【作者】 陈坡；

【导师】 杨天伦； 夏珂；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 第一章阿托伐他汀调节胰岛素抵抗大鼠血浆ADMA/NO,主动脉SREBP1、DDAH1表达的研究目的：内皮功能紊乱是心血管疾病发生的主要危险因素,同时也可导致机体对胰岛素敏感性下降,引起胰岛素抵抗(insulin resistance IR)。非对称性二甲基精氨酸(Asymmetric dimethylarginine ADMA)、内源性一氧化氮合酶(NOS)抑制剂,可损伤内皮功能,已被公认为心血管疾病的危险因子。二甲基精氨酸二甲胺水解酶(dimethylarginine dimethylaminohydrolase DDAH)可将内源性ADMA降解为L-瓜氨酸,从而增加NO的合成,改善内皮功能。他汀类药物已被公认为可改善内皮细胞功能,降低心血管事件发生。本研究第一部分通过建立高脂喂养胰岛素抵抗的大鼠模型,并给予阿托伐他汀干预治疗,检测主动脉DDAHl、SERBP1蛋白及基因的表达,以及血浆ADMA水平。旨在观察阿托伐他汀对胰岛素抵抗大鼠的DDAHl/ADMA/NO系统的影响,从而评价阿托伐他汀对于胰岛素抵抗状态下血管内皮细胞功能的作用和影响方法：30只SD大鼠(200-250g)随机分为2组：1)对照组(CON,N=10)：普通饮食喂养。2)高脂饮食组(HFD,N=20)：高脂饮食喂养8周。喂养第8周末,测空腹血糖,胰岛素水平,通过计算HOMA-IR(空腹胰岛素水平×空腹血糖水平/22.5,>2.69判断为有胰岛素抵抗)评估大鼠胰岛素敏感性。随后高脂喂养组进一步分为2组：胰岛素抵抗+阿托伐他汀治疗组(IR+A,N=10)：继续高脂饮食喂养,同时给予阿托伐他汀30mg/kg/d(辉瑞)灌胃8周；胰岛素抵抗组(IR,N=10),继续高脂喂养8周。实验终点检测血清学指标,Western blot、Real-time PCR检测主动脉DDAHl、SERBPl蛋白及基因的表达,高效液相色谱法检测血浆ADMA水平。结果：胰岛素抵抗组大鼠甘油三酯及C反应蛋白与对照组相比明显增高(分别为1.45±O.41vs.O.9±O.24mmol/L,P<0.05；0.3±O.1vs O.12±O.1mg/L,P<0.01)。与胰岛素抵抗组比较,阿托伐他汀组大鼠血甘油三酯、C反应蛋白水平明显降低(分别为0.82±0.3vs1.45±0.41mmol/l,P<0.01；0.03±O.01vs0.3±O.1mg/L,P<0.01.)通过高效液相色谱仪(HPLC)测定血ADMA的水平及主动脉DDAH活性,与对照组(CON)相比,胰岛素抵抗组(IR)血浆ADMA水平明显升高,而DDAH活性明显降低,(分别为1.1±0.24vs.0.52±0.1μmol/L,P<O.01；O.05±O.01vs.O.12±O.01μ/g protein,P<0.01),与胰岛素抵抗组比较,阿托伐他汀组大鼠血浆ADMA水平明显下降,DDAH活性明显升高(分别为O.87±O.22vs.1.1±O.24μmol/L,P<O.05；O.07±O.012vs.O.05±0.01μ/g protein,P<O.05)。胰岛素抵抗组大鼠HOMA指数较对照组增高(3.06±0.6vs.1.89±0.3,P<0.05),而阿托伐他汀可降低胰岛素抵抗大鼠HOMA(?)旨数(3.06±0.6vs.2.38±0.5,P<0.05)。通过Pearson(?)目关性分析发现,HOMA指数与DDAH活性呈明显的负性相关(r=-0.795,P<0.01)。通过乙酰胆碱诱导的主动脉舒张反应显示,与对照组对比,胰岛素抵抗组大鼠主动脉对乙酰胆碱的舒张反应明显减低,而阿托伐他汀可增强胰岛素抵抗组大鼠主动脉对乙酰胆碱舒张反应。胰岛素抵抗组大鼠主动脉DDAH1, SREBP1蛋白及mRNA水平较对照组均明显下降,而阿托伐他汀可增高DDAH1, SREBP1蛋白及]mRNA水平。结论：阿托伐他汀可能通过调节DDAH1/ADMA水平改善胰岛素抵抗大鼠内皮功能,而SREBP1作为转录因子,可能介导阿托伐他汀对DDAH1/ADMA的调节。第二章SREBP1介导阿托伐他汀调控胰岛素抵抗内皮细胞DDAH1/ADMA系统的表达背景：DDAH1/ADMA系统与内皮功能紊乱密切相关,前期动物实验发现：胰岛素抵抗状态下DDAH1、DDAH活性,SREBP1表达受抑制,阿托伐他汀作为内皮保护药物,可改善DDAH活性,增力(?)SREBP1表达。为进一步证实阿托伐他汀对胰岛素抵抗状态下内皮细胞DDAH1/ADMA系统调节的可能机制,本研究第二部分通过建立人脐血内皮细胞胰岛素抵抗模型,并采用siRNA转染技术抑制SREBP-1基因表达,旨在观察SREBP1抑制状态下内皮细胞表达DDAH1、ADMA及内皮细胞功能的变化,以评价SREBP1在阿托伐他汀改善血管内皮细胞功能中的作用机制,方法：1、高胰岛素培养诱导内皮细胞胰岛素抵抗模型：采用不同浓度胰岛素培养人脐血内皮细胞(Human Umbilical Vein Endothelial Cells, HUVECs)不同时间(12、24、36和48h)。分析不同浓度胰岛素,不同培养时间对内皮细胞摄取葡糖糖能力的影响,最后选择最佳浓度,最佳培养时间培养内皮细胞建立内皮细胞胰岛素抵抗模型。2、采用不同浓度阿托伐他汀(0.05,0.1,1.0,10μmol/L,)分别作用12、24、36和48h,通过分析内皮细胞摄取葡萄糖能力,确定最佳浓度及作用时间。3、siRNA转染抑制(?)SREBP1表达：使用LipofectamineTM2000转染siRNA。以250ul Opti-MEM(?)稀释5ul LipofectamineTM2000混匀后在室温下孵育5分钟。以250ul Opti-MEM(?) Ⅰ稀释7.5ul siRNA,轻轻混匀。孵育5分钟后将混合稀释的siRNA和LipofectamineTM2000混合,并在室温下孵育20分钟,以便允许复合物的形成。溶液可能出现混浊,但是这不会影响转染。将siRNA-LipofectamineTM2000复合物加入培养板中并通过前后摇动培养板使其混合。6小时后进行荧光检测转染率。4、实验分组：①正常对照组(CON)：正常培养HUVECs24小时；②胰岛素抵抗组(IR)：在含有100nmol/L胰岛素的RPMI1640培养基中培养内皮细胞24小时。