【作者】 尚敬；

【导师】 陈璐璐；

【作者基本信息】 华中科技大学 ， 内科学， 2008， 博士

【摘要】 第一部分大鼠正糖-高胰岛素钳夹术的改进目的:在经典正糖-高胰岛素钳夹术基础上对插管技术进行改进,利用尾动静脉插管技术实施清醒状态下大鼠高胰岛素-正糖钳夹实验并对该技术进行评价。方法:正常喂养和高脂喂养Wistar大鼠各5只,喂养4周后,清醒状态下鼠尾根局部麻醉,分别尾动静脉插管,由尾静脉输入胰岛素(速度0.25u/kg.h)和葡萄糖,尾动脉采血,进行高胰岛素-正糖钳夹实验。结果:尾动静脉插管术后大鼠血糖在正常范围内,术后血糖无明显波动;高脂喂养组大鼠的葡萄糖输注率显著低于对照组(P＜0.01),表明高脂喂养的大鼠有胰岛素抵抗。结论:尾动静脉插管技术基础上的高胰岛素-正糖钳夹实验是可行的,较传统插管技术,具有简便易行、缩短实验周期、大鼠死亡率低的优点。第二部分白藜芦醇改善高脂喂养大鼠胰岛素抵抗、脂肪肝及其机制的研究目的:观察白藜芦醇是否能够改善高脂喂养所导致大鼠的胰岛素抵抗和非酒精性脂肪肝(NAFLD)并探讨其机制。方法:急性实验:10只Wistar大鼠分成两组(每组5只),对照组和白藜芦醇处理组,处理组白藜芦醇(100mg/kg.day)灌胃,对照组生理盐水灌胃,4小时后均处死,检测肝脏内AMPK磷酸化水平。慢性实验:30只Wistar大鼠平均分为3组(每组10只):正常对照组(标准饲料喂养),高脂组(高脂饲料喂养),和白藜芦醇组,其中白藜芦醇组全程高脂饲料喂养,并从第7周开始用白藜芦醇干预治疗(100mg/kg.day),各组均喂养16周。以正糖-高胰岛素钳夹技术评价大鼠IR,肝脏石蜡切片HE染色观察病理学改变,肝脏冰冻切片油红O染色观察肝细胞内脂质沉积状况,WesternBlot免疫印迹检测肝脏内AMPK磷酸化水平,RT-PCR检测肝脏固醇调节元件结合蛋白-1C(SREBP-1c)和脂肪酸合成酶(FAS)的mRNA基因表达。结果:急性实验结果:白藜芦醇干预组肝脏组织内AMPK磷酸化水平增高到对照组的164.2%;慢性实验结果:高脂喂养组大鼠与对照组相比,内脏脂肪指数、肝指数增高(P＜0.05),空腹胰岛素水平增高、葡萄糖输注率下降(P＜0.01),光镜下肝脏出现明显脂肪变性,肝组织内AMPK磷酸化水平降低到对照组的45.8%(P＜0.01),同时脂质合成基因SREBP-1c和FAS的mRNA表达量显著增多;白藜芦醇治疗后的大鼠,内脏脂肪含量显著下降(P＜0.01),空腹胰岛素水平降低、葡萄糖输注率明显增加(P＜0.01),光镜下的肝脏脂肪变性得到明显改善,肝组织内的AMPK磷酸化水平提高到对照组的70.2%(P＜0.05),与此同时脂质合成基因SREBP-1c和FAS的mRNA表达量降低。结论:白藜芦醇可以在体内促进肝脏内AMPK活性;白藜芦醇治疗可以改善高脂喂养大鼠的IR同时减少肝脏内脂质沉积从而改善脂肪肝,其机制是通过激活AMPK而抑制下游脂质合成途径。第三部分白藜芦醇可通过激活AMPK途径减少肝细胞内脂质沉积目的:观察白藜芦醇是否能够直接作用于肝细胞而减少肝细胞内的脂质沉积,并深入探讨其机制。方法:HepG2细胞种植于6空板内,当细胞融合到80%单层贴壁细胞时血清饥饿过夜后,分别加入含0μM、10μM、25μM、50μM的白藜芦醇的低糖DMEM培养基孵育6小时和24小时后提取细胞蛋白检测细胞内AMPK水平;将HepG2培养于含25mmol/L葡萄糖和10nm/L的胰岛素培养基分别培养24小时诱导细胞脂肪变性模型,然后以50μM白藜芦醇浓度干预脂肪变性模型细胞24小时,提取蛋白后WesternBlot免疫印迹检测细胞内AMPK磷酸化水平;Trizol提取细胞内mRNA检测固醇调节元件结合蛋白-1C(SREBP-1c)和脂肪酸合成酶(FAS)的基因表达水平,同时油红O染色检测细胞内脂质沉积和提取细胞内脂质检测细胞内TG含量。结果:白藜芦醇呈剂量、时间依赖性促进细胞内AMPKα磷酸化。HepG2细胞脂肪变性模型组出现明显的红色脂滴聚集(油红O染色显示),细胞内TG含量显著高于对照组(P＜0.01),AMPK磷酸化水平降低到对照组的33.5%,SREBP-1c、FAS基因表达量分别增高到对照组的182%和167%;白藜芦醇处理组HepG2油红O染色和细胞内TG定量均显示脂肪变性得到明显改善,AMPK磷酸化水平恢复到对照组的72.6%,而SREBP-1c和FAS的基因表达量也显著下降。结论:白藜芦醇可以在体外提高肝细胞内AMPK活性;白藜芦醇可以直接作用于肝细胞减少肝细胞内TG沉积,其机制是激活AMPK后抑制下游的SREBP-1c、FAS的基因表达进而抑制脂质合成。第四部分白藜芦醇改善高脂诱导胰岛素抵抗大鼠氧化应激目的:观察白藜芦醇是否能够减轻高脂诱导IR模型大鼠体内氧化应激,并探讨氧化应激与IR关系。方法:动物造模分组同本文第二部分,高胰岛素-正糖钳夹术检测胰岛素敏感性,试剂盒分别检测血清标本羟自由基、MDA水平和SOD活性,ELISA法检测血清TNF-α浓度。结果:模型组大鼠血清羟自由基、MDA、TNF-α较对照组均显著增高,分别是对照组的1.2倍、2.1倍、1.69倍水平(P＜0.05或者0.01),SOD活性降低到对照组的86%(P＜0.01),且伴有胰岛素抵抗,提示模型组大鼠处于严重氧化应激和IR状态;经过白藜芦醇治疗后,羟自由基、MDA、TNF-α浓度降低,SOD活性恢复,IR也得到明显改善。结论:IR和氧化应激关系密切,氧化应激是发生IR的重要原因之一,白藜芦醇的抗氧化应激效应是白藜芦醇改善IR的机制之一。更多还原

【Abstract】 Aim To utilize tail arterial and vein catheterization technique to perform hyperinsulinemic-euglycemic clamp in conscious rats and evaluate this technique.Methods Wistar rats were fed with standard chow diet or high-fat diet(two groups and five rats each group).Four weeks later,conscious rats received local anesthesia on the root of tail,then tail arterial and vein catheterization respectively.Tail vein was used to inject