抵抗素对血管内皮功能的作用及不同干预方法保护内皮功能的实验研究
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摘要
研究背景及目的
动脉粥样硬化(Atherosclerosis,AS)是严重威胁人类健康和生命的心血管疾病。目前被普遍接受的Ross“损伤反应”学说认为,血管内皮功能障碍(endothelial dysfuction,ED)是动脉粥样硬化形成的早期始动环节,并且贯穿于动脉粥样硬化发生发展的全过程。研究证实,内皮功能障碍的程度还是预测急性心血管事件的重要标志。因此早期检测内皮功能障碍的危险因子及其引发内皮功能障碍的可能机制,对于预防动脉粥样硬化的发生发展有重要意义。
血管内皮细胞具有十分活跃的代谢与内分泌功能,能感受生理刺激并作出调节性反应,以维持血管内环境的平衡。在各种内外因素的作用下,内皮细胞合成和释放多种血管活性物质,细胞因子之间的平衡被打乱,导致其调节血管运动功能、抗血小板和白细胞黏附功能以及抗凝血功能等失调,造成内皮功能障碍。既往研究较多且证实关系密切的内皮功能障碍危险因子有血脂异常、高血压、糖尿病、肥胖、炎症与感染、高同型半胱氨酸血症、吸烟等。新近报道抵抗素(resistin)亦与早期血管内皮细胞功能失调相关。抵抗素系近年发现的一种由脂肪细胞特异分泌的蛋白质,因其具有抵抗胰岛素的作用,故被命名为抵抗素。目前关于抵抗素与血管内皮功能的研究主要集中在体外细胞培养和离体器官培养等方面。研究认为抵抗素在动脉粥样硬化发生前对内皮细胞就有激活作用,经抵抗素孵育的猪冠状动脉环发生了内皮依赖性血管舒张功能障碍,并伴有氧化应激增强和内皮型一氧化氮合酶表达下降。用抵抗素孵育人隐静脉内皮细胞的研究证实,抵抗素能够提高内皮素—1(endothelin—1,ET—1)编码基因启动子的活性,使ET—1释放增多,使血管细胞粘附分子—1(vascularcellular adhesion molecule—1,VCAM—1)与单核细胞趋化蛋白—1(monocyte chemotactic protein—1,MCP—1)的表达分泌增多,促进白细胞的趋化。
但是抵抗素在体内是否依然对血管内皮功能有影响?抵抗素对血管内皮功能作用的可能途经、机制是什么?调节抵抗素表达后血管内皮分泌功能有何变化?这些均是抵抗素研究中有待解决的关键问题,但迄今罕见研究报道。本实验分别观察了外源性、内源性高抵抗素对在体环境中大鼠血管内皮功能的作用,检测血管内皮细胞活化通路中磷脂酰激醇3激酶/蛋白激酶B(PI3K/AKT)信号蛋白表达的改变,比较研究不同干预方法对抵抗素、内皮功能、PI3K/AKT信号通路的影响,探讨抵抗素作用于内皮细胞的可能机制,并试图寻求能够调节抵抗素、保护内皮功能的安全有效之干预方法。
研究方法:
1、外源性高抵抗素血症模型建立,观察外源性高抵抗素对血管内皮功能的影响。4周龄SD大鼠16只,随机分为抵抗素(RE)组与生理盐水对照(NS)组,各8只。抵抗素组大鼠在禁食10小时后给予尾静脉注入重组大鼠抵抗素(Phoenix Pharmaceuticals Ltd,美国)10μg,2小时后第二次给予10μg抵抗素,作为短期高抵抗素血症模型。生理盐水对照组在相同时间给予等容量生理盐水尾静脉注射。于第二次注药2小时后腹腔麻醉,留取血液及组织标本。比较两组抵抗素、胰岛素、血管内皮分泌因子、血脂等血液指标以及主动脉内皮eNOS、ET、PI3K、AKT表达差异。
2、内源性高抵抗素血症模型建立,观察内源性高抵抗素对血管内皮功能的影响。4周龄SD大鼠130只,随机分为高脂组122只,正常对照组(NC组)8只。高脂组大鼠饲高脂饲料,正常对照组饲基础饲料。高脂组给予高脂饲料饲养8周后,高脂组与正常对照组大鼠空腹颈静脉采血,测血清抵抗素水平。①高脂组中血清抵抗素大于正常对照组(?)+2s的大鼠共52只,从中随机选取大鼠40只并分为饮食诱导高抵抗素血症组(HRE组,n=8)及其他四种干预组(32只,详见研究方法3)。②高脂组中血清抵抗素低于正常对照组(?)+s的大鼠共24只,从中随机选取8只作为高脂饮食正常抵抗素组(NRE组,n=8)。
将筛选出的高抵抗素血症组(HRE组)、正常抵抗素组(NRE组)与正常对照组(NC组)各自按原喂养方法继续喂养8周。8周后禁食麻醉,留取血液及组织标本。比较三组间抵抗素、胰岛素、血管内皮分泌因子、血脂等血液指标以及主动脉内皮eNOS、ET、PI3K、AKT表达差异。