③胰岛素抵抗+阿托伐他汀治疗组(IR+A)：胰岛素抵抗造模成功后换含有10-5mol/L阿托伐他汀的培养基培养24小时。④胰岛素抵抗+空白siRNA (IR+scramble):转染空白siRNA载体后在含有100nmol/L胰岛素的RPMI1640培养基中培养内皮细胞24小时。⑤胰岛素抵抗+siRNA组-SREBP1(IR+siRNA):转染siRNA-SREBPl后在含有100nmol/L胰岛素的RPMI1640培养基中培养内皮细胞24小时。⑥胰岛素抵抗+siRNA+阿托伐他汀组(IR+siRNA+A):转染siRNA-SREBPl后在含有100nmol/L胰岛素的RPMI1640培养基中培养内皮细胞24小时,再用含有10-5mol/L阿托伐他汀的培养基培养24小时。5、检测各组内皮细胞SREBP1, DDAH1, DDAH活性,ADMA, NO, NOS水平的表达。结果：1、内皮细胞置于含1%FBS和100nmol/L胰岛素的RPMI1640培养基中培养24h,细胞消耗葡萄糖最少,即胰岛素抵抗作用最为明显。2、阿托伐他汀对胰岛素抵抗内皮细胞表达ADMA和DDAH活性的影响：胰岛素抵抗内皮细胞上清液ADMA水平较对照组明显升高(0.94±0.11vs0.49±0.09u/mmol, P<0.01),而内皮细胞DDAH活性明显降低(0.052±0.01vs0.158±0.02u/g, P<0.01);与胰岛素抵抗内皮细胞相比较,加用阿托伐他汀处理组上清液ADMA水平明显降低(0.59±0.06vs0.94±0.11u/mmol P<0.01), DDAH活性明显增强(0.11±0.02vs0.052±0.01, P<0.01)。3、阿托伐他汀对胰岛素抵抗内皮细胞SREBP1和DDAH1表达的影响：胰岛素抵抗状态下内皮细胞表达SREBP1, DDAH1蛋白及基因均明显下降,阿托伐他汀干预组内皮细胞表达SREBP1和DDAH1明显增加(P<0.01)。4、siRNA阻断SREBP1基因对内皮细胞DDAH1表达的影响：阻断SREBP1基因表达后内皮细胞表达DDAH1基因和蛋白明显减少(P<0.01)。5、siRNA阻断SREBP1基因对内皮细胞DDAH活性和ADMA系统的影响：阻断SREBP1基因表达后内皮细胞DDAH活性明显降低(P<0.01),上清液ADMA增加、NO水平降低。6、siRNA阻断SREBP1基因对阿托伐他汀干预的胰岛素抵抗内皮细胞DDAH1表达的影响：阻断SREBP1基因表达后,阿托伐他汀干预的胰岛素抵抗内皮细胞DDAH1基因和蛋白表达明显减(p<0.01)。7、阻断SREBP1基因对阿托伐他汀干预的胰岛素抵抗内皮细胞DDAH活性和ADMA系统的影响：阻断SREBP1基因表达后,阿托伐他汀干预的胰岛素抵抗内皮细胞DDAH活性明显降低(P<0.01),上清液ADMA增加、NO水平降低。结论：1、阿托伐他汀改善胰岛素抵抗内皮细胞SREBP1、DDAH1表达及DDAH活性。2、siRNA沉默内皮细胞SREBP1基因,抑制了内皮细胞DDAH1蛋白和基因的表达。3、SREBP1介导阿托伐他汀调节胰岛素抵抗内皮细胞DDAH1/ADMA系统的表达。更多还原

【Abstract】 Chapter1. Atorvastatin modulates DDAH1/ADMA system in high-fat diet-induced insulin resistant rats with endothelial dysfunctionObjective:Endothelial dysfunction, a main risk factor of cardiovascular diseases, can be attributed to insulin resistance. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), plays an important role in endothelial function and has been considered as a biomarker for major cardiovascular events and mortality in cohorts with high, intermediate, and low overall cardiovascular risks. Dimethylarginine dimethyl-aminohydrolase1(DDAH1) is a metabolic enzyme for asymmetric dimethylarginine (ADMA), both of which are closely related to endothelial function. Atorvastatin has been widely used in cardiovascular disease to protect endothelial function. We established insulin resistant rats models and observed the effects of atorvastatin on DDAH1/ADMA system.Methods:Eight-week-old SD rats (weighed200-250g) were randomly divided into two groups:(1) the control group (CON; n=10), in which rats were fed with standard rodent chow and water adlibitum (protein,20kcal%; carbohydrate,70kcal%; and lipid,10kcal%) and (2) the high-fat diet group (HFD; n=20), in which rats were fed with fat-rich chow and water adlibitum (protein,20kcal%; carbohydrate,35kcal%; and lipid,45kcal%, predominantly in the form of lard). At