insulin(the infusion speed was 0.2 5u/kg.h) and tail arterial to obtain blood sample to perform hyperinsulinemic-euglycemic clamp.Result The glucose infusion rate(GIR) of high-fat diet group was significantly lower than control group,suggesting that the high-fat diet fed rats was in insulin resistance state.Conclusion Comparing with regularly jugular vein and carotid artery catheterization technique,hyperinsulinemic-euglycemic clamp in conscions rats based on tail arterial and vein catheterization technique has the advantages of stress-free,easy-to-perform,very low death rates of rats. Aim:To observe the effect of resveratrol on IR and the nonalcoholic fatty liver disease (NAFLD) and tentatively explore the mechanism.Methods Acute experiment:rats in fed state were orally administrated with RSV(100mg/kg) or saline(vehicle)(5 rats each group).Four hours after administration,the rats were sacrificed to determine the AMPK-αphosphorylation level;Chronic experiment:thirty Wistar rats were randomly allocated to three groups(10 rats for each group):normal control group(NC group,fed with standard chow),high fat feeding group(HF group,fed with high fat diet) and resveratrol treated group(HR group).The resveratrol-treated group was fed with high fat diet all the time and received resveratrol administration(100mg/kg.day) except the first 6 weeks.Each group rats were kept 16 weeks before killed.At the end of animal experiment,hyperinsulinemic-euglycemic clamp was performed to evaluate insulin sensitivity.Liver histology was detected by HE staining.Phosphorylation of AMP-activated protein kinase(AMPK) levels were determined by WesternBlot technique.SREBP-1c and FAS gene expressions,two key lipogenesis regulators,were detected by RT-PCR.Results Compared to NC group,visceral fat index and liver mass index increased in HF group.Fasting serum insulin(FINS) was elevated and glucose infusion rate(GIR) decreased in HF group.Pathology observation by light microscope showed liver steatosis in HF group.Phosphorylation of AMPK levels in HF group decreased to 45.8%with the down regulation of SREBP-1c and FAS gene expressions.However,resveratrol administration greatly improved the visceral fat index,liver mass index,FINS,GIR as well as liver steatosis compared with HF group.Phosphorylation of AMPK was also elevated to 70.2% in HR group compared with NC group with the subsequent up-regulation of SREBP-1c and FAS gene expressions.Conclusion Administration of RSV can promote AMPK activation in vivo;Resveratrol administration markedly improved high-fat feeding induced insulin resistance and liver fat accumulation,with the resultant improvement of NAFLD.Elevated phosphorylation of AMPK contributed to the improvement of NAFLD. Aim:To investigate whether resveratrol(RSV) can directly