3、不同干预方法的效果比较。高脂组中(见研究方法2)血清抵抗素大于正常对照组(NC)(?)+2s的大鼠共52只,从中随机选取大鼠40只并分为饮食诱导高抵抗素血症组(HRE组,n=8)及四种干预组,分别为单纯调整饮食组(DC组,n=8)、运动+调整饮食组(SW组,n=8)、罗格列酮+调整饮食组(RO组,n=8)、抗体+调整饮食组(AB组,n=8)。正常对照组采用研究方法2中正常对照组(NC组,n=8)。
DC组将高脂饲料改为基础饲料,不采取其他措施;SW组采用无负重游泳方式,每周游泳5次,每次1小时,水温29±2℃,喂基础饲料;RO组给马来酸罗格列酮(Rosiglitazone)(葛兰素史克公司生产,文迪雅)2mg/(Kg·d),灌胃,喂基础饲料。AB组每日给予抵抗素抗体(Biovendor Laboratory Medicine,Inc.德国)500ug尾静脉注射,共3天,饮食改为基础饲料;高抵抗素血症组不采取干预措施,继续高脂饮食至试验结束。干预持续8周后,禁食麻醉,留取血液及组织标本待测。比较上述各组抵抗素、胰岛素、血管内皮分泌因子、血脂等血液指标以及主动脉内皮eNOS、ET、PI3K、AKT表达差异。
4、血液指标测定:
采用ELISA法测量外周血抵抗素、血浆纤溶酶原激活物抑制剂1(PAI—1)、von Willebrand因子(vWF);放免法测内皮素(ET)、胰岛素(INS);硝酸还原酶法测一氧化氮(NO);利用全自动生化分析仪测定血脂:胆固醇(CH)、甘油三酯(TG)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL),并计算动脉粥样硬化指数(AI):AI=(TC—HDL)/HDL。
5、逆转录—聚合酶链式反应(RT—PCR)方法测定胸主动脉血管内皮eNOS、ETmRNA表达;分别以eNOS、ET扩增条带吸光度比值(eNOS/actin、ET/actin)作为其表达强度。
6、免疫组化法检测胸主动脉血管内皮磷脂酰激醇3激酶(PI3K)p85a亚基、丝氨酸/苏氨酸激酶蛋白激酶B(Akt)—1蛋白表达。
7、原位杂交法检测胸主动脉血管内皮PI3Kp85a、Akt—1mRNA表达。
8、采用计算机显微图像分析系统计算原位杂交及免疫组化图像中血管内皮层阳性表达细胞积分光密度(IOD),作为PI3K与Akt的表达强度。
9、统计学处理:所有数据均以(?)±s表示,采用SPSS14.0软件分析;两组之间比较用t检验,多组数据比较用方差分析。试验指标均经正态性检验,符合正态分布的指标之间进行person相关分析与partial相关分析(控制混杂因素后进行相关分析)。P＜0.05认为有统计学意义。
研究结果:
1、外源性抵抗素对血管内皮功能的作用:
1.1注入外源性重组抵抗素后,抵抗素组(RE)大鼠血清抵抗素与胰岛素均明显高于生理盐水对照组(NS),胰岛素敏感指数在RE组降低。RE组大鼠血管内皮舒缩因子ET明显升高,NO降低;促凝因子PAI与vWF明显升高。两组体格测量指标与血脂无统计学差异。
1.2 RE组与NS组脂肪组织抵抗素表达无差异。RE组血管内皮eNOSmRNA表达降低,而ETmRNA表达升高。
1.3原位杂交与免疫组化均显示,RE组内皮细胞阳性着色颗粒少,着色浅,分布稀疏;RE组PI3K、Akt免疫组化及原位杂交图像分析积分光密度值均较NS组低,差异有统计学意义。
2、内源性抵抗素对血管内皮功能的作用:
2.1高脂喂养高抵抗素血症组(HRE组)与正常抵抗素组(NRE组)体重、Lee指数、TC、TG、LDL均显著高于正常对照组,HDL较正常对照绢(NC组)降低。
2.2 HRE组血清抵抗素显著高于NRE组和正常对照组。HRE组、NRE组比NC组胰岛素升高、胰岛素敏感指数下降,均有统计学差异。HRE组、NRE组均较NC组出现明显内皮功能紊乱,并且HRE组比NRE组改变更明显,表现为血管内皮因子NO显著降低,ET升高;促凝因子PAI与vWF明显升高。
2.3 HRE组脂肪组织抵抗素表达明显高于NC组与NRE组,而后两者抵抗素表达无统计学差异。在血管内皮eNOSmRNA表达方面,HRE组低于NRE组,NRE组低于正常对照组,差异均有统计学意义。内皮ETmRNA表达则在HRE组最高,其次为NRE组,NC组最低,三组间差异均有显著意义。
2.4免疫组化与原位杂交结果均显示,NC组内皮细胞染色呈强阳性,细胞内有密集、强染、着棕黄色颗粒。NRE组内皮层着色明显,部分内皮细胞内有棕黄色阳性颗粒。HRE组内皮层着色较浅,只有少数内皮细胞内有阳性表达颗粒。PI3K、Akt图像分析积分光密度值HRE组最低,NRE组居中,NC组最高,差异有统计学意义。