the end of the8th week, fasting plasma glucose and insulin levels were measured, and insulin sensitivity was evaluated by calculating the Homeostatic Model Assessment-Insulin Resistance (HOMA-IR) index as [(fasting insulin, mU/ml)×(fasting glucose, mmol/L)/22.5]. Studies have previously demonstrated that high-fat diet induced insulin resistance in rats. Next, the HFD group was further divided into two group. The first group (IR+A; n=10) received atorvastatin (30mg/kg/day; Pfizer Pharmaceuticals) and the second group (IR; n=10) was kept on vehicle (water) for additional8weeks. Body weights were measured weekly. Fasting glucose and insulin levels were measured at the beginning of the8th and16th week. Finally, the aorta and plasma were collected and stored at-80℃. At the end of study, protein and mRNA were examined by using Western-blot and Real-time PCR, Plasma ADMA concentrations were measured by high-performance liquid chromatography.Results:1. Higher levels of plasma triglycerides and C reactive protein (CRP) were observed in insulin resistant rats than those in the control (1.45±0.41vs.0.9±0.24mmol/L, P<0.05;0.3±0.1vs.0.12±0.1mg/L, P<0.01; respectively). Significantly, atorvastatin treatment reduced both plasma triglycerides and CRP levels in insulin resistant rats (0.82±0.3mmol/L and0.03±0.01mg/L, respectively; P<0.01).2. High-fat diet-fed rats showed the significant higher HOMA-IR than the control group (3.06±0.6vs.1.89±0.3, P<0.05), and such high insulin sensitivity in insulin resistance rats was reduced by atorvastatin treatment (3.06±0.6vs.2.38±0.5, P<0.05).3. Plasma ADMA concentrations were found significantly increased in insulin resistance rats, compared to the control (1.1±0.24vs.0.52±0.1μmol/L, P<0.01), whereas aortic DDAH activity was reduced (0.05±0.01vs.0.12±0.01μ/g protein, P<0.01). Further atorvastatin treatment of insulin resistance rats not only significantly decreased plasma ADMA levels (0.87±0.22vs.1.1±0.24μmol/L, P<0.05) but also enhanced aortic DDAH activity by18%(0.07±0.012vs.0.05±0.01μ/g protein, P<0.05).4. A significant negative correlation between HOMA-IR and aortic DDAH activity (r=-0.795, P<0.01) was observed, which in turn indicates a positive correlation between insulin sensitivity and aortic DDAH activity.5. Insulin resistant rats showed decreased plasma NO levels and NOS activity, compared to the control group (25.6±7.4vs.62.4±4.9μmol/L,19±3.2vs.35±4.8μ/ml, respectively; P<0.01). Significantly, atorvastatin treatment of insulin resistance rats was able to increase the plasma NO levels (43.53±8.2vs.25.6±7.4μmol/L, P<0.01) and NOS activity (26±3.5vs.19±3.2μ/ml, P<0.05).6.Insulin resistance in high-fat diet-fed rats significantly attenuated