improve steatosis in hepatocytes and try to find the possible mechanism.Methods:HepG2 cells exposed to increasing concentrations of RSV for various times on 6-well plate after serum starvation overnight when they come to 80%cell confluence.A cell steatosis model were established by exposing HepG2 cells to a high concentration of glucose(25mmol/l) and insulin(100nm/L) and then were treated with resveratrol for 24 hours.Phosphorylation levels of AMPK were detected by WesternBlot analysis.Gene expression of SREBP-1c and FAS were determined by RT-PCR.Oil red O staining was performed and intracellular triglyceride contents were quantified to observe the lipid accumulation in cell steatosis model treated with or without RSV.Results:RSV can promote AM PK phosphorylation in a dose and time dependent manner in HepG2 cells.Cell steatosis model was established,as confirmed by Oil red O staining and quantization of total intracellular TG contents.In the model,phosphorylation levels of AMPK were reduced to the 33.5%of the control cells;gene expression of SREBP-1c and FAS were elevated to the 182%and 167%of control,respectively.However,when challenged with RSV treatment,cell steatosis was greatly improved,as confirmed by Oil red O staining and quantization of total intracellular TG contents,phosphorylation levels of AMPK returned to the 72.6%of the control cells;gene expression of SREBP-1c and FAS were decreased,respectively.Conclusion:RSV can stimulate AMPK in vitro in hepatocytes.RSV can directly act on hepatocytes to improve hepatic steatosis.The possible mechanism is that RSV can stimulate AMPK and prevent the lipogenesis process by inhibit its downstream key fators such as SREBP-1c and FAS. Aim:To investigate whether resveratrol can ameliorate oxidative stress in the high fat induced insulin resistance(IR) rat model and explore the relationship between oxidative stress and IR.Methods:The animal model was established according to the second part of this article. Hyperinsulinemic-euglycemic clamp was performed to evaluate the IR state.Serum hydroxy radical level,serum malondialdehyde(MDA)level and activity of superoxide dismutase(SOD) were measured.Serum TNF-αlevel was detected by ELISA.Results:Compared with control group,serum hydroxy radical,MDA,TNF-αlevels of model rats increased significantly(P＜0.05 or 0.01),while activity of SOD decreased markedly.Beside,model rats developed IR.The indicators suggested that the model rats were in serious oxidative stress and IR state.However,resveratrol recovered the the indicators above,respectively.Conclusion:There is a close relationship between IR and oxidative stress.Oxidative stress has a causal role in the development of IR.Antioxidant property of resveratrol contributed to the mechanism by which resveratrol improve IR.更多还原