2±0,t=5.715,P＜0.01)表达明显降低;SIRT1蛋白(0.8±0.2比0.3±0,t=5.941,P＜0.01)和磷酸化AMPK蛋白(0.5±0.1比0.1±0,t=9.608,P＜0.01)水平明显降低.结论 HCV核心蛋白能下调SIRT1-AMPK信号通路活性,导致肝细胞能量代谢紊乱.     

糖脂代谢异常对丙型肝炎病毒感染肝细胞SIRT1-AMPK信号通路的影响

目的探讨不同浓度的葡萄糖、油酸对丙型肝炎病毒(HCV)感染肝细胞沉默信息调节因子1(SIRT1)-AMP激活蛋白激酶(AMPK)信号通路的影响。方法表达HCV核心蛋白的质粒转染HepG2细胞。表达HCV核心蛋白的HepG2细胞在不同终浓度的葡萄糖或油酸的无血清DMEM培养液中孵育24h。分别应用液体闪烁计数仪和蛋白质印迹检测SIRT1和AMPK活性及蛋白的表达。结果随着葡萄糖浓度(1、2、4.5g/L)不断升高,SIRT1活性(1.0±0.2、0.6±0.15、0.2±0.05)及SIRT1蛋白表达(0.9±0.2、0.4±0.1、0.1±0.05)下降,AMPK活性(1.0±0.2、0.5±0.08、0.22±0.05)及p-AMPK蛋白表达(0.9±0.2、0.4±0.1、0.2±0.05)下降;随着油酸浓度(0、300、500uM/L)不断升高,SIRT1活性(1.0±0.2、0.6±0.1、0.2±0.05)及SIRT1蛋白表达(0.9±0.2、0.5±0.1、0.2±0.05)下降,AMPK活性(1.0±0.2、0.5±0.1、0.1±0.05)及p-AMPK蛋白表达(0.9±0.2、0.5±0.1、0.2±0.05)下降。结论高糖、高油酸可下调HCV感染肝细胞SIRT1-AMPK信号通路的活性及表达。     

胰岛素抵抗的慢性丙型肝炎患者外周血及肝组织内源性大麻素系统的变化

目的研究胰岛素抵抗(IR)的慢性丙型肝炎(CHC)患者外周血及肝组织内源性大麻素系统的变化。方法采用高效液相色谱-质谱联用技术检测CHC患者和CHC合并IR患者外周血内源性大麻素花生四烯酸乙醇胺(AEA)和花生四烯酰甘油(2-AG)水平,应用RT-PCR及Western印迹检测肝组织大麻素受体1(CB1R)及脂肪酸酰胺水解酶(FAAH)的表达,数据行t检验。结果与CHC患者相比,CHC合并IR患者外周血AEA水平升高[(6.80±1.70)pmol/mL比(3.88±0.63)pmol/mL,t=8.053,P0.01];肝组织CB1R mRNA水平(1.7±0.4比1.0±0.2,t=4.696,P0.01)和CB1R蛋白水平(0.8±0.2比0.4±0.1,t=5.367,P0.01)增加;FAAH mRNA水平(0.5±0.1比1.0±0.3,t=4.743,P0.01)和FAAH蛋白水平(0.5±0.1比0.8±0.3,t=2.846,P0.01)下降。与胰岛素抵抗指数(HOMA-IR)4的患者相比,HOMA-IR指数≥4的患者AEA水平升高[(7.53±1.85)pmol/mL比(5.56±1.40)pmol/mL(t=2.986,P0.05)];肝组织CB1 mRNA水平(2.3±0.65比1.4±0.35,t=2.986,P0.05)和CB1R蛋白水平(1.2±0.3比0.9±0.2,t=2.038,P0.05)升高,FAAH mRNA水平(0.3±0.08比0.6±0.15,t=4.076,P0.01)和FAAH蛋白水平(0.2±0.05比0.4±0.1,t=3.464,P0.05)下降。结论随着IR程度加重,CHC患者外周血AEA水平升高;肝组织CB1R表达增加而FAAH表达减少。     

