目的观察高脂、高糖喂养对大鼠非酒精性脂肪肝(NAFLD)的影响,探讨其沉默信息调节因子2相关酶1(Sirt1)及脂肪酸合成酶(FAS)的表达变化,进而揭示可能引起NAFLD发病的分子途径。方法 Wistar雄性大鼠分为正常组和模型组,分别饲以基础饲料和...目的观察高脂、高糖喂养对大鼠非酒精性脂肪肝(NAFLD)的影响,探讨其沉默信息调节因子2相关酶1(Sirt1)及脂肪酸合成酶(FAS)的表达变化,进而揭示可能引起NAFLD发病的分子途径。方法 Wistar雄性大鼠分为正常组和模型组,分别饲以基础饲料和高脂、高糖饲料。13周后处死,肝脏行苏木精-伊红(HE)染色和油红O染色,检测血清及肝匀浆总胆固醇(TC)、三酰甘油(TG)含量,应用实时逆转录聚合酶链反应(Real-time RT-PCR)和Western blot检测大鼠肝组织FAS、Sirt1的m RNA和蛋白表达水平。结果模型组大鼠体重、能量利用率、血清及肝脏TC和TG明显高于正常组。HE染色和油红O染色观察到模型组大鼠肝细胞脂肪明显变性。模型组肝脏Sirt1 m RNA水平与正常组比较,差异无统计学意义,但蛋白水平明显低于正常组;模型组肝脏中FAS m RNA和蛋白水平明显低于正常组。结论高脂、高糖喂养能使大鼠形成NAFLD,增加血清及肝脏的脂肪浓度。Sirt1表达降低提示其与NAFLD的发生有关,而FAS表达降低与以往部分研究结果截然不同,还需要更深层次的研究。展开更多
Objective To study the role of sirtuin 1 (SIRT1) in Fas ligand (FasL) expression regulation during vascular lesion formation and to elucidate the potential mechanisms.Methods SIRT1 and FasL protein levels were detecte...Objective To study the role of sirtuin 1 (SIRT1) in Fas ligand (FasL) expression regulation during vascular lesion formation and to elucidate the potential mechanisms.Methods SIRT1 and FasL protein levels were detected by Western blotting in either mouse arteries extract or the whole rat aortic vascular smooth muscle cell (VSMC) lysate.Smooth muscle cell (SMC)-specific human SIRT1 transgenic (Tg) C57BL/6 mice and their littermate wild-type (WT) controls underwent complete carotid artery ligation (ligation groups) or the ligation-excluded operation (sham groups).The carotid arteries were collected 1 day after operation.Reverse transcription-polymerase chain reaction was performed to detect the mRNA levels of SIRT1 and FasL.Luciferase reporter assays were performed to detect the effect of WT-SIRT1,a dominant-negative form of SIRT1 (SIRT1H363Y),and GATA-6 on the promoter activity of FasL.Flow cytometry assay was applied to measure the hypodiploid DNA content of VSMC so as to monitor cellular apoptosis.Results SIRT1 was expressed in both rat aortic VSMCs and mouse arteries.Forced SIRT1 expression increased FasL expression both in injured mouse carotid arteries 1 day after ligation (P<0.001) and VSMCs treated with serum (P<0.05 at the transcriptional level,P<0.001 at the protein level).No notable apoptosis was observed.Furthermore,transcription factor GATA-6 increased the promoter activity of FasL (P<0.001).The induction of FasL promoter activity by GATA-6 was enhanced by WT-SIRT1 (P<0.001),while SIRT1H363Y significantly relieved the enhancing effect of WT-SIRT1 on GATA-6 (P<0.001).Conclusions Overexpression of SIRT1 up-regulates FasL expression in both flow-restricted mouse carotid arteries and serum-stimulated VSMCs.The transcription factor GATA-6 participates in the transcriptional regulation of FasL expression by SIRT1.展开更多