ACh-induced endothelium-dependent relaxation; in contrast, atorvastatin treatment preserved the control-level relaxation7. The mRNA and protein expression of SREBP1and DDAH1in thoracic aorta of insulin resistant rats were significantly lower than those in the control; however, all levels were restored by further atorvastatin treatment.Conclusion:High-fat diet-induced insulin resistance was able to not only downregulate aortic DDAH1expression and DDAH activity but also increase plasma ADMA concentrations in rats. Furthermore, atorvastatin treatment increased the expression of both SREBP1and DDAH1in thoracic aorta and decreased plasma ADMA levels in insulin resistant rats. These results suggest that atorvastatin may protect endothelial function by modulating the DDAH1/ADMA system, specifically, by restoring DDAH activity in insulin resistant rats. Chapter2Atorvastain modulates DDAH1/ADMA system via SREBP1pathway in insulin resistant HUVECsObjective:Dimethylarginine dimethyl-aminohydrolase1(DDAH1)/Asymmetric dimethylarginine (ADMA) system is closely related to endothelial function. Atorvastatin performed as an endothelium-protective drug, and in vivo study we have proved that atorvastatin modulated DDAH1/ADMA system in insulin resistant rats. However, the possible mechanism is not clear. Sterol regulatory element binding protein-1(SREBP1), a transcription factor regulating the expression of genes involving in lipid homeostasis and glucose metabolism, was reported to be regulated by Atorvastatin in the progress of lipid lowering. Considering the promoter of DDAH1contains SREBP1binding sites, we make a hypothesis that atorvastatin can modulate DDAH1/ADMA system via SREBP1pathway. We aimed to determine the possible mechanism of atorvastatin on DDAH1/ADMA in Human umbilical vein endothelial cells (HUVECs)Methods:Human umbilical vein endothelial cells (HUVECs) were treated in RPMI1640medium supplemented with1%FBS and10,50,100,500,1000nmol/L insulin for12,24,36,48hours respectively. The glucose consumption were used to determine the insulin sensitivity. After the establishment of insulin resistant cell model, siRNA for silencing SREBP-1were transfected into HUVEC cells at a final concentration of50nM according to the manufacturer’s protocol. a master mix of Lipofectamine2000was diluted with1ml of OPTI-MEM (Invitrogen) and incubated for5min. Lipofectamine2000dilution was added to the DNA/siRNA dilution, incubated for20min and added drop-wise to the cells. Five hours after transfection, the media was changed and the cells were allowed to recover overnight The gene and protein of SREBPlwere tested to evaluate the transfect efficiency. The total content of nitrite and nitrate were