过氧化物酶体增殖活化受体-α激活对HepG2细胞脂肪变性及血红素加氧酶-1表达的影响

目的 研究过氧化物酶体增殖活化受体-α(PPAR-α)激活对油酸诱导的HepG2细胞脂肪变性及血红素加氧酶-1 (HO-1)表达的影响.方法 以油酸(OA)诱导人肝癌HepG2细胞脂肪变性为模型组,采用不同浓度的PPAR-α激动剂非诺贝特(FF)处理HepG2细胞24h,油红O染色观察细胞内脂滴数量,甘油-3-磷酸氧化酶法检测细胞内甘油三酯(TG)含量,采用实时荧光定量PCR检测各组HepG2细胞PPAR-α、HO-1 mRNA水平,采用免疫细胞化学法检测PPAR-α与HO-1蛋白表达.采用SPSS13.0软件,采用单因素方差分析及Pearaon直线相关进行相关分析.结果 (1)模型组HepG2细胞内TG含量为(379.98±23.19) mg/g,对照组为(185.03±12.68) mg/g,模型组HepG2细胞内TG含量明显增加,t=24.385,P＜0.01.PPAR-α的mRNA及蛋白相对表达水平模型组分别为0.42±0.38和0.47±0.14,对照组分别为1.00±0.00和1.85±0.12,模型组明显降低,t=0.583和1.382P值均＜0.01.HO-1的mRNA及蛋白相对表达水平模型组分别为0.36±0.66和0.26±0.10,对照组分别为1.00±0.00和1.22±0.12,模型组明显降低,t=0.637和t=0.967,P值均＜0.01;(2)FF浓度在5、10、50μmol/L时,HepG2细胞内TG值分别为(294.00±19.80) mg/g、(250.33±9.96)mg/g和(196.99±9.14) mg/g,t值分别为10.747、16.200和22.873, F=148.555;P值均＜0.01 ;PPAR-α的mRNA相对表达量分别为0.55±0.65,0.85±0.61,1.31±0.36,t值分别为0.137、0.430和0.893,F=177.637,P值均＜0.01;PPAR-α蛋白累积光密度值分别为0.82±0.11、1.31±0.16和1.75±0.13,t值分别为0.352,0.840,1.280,F=120.764,P值均＜0.01;HO-1的mRNA相对表达量分别为0.62±0.05、0.84±0.07和1.30±0.11,t值分别为0.257,0.480,0.937,F=74.768,P值均＜0.01;HO-1蛋白累积光密度值分别为0.44±0.08、0.81±0.08和1.20±0.10,t值分别为0.180,0.553,0.943,F=119.903,P值均＜0.01.结论 PPAR-α的激活可以抑制HepG2细胞脂肪变性,HO-1可能是其重要下游因子.     

Roux-en-Y胃转流术后长期维持减重效应和血糖稳态的机制研究

目的探究Roux-en-Y胃转流术(RYGB)后长期维持减重效应和血糖稳态的机制。方法选用12周龄雄性Sprague Dawley(SD)大鼠作为动物模型,采用完全随机法分为假手术组(n=22)、RYGB组(n=12)和胰腺大部切除术组(n=10),以普通饲料和45%高脂饲料干预8周,检测各组大鼠胰岛素分泌水平、葡萄糖输注率、体重和血糖水平,并通过基因芯片和免疫印迹试验检测各组大鼠脂肪组织代谢通路等的变化。组间比较采用t检验及方差分析。结果与假手术相比,RYGB组大鼠术后空腹及葡萄糖刺激后血浆胰岛素水平均显著降低[(1.3±0.2)比(0.6±0.1)ng/ml,t=3.6,P<0.01;(4.1±0.4)比(2.1±0.3)ng/ml,t=4.3,P<0.01],且在45%高脂饲料喂养的情况下可长期维持空腹胰岛素及体重在较低水平[(3.1±0.3)比(0.7±0.1)ng/ml,t=8.1,P<0.01;(453±11)比(349±6)g,t=8.5,P<0.01]。机制研究发现,大鼠RYGB术后脂肪组织胰岛素信号通路受到抑制(1.0±0.1比0.5±0.1,t=6.4,P<0.01),脂肪酸合成减少,脂肪酸分解增加,腺嘌呤核糖核苷酸依赖的蛋白激酶表达增强(1.0±0.1比1.4±0.1,t=4.0,P<0.01)。与假手术组比较,胰腺大部切除术组空腹血浆胰岛素和胰高血糖素水平分别降低71.7%和68.0%[(1.10±0.10)比(0.30±0.04)ng/ml,t=7.5,P<0.01;(16.2±0.8)比(5.2±0.9)pmol/L,t=9.1,P<0.01],但空腹血糖无明显升高,在普通饲料和45%高脂饲料喂养时体重均显著低于假手术组[(434±13)比(389±5)g,t=3.1,P<0.05;(548±16)比(433±11)g,t=6.0,P<0.01]。结论RYGB术后通过减少能量摄入及避免高胰岛素血症可长期维持低体重和血糖稳态,其机制与脂肪合成代谢抑制,而分解代谢增强密切相关。     