[目的]探究缬草酸对骨折后深静脉血栓形成及Sirtuin1/NF-κB通路的影响。[方法]所有Sprague Dawley大鼠被随机分为3组(每组n=10):假手术组、模型组和缬草酸组。通过HE染色分析下腔静脉组织病理变化。通过蛋白免疫印迹分析Sirtuin1/NF-κ...[目的]探究缬草酸对骨折后深静脉血栓形成及Sirtuin1/NF-κB通路的影响。[方法]所有Sprague Dawley大鼠被随机分为3组(每组n=10):假手术组、模型组和缬草酸组。通过HE染色分析下腔静脉组织病理变化。通过蛋白免疫印迹分析Sirtuin1/NF-κB通路的变化。通过酶联免疫吸附剂测定分析炎症因子IL-6、IL-1β和TNF-α的水平。通过全自动凝血分析仪检测凝血酶原时间、活化部分凝血活酶时间、凝血酶时间。[结果]病理分析发现,缬草酸治疗后,模型组大鼠血栓染色变淡,血管再通,血管壁有少数炎症细胞。此外,缬草酸治疗后,模型组大鼠血栓重量逐渐减小(6.36±0.02 vs 11.03±0.02,P<0.05),长度逐渐缩短。此外,模型组大鼠在经过缬草酸治疗后炎症因子水平明显降低(4.67±0.08 vs 7.02±0.01;5.99±0.05 vs 11.02±0.03;5.81±0.06 vs 10.02±0.03;P<0.05),凝血酶原时间、活化部分凝血活酶时间、凝血酶时间水平明显上升。蛋白免疫印迹结果发现,与模型组相比,缬草酸组大鼠的下腔静脉组织中NF-κB p65磷酸化水平降低(0.58±0.02 vs 0.92±0.03;P<0.05),Sirtuin1水平增加(0.69±0.01 vs 0.16±0.03;P<0.05)。[结论]缬草酸能够改善下腔静脉血栓导致的病理损伤、抑制下腔静脉血栓形成,并能抑制血浆中炎症因子的水平。这一作用可能与Sirtuin1表达上调而NF-κB的抑制相关。展开更多
目的研究沉默信息调节因子2相关酶1（silentmating—typeinformationregulation2homologue1，Sirtuinl或SIRT1）在野生型及突变型p53肝细胞肝癌中差异性调节肿瘤形成的机制。方法对PLC5（249位点突变型r63），HepG2（野生型p53）细胞株...目的研究沉默信息调节因子2相关酶1（silentmating—typeinformationregulation2homologue1，Sirtuinl或SIRT1）在野生型及突变型p53肝细胞肝癌中差异性调节肿瘤形成的机制。方法对PLC5（249位点突变型r63），HepG2（野生型p53）细胞株进行shSIRT1慢病毒转染，通过Westernblot检测信号转导通路，通过细胞生长和增殖实验，集落形成实验，动物实验等检测HepG2细胞，HepG2一shSIRTI细胞，PLC5细胞和PLC5-shSIRT1细胞肿瘤形成能力。结果抑制SIRT1表达可降低HepG2（野生型p53）肝癌细胞的增殖，提高PLC5（突变型p53）肝癌细胞的增殖（f=3．595，P〈0．01）；敲除HepG2细胞中的SIRT1使得p53活化，激活p53下游蛋白p21的表达，敲除PLC5细胞中SIRT1能使乙酰化p53蛋白表达升高，但其下游蛋白p21不能被激活；在HepG2细胞中抑制SIRT1不能改变AMP依赖的蛋白激酶[adenosine5’-monophosphate（AMP）-activated protein kinase，AMPK]的功能（磷酸化AMPK蛋白），而在PLC5细胞（突变型p53）中抑制SIRT1可降低AMPK的功能（t=4．268，P〈0．叭）。结论在肝细胞肝癌中SIRT1激活后的作用及功能主要受到p53突变状态的影响。展开更多
文摘目的观察高脂、高糖喂养对大鼠非酒精性脂肪肝(NAFLD)的影响,探讨其沉默信息调节因子2相关酶1(Sirt1)及脂肪酸合成酶(FAS)的表达变化,进而揭示可能引起NAFLD发病的分子途径。方法 Wistar雄性大鼠分为正常组和模型组,分别饲以基础饲料和高脂、高糖饲料。13周后处死,肝脏行苏木精-伊红(HE)染色和油红O染色,检测血清及肝匀浆总胆固醇(TC)、三酰甘油(TG)含量,应用实时逆转录聚合酶链反应(Real-time RT-PCR)和Western blot检测大鼠肝组织FAS、Sirt1的m RNA和蛋白表达水平。结果模型组大鼠体重、能量利用率、血清及肝脏TC和TG明显高于正常组。HE染色和油红O染色观察到模型组大鼠肝细胞脂肪明显变性。模型组肝脏Sirt1 m RNA水平与正常组比较,差异无统计学意义,但蛋白水平明显低于正常组;模型组肝脏中FAS m RNA和蛋白水平明显低于正常组。结论高脂、高糖喂养能使大鼠形成NAFLD,增加血清及肝脏的脂肪浓度。Sirt1表达降低提示其与NAFLD的发生有关,而FAS表达降低与以往部分研究结果截然不同,还需要更深层次的研究。
基金Supported by the National Natural Science Foundation of China(81102444)Special Fund of the National Laboratory of China(2060204)
文摘Objective To study the role of sirtuin 1 (SIRT1) in Fas ligand (FasL) expression regulation during vascular lesion formation and to elucidate the potential mechanisms.Methods SIRT1 and FasL protein levels were detected by Western blotting in either mouse arteries extract or the whole rat aortic vascular smooth muscle cell (VSMC) lysate.Smooth muscle cell (SMC)-specific human SIRT1 transgenic (Tg) C57BL/6 mice and their littermate wild-type (WT) controls