3、不同方法干预效果的比较:
3.1单纯调整饮食组(DC)与运动+调整饮食组(SW)干预8周后,大鼠体重、Lee指数显著低于高抵抗素血症组(HRE),罗格列酮+调整饮食组(RO)、抵抗素抗体+调整饮食组(AB)则与HRE组无明显差异。SW组血脂改善最显著,DC组、RO组次之,AB组血脂各项指标与HRE组无显著差异。
3.2四种干预方法均使血清抵抗素、胰岛素显著低于高抵抗素血症对照组,胰岛素敏感指数显著升高。其中,RO组与SW组抵抗素、胰岛素下降明显,而DC组和AB组抵抗素、胰岛素虽已有统计学改变,但与正常对照组仍然有统计学差异。
3.3 RO组、SW组与AB组均使血清ET、PAI、vWF均较HRE组明显降低,NO显著升高;调整饮食组只有ET、PAI较HRE组显著降低,而vWF与NO的改变没有统计学意义。
3.4 SW组与RO组脂肪组织抵抗素表达下降最明显,DC组抵抗素表达有下降,但仍高于对照组。AB组抵抗素表达与HRE组无统计学差异。四个干预组均较HRE组内皮eNOSmRNA表达增加,ETmRNA表达降低,差异有统计学意义,其中RO组已经达到正常对照组水平。
3.5原位杂交与免疫组化显示,四种干预措施均使血管内皮PI3Kp85a、AktmRNA及蛋白表达升高,内皮层棕黄色阳性颗粒增多,染色加深,图像分析积分光密度值明显升高,有统计学意义。其中RO组与SW组改变程度较大。
4、相关分析:血清抵抗素、抵抗素mRNA与血清NO、内皮eNOSmRNA表达、内皮PI3Kp85a、AktmRNA及蛋白表达呈显著负相关,与血清ET、PAI、vWF、内皮ETmRNA表达显著正相关。控制体重、Lee指数、胰岛素、胰岛素敏感指数、血脂等混杂因素后,血清抵抗素、抵抗素mRNA仍然与内皮分泌因子、PI3K、AKT表达显著相关。
研究结论
1、外源性注射大鼠重组抵抗素后,试验大鼠出现明显血管内皮分泌功能紊乱,伴有血管内皮细胞活化通路信号PI3K、AktmRNA与蛋白表达的下降。
2、高脂饮食喂养大鼠可以诱导内源性高抵抗素血症模型及正常抵抗素大鼠。高抵抗素血症大鼠血清抵抗素水平与脂肪组织抵抗素表达均显著高于正常抵抗素大鼠。二者都有肥胖、血脂紊乱、动脉粥样硬化指数升高及不同程度的胰岛素抵抗现象。
3、高脂饮食诱导高抵抗素大鼠血管内皮分泌功能紊乱的程度显著高于高脂饮食正常抵抗素大鼠。高脂饮食诱导高抵抗素大鼠与高脂饮食正常抵抗素大鼠比较,血管内皮细胞PI3K、Akt的mRNA与蛋白表达均显著降低。
4、肥胖个体中抵抗素升高与血管内皮功能障碍同步发生,提示血清抵抗素检测可以作为肥胖个体血管内皮功能障碍的早期预测指标。
5、控制体重、Lee指数、胰岛素、血糖、血脂等因素的影响后,血清抵抗素、抵抗素mRNA与内皮分泌因子显著相关,与PI3K、Akt的表达显著负相关。提示抵抗素通过PI3K/Akt信号通路引起了血管内皮分泌功能紊乱。
6、调整饮食、运动、罗格列酮、抗体四种方法均可以使血管内皮分泌功能有不同程度改善,伴有血管内皮PI3K、Akt的mRNA与蛋白表达升高。其中运动+调整饮食或罗格列酮+调整饮食的干预效果最好。
创新和意义:
1、在体证实了抵抗素有引发血管内皮分泌功能障碍的作用。首次比较了高脂饮食状态下,不同抵抗素水平个体血管内皮分泌功能的差异;证实了在排除混杂因素后抵抗素是诱导血管内皮分泌功能障碍的独立危险因素。提出早期检测并干预抵抗素是预防血管内皮功能障碍的重要环节。
2、首次在体研究了抵抗素对内皮细胞活化通路PI3K/AKT蛋白的作用,比较了不同抵抗素水平个体PI3K/AKT表达的差异。排除混杂因素后,相关分析证实抵抗素、血管内皮分泌功能、PI3K/AKT三者有显著相关性;提示PI3K/AKT信号通路可能参与了抵抗素引起血管内皮分泌功能障碍的过程。
3、首次比较了不同干预措施对抵抗素表达及血管内皮功能的影响。提出运动与调整饮食结构是适用于高脂肥胖个体早期降低抵抗素表达、改善血管内皮功能的安全有效措施。
Background and objectives:
Atherosclerosis(AS),one of the cardiovascular diseases, is the most significant cause of mortality.Investigations in recent years have demonstrated that endothelial dysfunction is the initiating and early event in the development of atherosclerosis.Moreover,endothelial dysfunction exists in all periods of the development of AS and is a important marker for acute cardiovascular diseases.Therefore,it is crucial to detect the risk factor and mechanism of endothelial dysfunction earlier for the prevention of AS.
Vascular endothelial cells play a central role in maintaining cardiovascular homeostasis in health.In addition to providing a physical barrier between the vessel wall and lumen, the endothelium secretes a number of mediators that regulate platelet aggregation,coagulation,fibrinolysis and vessel tone. The term endothelial dysfunction refers to an imbalance in the production of these mediators,which may promote vasospasm, thrombosis or vessel occlusion in experimental models and has been implicated in the pathogenesis of acute coronary syndromes and other cardiovascular disorders.The high risk factors for endothelial dysfunction include hyperlipidemia,hypertension, diabetes mellitus,inflammation,infection and smoking. Resistin was recently suggested to be involved in the development of endothelial dysfunction.Resistin is a recently described novel adipokine that has been suggested to play a role in the development of insulin resistance.Recent studies on the relationship between resistin and endothelial dysfunction have been predominantly in vitro.Some studies indicated resistin exerted direct effects to promote endothelial cell activation before the onset of overt AS.Incubation of porcine coronary rings with resistin impaired endothelium-dependent vasorelaxation,increased oxidative stress,and decreased eNOS expression.Endothelial cells(ECs)were activated by promoting ET-1 release,in part by inducing ET-1 promoter activity after incubated with human recombinant resistin.Furthermore, resistin upregulates adhesion molecules and monocyte chemotactic protein-1.