小泛素样修饰物特异性蛋白酶3通过调节脂滴水平体外促进HepG2细胞丙型肝炎病毒复制

目的 探究丙型肝炎病毒(HCV)感染细胞小泛素样修饰物特异性蛋白酶3(SENP3)对脂滴水平的调节及其对HCV复制的影响。方法 以感染性HCV病毒颗粒感染人HepG2细胞,采用Western blot法检测感染前后SENP3蛋白表达水平。采用脂质体法转染SENP3-siRNA至HepG2细胞,在感染HCV后采用Western blot法检测HCV核心蛋白表达量,采用qRT-PCR法检测HCV RNA水平,采用油红O染色观察细胞脂滴水平。以软脂酸处理敲低SENP3后的HepG2细胞,采用qRT-PCR法检测感染HCV后HCV RNA水平。结果 在HCV感染HepG2细胞后1 d、3 d和6 d,HCV 核心蛋白相对表达量分别为0.01±0.00、0.17±0.02和0.43±0.04,SENP3蛋白相对表达量分别为0.23±0.04、0.42±0.03和0.46±0.04,差异显著(P<0.05);转染SENP3-siRNA可有效降低SENP3表达并降低HCV感染后细胞HCV核心蛋白表达水平;SENP3敲低细胞HCV RNA相对水平为54.2±11.4%,较转染非特异性siRNA细胞显著降低(P<0.01);敲低SENP3蛋白表达后细胞脂滴数量显著减少;敲低SENP3的HepG2细胞感染HCV后HCV RNA相对水平为58.2±5.2%,较转染非特异性siRNA细胞显著降低(P<0.01),而以软脂酸处理敲低SENP3的HepG2细胞,在感染HCV后HCV RNA相对水平为74.6±6.4%,较未经软脂酸处理细胞显著升高(P<0.01)。结论 在HCV感染肝细胞时SENP3可通过调节脂滴代谢促进HCV复制。     

PHB1对人肝癌细胞增殖的影响及其作用机制

目的探讨抗增殖蛋白1(prohibitin1,PHB1)对人肝癌细胞增殖的影响及其作用机制。方法构建PHB1真核表达重组质粒(pEGFP-PHB1)和PHB1靶向siRNA质粒(shRNA-PHB1)转染人肝癌细胞HepG2和SMMC-7721,诱导PHB1在人肝癌细胞中表达上调和下调,采用MTT、流式细胞学、荧光定量PCR和免疫印迹等技术检测人肝癌细胞增殖、细胞周期,以及细胞周期关键调控分子的表达情况。结果高表达PHB1可阻滞HepG2和SMMC-7721细胞于G0/G1期[(67.28±2.94)%比(56.71±2.56)%,t=6.64,P=0.00;(69.48±3.82)%比(60.43±2.59)%,t=4.80,P=0.00],使S期比例下降[(14.74±1.45)%比(24.13±1.92)%,t=9.54,P=0.00;(13.73±1.26)%比(25.50±2.30)%,t=10.99,P=0.00],抑制细胞增殖;周期调控分子p53和p21CIP1mRNA和蛋白质水平显著升高,而Cyclin A2、Cyclin E1和CDK2 mRNA和蛋白质水平显著降低(P0.01)。低表达PHB1可促使HepG2和SMMC-7721细胞趋于S期[(31.65±2.71)%比(24.68±1.28)%,t=5.69,P=0.00;(31.02±2.49)%比(25.88±2.40)%,t=3.64,P=0.005],促进细胞增殖;p53、p21CIP1、Cyclin A2、Cyclin E1、CDK2 mRNA和蛋白质水平与PHB1高表达时相反(P0.01)。结论高表达PHB1可以阻滞人肝癌细胞周期于G0/G1期,进而抑制细胞增殖,其作用机制可能与p53介导的G0/G1期相关细胞周期蛋白有关。     