measured to reflect NO level. ADMA concentrations in medium were measured by high-performance liquid chromatography (HPLC) using precolumn derivatization with ophthaldialdehydeas. The cell DDAH activity was measured by determining L-citrulline formation. Western immunoblotting and Realtime PCR were carried out to determine the protein and mRNA expressed in endothelial cells.Results:1. Endothelial cells treated with100nmol/L insulin for24hours showed the least glucose consumption, which indicate an insulin resistant cell model.2. The medium ADMA concentrations were significantly increased in insulin resistant group (IR) compared with control group (CON), whereas both NO concentration DDAH activity were reduced in insulin resistant groups. Treatment with atorvastatin significantly decreased plasma ADMA level and enhanced DDAH activity and NO production in HUVECs.3. SREBP1and DDAH1mRNA expression were decreased in insulin resistant HUVECs. In consistent with gene expression, SREBP1and DDAH1protein expression were also reduced significantly. With atorvastatin treatment, both mRNA and protein expression of SREBP1and DDAH1were increased in HUVECs.4. SREBP-1siRNA successfully knocked down SREBP-1mRNA and protein levels in HUVECs in insulin-resistant condition. Whereas, the scramble siRNA had no effect on the expression of SREBP-1. Treatment with siRNA targeting SREBP-1caused a73%decrease in SREBP-1mRNA and a79%decrease in protein in comparison with scramble siRNA transduced cells in insulin-resistant condition. And SREBP-1knockdown is associated with a75%decrease in DDAH1mRNA and a69%decrease in protein expression in high glucose cultured HUVECs. Moreover, SREBP1knockdown decreased the expression of DDAH1in HUVECs treated with atorvastatin significantly.5. SREBP-1knockdown resulted a64%decrease in DDAH activity compared with scramble siRNA transduced cells, accompanying a2.9-fold increase in ADMA concentration. atorvastatin treatment induced an additional increase in DDAH activity in SREBP-1knockdown cells, but there is no statistical significance. Levels of NO indicated the activity of NOS isoforms. SREBP-1knockdown was associated with a62%decrease in NO levels. Moreover, SREBP1knockdown led to a significant decrease of DDAH activity and NO level in HUVECs treated with atorvastatin, accompanying an increase of ADMA level.Conclusion:1. Atorvastatin benefits endothelial function by modulating DDAH1/ADMA/NO axis in insulin resistant HUVECs.2. SREBP1acts as a mediator in the regulation of DDAH1/ADMA system by atorvastatin. Further efforts are required to investigate the concrete mechanism of SREBP1in modulating DDAH1expression and to validate the effects of SREBPs on DDAH1.更多还原