However,the researches on the relationship between resistin and endothelial dysfunction were inadequate.Invivo, there are few investations and reports on whether resistin have regulating effects on endothelial function.And little is known about the mechanisms of how resistin signals in the endothelium. These problems are to be solved in the research of resistin. Whether the endothelium secretion factors changed with the variation of resistin? In this study,we established hyper-resistin model in rats by high-fat diet and resistin injection to observe the effect of resistin on the endothelial function invivo.At the same time,the present study was designed to investigate the protein and gene expression of phosphatidylinositol 3 kinase in the hyper-resistinmodel.The effects of rosiglitazone,swimming,diet control and resistin antibody on the resistin,endothelial function and PI3K were contrasted.
Methods
1.The establishment of extrinsic hyper-resistin model
This study was conducted on 16 male 4 week old Sprague Dawley rats.The rats were divided into two groups randomly:resistin group(n=8 RS),normal saline group(n=8 NS).The rats in the resistin group received an infusion of rat recombinant resistin (Phoenix Pharmaceuticals Ltd,American)(10μg/rat)through the tail vein after 10 hours of fasting.A second infusion of rat recombinant resistin was given to the resistin group two hours after the first infusion.Sham operations were conducted on the NS group at the same time.Two hours after the second dose of resistin,the resistin group and the NS group were anesthetized and blood samples and thoracic arteries were collected for different detections.
2.The establishment of intrinsic hyper-resistin model
This study was conducted on 130 male 4 week old Sprague Dawley rats.The rats were divided into two groups randomly: high-fat diet group(n=122)and normal control group(NC n=8). All rats received high-fat chow except the rats in the normal control group,which received standard chow.After the high-fat diet group was fed for 8 weeks,blood samples of the two groups were collected from the right internal jugular vein for the measure of resistin.The rats with resistin levels 2 S.D.greater than the mean of resistin in the control group were defined as having diet-induced hyper-resistinemia model.There were 52 rats in the diet-induced hyper-resistinemia model.The 52 rats were divided randomly into hyper-resistinemia model group(HRE n=8) and other four groups(see method 3).The rats with resistin levels lower than X+1S.D.of resistin in the control group were defined as having diet-induced normal resistin model(24 rats). Eight rats were selected randomly from the diet-induced normal resistin model and defined as normal resistin goup(NRE n=8). Then the rats in the diet-induced hyper-resistinemia group(HRE), diet-induced normal resistin group(NRE)and normal control group(NC)were fed as before for 8 weeks.After 8 weeks,all rats in the three groups were anesthetized and blood samples and thoracic arteries were collected for different detections.
3.The effects in different treatment methods
Forty rats were selected randomly from diet-induced hyper-resistinemia model(see method 2)and randomized into diet-control group(DC n=8),swimming with diet-control group (SW n=8),rosiglitazone with diet-control group(RO n=8) antibody with diet-control group(AB n=8)and hyper-resistinemia group(HRE n=8).The rats in DC group were given standard chow without other treatments.The rats in SW group swam one hour per day,five days per week.The rats in RO group were received rosiglitazone 2mg/(Kg·d)The antibody group received two injections of resistin antibody(Biovendor Laboratory Medicine, Inc.Germany)through the tail vein for 3 days.The rats in SW, RO and AB group were given standard chow and the rats in HRE group were given high-fat diet as before.The treatments in RO,SW and DC group were lasted for 8 weeks.After the treatments,all rats in NC,DC,SW,RO,AB and HRE groups were anesthetized and blood samples and thoracic arteries were collected for different detections.
4.Measurement of variables in serum
Serum levels of resistin,PAI-1,and vWF were measured by using ELISA kits.Insulin(INS)and endothelin(ET)were measured by radioimmunoassay.Serum nitric oxide(NO)production was detected by measuring the final stable equimolar degradation products nitrite and nitrate by using a standard colormetric assay.Total cholesterol(TC)and triglycerides(TG)were determined by enzymatic method and low-density lipoprotein(LDL) and high-density lipoprotein(HDL)cholesterol were determined by immuno—turbidimetry assay respectively.Atherosclerotic index(AI)was calculated from TC and HDL:AI=(TC—HDL)/HDL.