SIRT1对高糖高脂培养胰岛微血管内皮细胞炎性反应激活的影响

目的 分析高糖、高脂环境下胰岛微血管内皮细胞(IMVC)分泌E-选择素、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β的变化,研究沉默信息调节蛋白1(SIRT1)/核因子-κB信号途径异常在内皮细胞炎性反应激活中的作用.方法 合成含绿色荧光蛋白报告基因的小鼠SIRT1基因重组质粒,以Lipofectamine 2000转染IMVC.之后将IMVC分为4组:正常对照组、高糖高脂组、高糖高脂+ SIRT1基因转染组、高糖高脂+基因转染对照组.高糖高脂组以33.3 mmol/L葡萄糖+0.5mmol/L棕榈酸处理48 h.高糖高脂+SIRT1基因转染组在高糖高脂处理前以SIRT1基因重组质粒预处理48 h;高糖高脂+基因转染对照组在高糖高脂处理前以空质粒进行预处理48 h.应用实时荧光定量PCR法检测SIRT1基因转染效果及各组核因子-κB的mRNA水平,应用酶联免疫吸附法测定细胞培养上清中TNF-α、IL-1β、E-选择素的水平.结果 与正常对照组相比,高糖高脂组SIRT1 mRNA表达明显下降(0.58 ±0.12,P＜0.01);而高糖高脂+SIRT1基因转染组的SIRT1基因表达明显升高,与高糖高脂组相比差异显著[(1.55±0.43)比(0.65±0.37),P＜0.01].与正常对照组相比,高糖高脂组核因子-κB mRNA(1.59 ±0.32,P＜0.01)、E-选择素[(48.46±1.04)比(67.12±0.57) ng/L,P＜0.01]、TNF-α[(467.46±8.98)比(621.14±11.26) ng/L,P＜0.01]、IL-1β[(63.32±1.48)比(118.43±1.40) ng/L,P＜0.01]的水平明显升高(P＜0.01).与高糖高脂组相比,高糖高脂+SIRT1基因转染组核因子-κB mRNA (0.95±0.31,P＜0.01)及TNF-α[(451.38±15.91)比(618.89±7.23) ng/L,P＜0.01]明显下降(P＜0.01),E-选择素、IL-1β无明显变化(P＞0.05).结论 高糖高脂环境下,IMVC存在炎性反应激活,可释放大量炎性反应细胞因子.SIRT1-核因子-κB-TNF-α通路异常与IMVC的炎性反应激活密切相关,早期干预该信号通路可能在预防及治疗2型糖尿病的胰岛炎性反应中具有重要意义.     

Adiponectin, skeletal muscle adiponectin receptor expression and insulin resistance following dexamethasone