underwent complete carotid artery ligation (ligation groups) or the ligation-excluded operation (sham groups).The carotid arteries were collected 1 day after operation.Reverse transcription-polymerase chain reaction was performed to detect the mRNA levels of SIRT1 and FasL.Luciferase reporter assays were performed to detect the effect of WT-SIRT1,a dominant-negative form of SIRT1 (SIRT1H363Y),and GATA-6 on the promoter activity of FasL.Flow cytometry assay was applied to measure the hypodiploid DNA content of VSMC so as to monitor cellular apoptosis.Results SIRT1 was expressed in both rat aortic VSMCs and mouse arteries.Forced SIRT1 expression increased FasL expression both in injured mouse carotid arteries 1 day after ligation (P<0.001) and VSMCs treated with serum (P<0.05 at the transcriptional level,P<0.001 at the protein level).No notable apoptosis was observed.Furthermore,transcription factor GATA-6 increased the promoter activity of FasL (P<0.001).The induction of FasL promoter activity by GATA-6 was enhanced by WT-SIRT1 (P<0.001),while SIRT1H363Y significantly relieved the enhancing effect of WT-SIRT1 on GATA-6 (P<0.001).Conclusions Overexpression of SIRT1 up-regulates FasL expression in both flow-restricted mouse carotid arteries and serum-stimulated VSMCs.The transcription factor GATA-6 participates in the transcriptional regulation of FasL expression by SIRT1.
文摘[目的]探究缬草酸对骨折后深静脉血栓形成及Sirtuin1/NF-κB通路的影响。[方法]所有Sprague Dawley大鼠被随机分为3组(每组n=10):假手术组、模型组和缬草酸组。通过HE染色分析下腔静脉组织病理变化。通过蛋白免疫印迹分析Sirtuin1/NF-κB通路的变化。通过酶联免疫吸附剂测定分析炎症因子IL-6、IL-1β和TNF-α的水平。通过全自动凝血分析仪检测凝血酶原时间、活化部分凝血活酶时间、凝血酶时间。[结果]病理分析发现,缬草酸治疗后,模型组大鼠血栓染色变淡,血管再通,血管壁有少数炎症细胞。此外,缬草酸治疗后,模型组大鼠血栓重量逐渐减小(6.36±0.02 vs 11.03±0.02,P<0.05),长度逐渐缩短。此外,模型组大鼠在经过缬草酸治疗后炎症因子水平明显降低(4.67±0.08 vs 7.02±0.01;5.99±0.05 vs 11.02±0.03;5.81±0.06 vs 10.02±0.03;P<0.05),凝血酶原时间、活化部分凝血活酶时间、凝血酶时间水平明显上升。蛋白免疫印迹结果发现,与模型组相比,缬草酸组大鼠的下腔静脉组织中NF-κB p65磷酸化水平降低(0.58±0.02 vs 0.92±0.03;P<0.05),Sirtuin1水平增加(0.69±0.01 vs 0.16±0.03;P<0.05)。[结论]缬草酸能够改善下腔静脉血栓导致的病理损伤、抑制下腔静脉血栓形成,并能抑制血浆中炎症因子的水平。这一作用可能与Sirtuin1表达上调而NF-κB的抑制相关。
文摘目的研究沉默信息调节因子2相关酶1（silentmating—typeinformationregulation2homologue1，Sirtuinl或SIRT1）在野生型及突变型p53肝细胞肝癌中差异性调节肿瘤形成的机制。方法对PLC5（249位点突变型r63），HepG2（野生型p53）细胞株进行shSIRT1慢病毒转染，通过Westernblot检测信号转导通路，通过细胞生长和增殖实验，集落形成实验，动物实验等检测HepG2细胞，HepG2一shSIRTI细胞，PLC5细胞和PLC5-shSIRT1细胞肿瘤形成能力。结果抑制SIRT1表达可降低HepG2（野生型p53）肝癌细胞的增殖，提高PLC5（突变型p53）肝癌细胞的增殖（f=3．595，P〈0．01）；敲除HepG2细胞中的SIRT1使得p53活化，激活p53下游蛋白p21的表达，敲除PLC5细胞中SIRT1能使乙酰化p53蛋白表达升高，但其下游蛋白p21不能被激活；在HepG2细胞中抑制SIRT1不能改变AMP依赖的蛋白激酶[adenosine5’-monophosphate（AMP）-activated protein kinase，AMPK]的功能（磷酸化AMPK蛋白），而在PLC5细胞（突变型p53）中抑制SIRT1可降低AMPK的功能（t=4．268，P〈0．叭）。结论在肝细胞肝癌中SIRT1激活后的作用及功能主要受到p53突变状态的影响。