Objective Skeletal muscle is a major site of adiponectin action and of glucocorticoid‐induced insulin resistance. Little human data exist however, regarding the impact of exogenous glucocorticoids on adiponectin receptors in skeletal muscle. Design and patients Twelve subjects with type 2 diabetes and 12 controls underwent blood sampling and muscle biopsy of vastus lateralis before and after 4 days of 4 mg dexamethasone. Measurements (i) Total and high molecular weight (HMW) plasma adiponectin, glucose and insulin; (ii) Skeletal muscle adiponectin receptor AdipoR1 and AdipoR2 mRNA levels by quantitative real time RT‐PCR. Results Baseline total adiponectin (8·0 ± 0·89 vs. 12·5 ± 1·46 µg/ml, P = 0·013), HMW adiponectin (2·8 ± 0·44 vs. 5·9 ± 1·04 µg/ml, P = 0·014) and AdipoR2 mRNA levels (mean ΔC_T 14·71 ± 0·35 vs. 13·37 ± 0·28, P = 0·017) were significantly lower in diabetic subjects. After dexamethasone, AdipoR2 mRNA fell in the controls but there was no change in the diabetic group, while there was a significant increase in total (P = 0·002) and HMW adiponectin (P < 0·001) across both groups. Total and HMW plasma adiponectin correlated with clinical and biochemical measures of insulin sensitivity. However following dexamethasone which increased insulin resistance, the relationship between adiponectin and the biochemical measures was lost. Conclusions Plasma adiponectin and skeletal muscle AdipoR2 mRNA expression are reduced in subjects with diabetes; both are likely to contribute to the observed insulin resistance. Dexamethasone inhibits AdipoR2 mRNA expression in nondiabetic subjects, while there is a small rise in plasma adiponectin levels. The close relationship between plasma adiponectin and biochemical measures of insulin sensitivity is lost in the setting of glucocorticoid‐induced insulin resistance.     

高血压病超重及肥胖患者血清脂联素水平和IL-6含量的变化

目的研究高血压病超重及肥胖患者血清脂素水平、白介素-6(IL-6)含量的变化及其临床意义。方法63名正常非肥胖对照(男31名,女32名)和57名体重指数>25kg/m2的单纯性超重肥胖个体和41名高血压病超重肥胖患者进入研究,测量血压(BP)、体重指数(BMI)、腰围(WC)、腰臀围比(WHR),用ELISA法检测所有研究对象血清脂联素浓度、血清IL-6含量。结果单纯性超重肥胖患者与高血压超重病肥胖患者血清脂联素水平显著低于正常对照者[单纯性超重肥胖男性(7.03±1.67)mg/L,高血压病超重肥胖男性(6.13±1.74)mg/Lvs正常对照男性(8.21±0.93)mg/L,t=3.868,t=5.388,均P<0.01;单纯性超重肥胖女性(7.90±1.35)mg/L,高血压病超重肥胖女性(6.97±1.16)mg/Lvs正常对照女性(9.22±0.68)mg/L,t=4.612,t=7.167,均P<0.01]。高血压病超重肥胖男性血清IL-6含量显著高于正常对照男性[高血压病超重肥胖男性(394.86±353.9)pg/mlvs正常对照男性(248.02±223.2)pg/ml,t=2.368,P<0.05;高血压病超重肥胖女性(303.2±283.6)pg/mlvs正常对照女性(230.5±214.2)pg/ml,t=0.9046,P>0.05]。在高血压病超重肥胖患者中,脂联素与体重指数、腰围、腰臀围比、收缩压之间呈显著负相关;IL-6与收缩压之间呈显著正相关。结论脂联素水平下降与超重肥胖、血压之间存在一定的相关性;IL-6含量升高可能与高血压病有关。     

淫羊藿总黄酮对高脂血症大鼠主动脉脂联素受体表达的影响

目的 探讨淫羊藿总黄酮对高脂血症大鼠主动脉脂联素受体1和2表达的影响.方法 清洁级SD大鼠35只,随机分为高脂饮食组(26只)和对照组(9只).高脂饮食组高脂饮食喂养12周后,随机分为模型组(8只)和高脂饮食加淫羊藿总黄酮低剂量组(9只)、高脂饮食加淫羊藿总黄酮高剂量组(9只),检测血脂水平及观察主动脉病理变化,通过ELISA检测血清脂联素水平,通过荧光定量PCR法检测主动脉脂联素受体1和2基因的表达,Western boltting检测脂联素受体1和2蛋白的表达.结果 造模12周后,高脂饮食组甘油三酯、总胆固醇和低密度脂蛋白胆固醇水平明显升高;给药4周后,与模型组比较,淫羊藿总黄酮低剂量组和高剂量组甘油三酯和总胆固醇水平明显降低(P<0.05或0.01),淫羊藿总黄酮能减轻高脂饮食所致的内膜增厚和平滑肌细胞增殖,淫羊藿总黄酮组血清脂联素水平明显升高(P<0.05),且淫羊藿总黄酮高剂量组较淫羊藿总黄酮低剂量组明显升高(P<0.05);淫羊藿总黄酮组脂联素受体1和2 mRNA的表达较模型组明显升高(P<0.05或0.01),且淫羊藿总黄酮高剂量组脂联素受体1 mRNA的表达较低剂量组明显升高 (P<0.05);淫羊藿总黄酮组脂联素受体1和2蛋白的表达较模型组明显升高(P<0.05或0.01).结论 淫羊藿总黄酮能降低高脂血症大鼠血脂水平,并升高血清脂联素水平及主动脉脂联素受体1和2的表达.     

Pattern of expression of adiponectin receptors in human adipose tissue depots and its relation to the metabolic state

OBJECTIVE: To investigate whether adiponectin receptor genes (AdipoR1 and AdipoR2) expression in human subcutaneous (SAT) and visceral (VAT) adipose tissue in severely obese patients with or without diabetes is related to adiponectin gene (APM1) expression and in vivo metabolic parameters. DESIGN: Cross-sectional, clinical research study. SUBJECTS: Total RNA was extracted from SAT and VAT tissue obtained during surgery from 13 lean controls, 30 obese diabetic patients, 19 obese glucose-intolerant patients and 54 obese subjects with normal glucose tolerance. MEASUREMENTS: Tissue expression of APM1, AdipoR1 and AdipoR2, tissue concentration of adiponectin (ApN), and metabolic variables. RESULTS: APM1 expression was higher in SAT than VAT (1.06+/-0.76 vs 0.69+/-0.52, P<0.0001) as was AdipoR1 (1.17+/-0.70 vs 0.66+/-0.38, P<0.0001) and AdipoR2 (7.02+/-6.19 vs 0.75+/-0.64, P<0.0001). In SAT, APM1 and AdipoR1 expression tended to be lower - by 0.38+/-0.22 and 0.35+/-0.22, respectively - and AdipoR2 expression was markedly depressed - by 4.82+/-1.93 - in association with obesity, whereas presence of diabetes had no additional effect. In VAT, APM1 and AdipoR1 expressions were also reduced - by 0.36+/-0.16 and 0.30+/-0.11, respectively - in association with obesity. Within both SAT and VAT, expression levels of APM1, AdipoR1 and AdipoR2 were all positively interrelated. Tissue ApN concentrations in SAT were similar across groups, whereas ApN levels in VAT were substantially lower in association with obesity (by an average of 63+/-12 ng/mg total protein, P<0.0001). In multivariate models adjusting for sex, age and obesity, serum triglyceride concentrations were reciprocally related to APM1 (r=-0.27, P<0.02), AdipoR1 (r=-0.37, P<0.002 and AdipoR2 expression (r=-0.37, P<0.002) in VAT. Likewise, plasma insulin concentrations were inversely related only to APM1 in VAT (r=-0.25, P<0.03). CONCLUSIONS: Severe obesity is associated with suppressed expression of both ApN and its receptors in both SAT and VAT, the expression levels in VAT are specifically linked with hyperinsulinemia and dyslipidemia.     

Hepatitis C virus infection down-regulates the expression of peroxisome proliferator-activated receptor α and carnitine palmitoyl acyl-CoA transferase 1A

AIM: To elucidate the role of the peroxisome proliferator-activated receptor α (PPARα) and its target gene carnitine palmitoyl acyl-CoA transferase 1A (CPT1A)in the pathogenesis of hepatitis C virus (HCV) infection.METHODS: Liver samples were collected from the patients with chronic HCV infection and controls. HepG2cells were transfected with vector pEF352neo carrying.Two independent clones (clone N3 and N4) stably expressing HCV core protein were analyzed. Total RNA was extracted from cells and liver tissues. PPARα and CPT1A mRNAs were quantified by real-time polymerase chain reaction (PCR) using SYBR Green Master. Total extracted proteins were separated by polyacrylamide gel electrophoresis, and electroblotted. Membranes were incubated with the anti-PPARα antibody, then with a swine anti-rabbit IgG conjugated to horseradish peroxidase for PPARα. Protein bands were revealed by an enhanced chemiluminescence reaction for PPARα. For immunohistochemical staining of PPARα, sections were incubated with the primary goat polyclonal antibody directed against PPARα at room temperature.RESULTS: Real-time PCR indicated that the PPARα level and expression level of CPT1A gene in hepatitis C patients lowered significantly as compared with the controls (1.8±2.8 vs 13±3.4, P = 0.0002; 1.1±1.5 vs 7.4+1, ,P = 0.004). Western blot results showed that the level of PPARα protein in the livers of hepatitis C patients was lower than that in controls (2.3±0.3 vs 3.6±0.2,P = 0.009). The immunohistochemical staining results in chronic hepatitis C patients indicated a decrease in PPARα staining in hepatocytes compared with those in the control livers. The in vitro studies found that in the N3 and N4 colon stably expressing HCV core protein, the PPARα mRNA levels were significantly lower than that in the controls.CONCLUSION: The impaired intrahepatic PPARα expression is associated with the pathogenic mechanism in hepatic injury during chronic HCV infection. HCV infection reduced the expression of PPARα and CPT1A at the level of not only mRNAs but also proteins. PPARα plays an important role in the pathogenesis of chronic HCV infection, but the impaired function of this nuclear receptor in HCV infection needs further studies.     

丙型肝炎病毒NS5A对p53活性调控机制研究

目的 研究HCV NS5A对抑癌蛋白p53活性的抑制作用及其作用机制。 方法 采用荧光素酶报告基因系统观察pwwp-luc,pwwp-mut-luc,pc53-NS3及pCNS5A分别转染或共同转染HepG2、Huh7细胞的荧光素酶活性。应用凝胶滞留试验观察HCV NS5A是否影响p53与其特异DNA序列的结合。 结果 内源性p53能激活p21启动子转录功能,使荧光素酶活性明显增加(3.49×10~5与0.60×10~5,t=5.92,P<0.01)。外源性p53也能激活p21启动子转录功能,荧光素酶活性为5.63×10~5,与对照组(0.47×10~5)比较差异具有显著性(t=10.12,P<0.01)。HCV NS5A能抑制内源性和外源性p53对p21启动子的激活作用,并呈剂量依赖性(F≥20.71,P<0.01)。凝胶滞留试验显示HCV NS5A能阻碍p53与其特异DNA序列的结合。 结论 HCV NS5A能抑制p53的反式激活功能使其目的基因p21启动子的转录功能下降,其机制是HCV NS5A能阻碍p53与其特异DNA序列的结合。     

Microsomal triglyceride transfer protein polymorphism (−493G/T) is associated with hepatic steatosis in patients with chronic hepatitis C

Background: Hepatic steatosis may promote progression of chronic hepatitis C (CHC). Microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of ApoB lipoprotein and is implicated in hepatitis C virus (HCV)‐related steatosis. The MTP ?493G/T polymorphism may promote liver fat accumulation, but its role in HCV‐related steatosis is still unclear. Methods: Two hundred ninety‐eight CHC patients were studied and genotyped for MTP ?493G/T variants. Hepatic MTP mRNA expression and activity were determined in a subgroup. Results: Patients with grades 2/3 steatosis were older, had a higher body mass index (BMI), more advanced fibrosis and lower MTP mRNA expression and carried more often HCV genotype 3 and the MTP T allele. Age, BMI, HCV‐3 and MTP T allele [odds ratio (OR) 2.05; 95% confidence interval (CI) 1.2–3.53; P=0.009] were independent risk factors for steatosis grades 2/3, and in HCV genotype non‐3 patients, the MTP T allele was the strongest predictor for steatosis grade 2/3 (OR 2.17; 95% CI 1.22–3.86; P=0.008). Moreover, TT carriers had higher high‐density lipoprotein (65.6±14.6 vs 56.1±16.2 mg/dl; P=0.003) and apolipoprotein AI (1.80±0.3 vs 1.60±0.3 g/L; P=0.005) levels than G allele carriers. Conclusions: Chronic hepatitis C patients with the MTP ?493T allele reveal higher grades of steatosis, indicating a relevant contribution to liver fat accumulation, particularly in HCV non‐3 patients.