5.Determination of the mRNA expression of eNOS and ET by RT-PCR method:The relative expressing level was defined by optical density ratio of eNOS and ET to actin respectively(eNOS /actin、ET/actin).
6.Immunohistochemistry was employed to examine the expression of phosphatidylinositol 3kinase(PI3K)p85αsubunit and protein kinase B(Akt)in thoracic artery endothelium.
7.In situ hybridization was employed to examine the mRNA expression of PI3K p85αsubunit and Akt—1 in thoracic artery endothelium.
8.Quantification was performed through a microscope with a computer-assisted image analysis system(image-pro plus 5.0).
9.The data were indicated as mean±standard deviation((?)±s)and were analyzed by Student t test and one-way ANOVA using SPSS14.0 statistical software.The correlation between variables,which were considered to be normally distributed by normality plots,was analyzed using Person' s correlation and partial correlation.Value of P＜0.05 was considered significant.
Results
1.The effect of extrinsic resistin on endothelial function
1.1 After two injections of rat recombinant resistin,the levels of insulin and resistin in the resistin group were increased significantly,while the insulin sensitive indexs were lower in the resistin group than those in the NS group.In the resistin group,the levels of ET,PAI,and vWF were higher while the level of NO was lower than those in the NS group.There was no difference in body weight and lipids of the two groups.
1.2 There was no difference in resistin mRNA expression of RE and NS group.The eNOS mRNA expression in RE group was lower and ET mRNA higher than those in NS group.
1.3 The results in immunohistochemistry and In situ hybridization indicate that the expression levels of PI3K and Akt in the resistin group were much lower than that in the NS group,which had strong positive brown granules in the cytoplasm.
2.The effect of intrinsic resistin on endothelium function
2.1 The body weight,Lee index,TC、TG、LDL were much higher in high resistin group(HRE)and normal resistin group(NRE)than those in normal control group(NC),while the level of HDL in HRE and NRE group decreased.
2.2 The level of resistin in HRE group was the highest among HRE,NRE and NS group.The level of insulin increased and the level of ISI decreased in HRE and NRE group compared with normal control group.There was markedly endothelial dysfunction in HRE and NRE group.Moreover,the rats in HRE group had worse endothelial dysfunction than NRE group.The level of NO decreased significantly and the levels of ET,PAI and vWF increased in HRE group.
2.3 The resistin expression in HRE group was the highest among HRE,NRE and NC group.There was no difference between NRE and NC group in resistin expression.The eNOSmRNA expression in NRE group was higher than that in HRE group and lower than that in NC group.The ETmRNA expression in HRE group was the highest among HRE,NRE and NC group and the ETmRNA expression in NRE group was the second.
2.4 The results in immunohistochemistry and In situ hybridization indicated that the expression levels of PI3K and Akt in the HRE group were much lower than that in the NRE group and NC group,which had strong positive brown granules in the cytoplasm.The IOD of PI3K and Akt in the HRE group were the lowest among HRE,NRE and NC group and those in NC group were the highest.
3.The effects in different treatment methods
3.1 After the treatments in RO and SW group lasted for 8 weeks, the body weight,Lee index were much lower in DC and SW group than those in HRE group.There was no difference in body weight, Lee index among RO,AB and HRE group.The lipids in SW group improved most and those in DC and RO group the second.There was no difference in lipids between HRE and NC group.
3.2 The four treatments all decreased the level of resistin and insulin and increased ISI markedly.The levels of resistin and insulin in RO and SW decreased most among the four treatment groups and those in DC and AB group decreased secondly.
3.3 The levels of ET、PAI、vWF in RO,SW and AB group were much lower and NO higher than those in HRE group.The levels of ET and PAI in De group decreased compared with the HRE group. There no difference in vWF and NO between DC and HRE group.
3.4 The resistin mRNA expression in SW and RO group were decreased mostly.There was no difference in resistin mRNA expression between AB and HRE group.The four treatments all increased markedly the expression of eNOSmRNA and decreased the expression of ETmRNA.There was no difference between HRE and NC group.
3.5 The results in immunohistochemistry and In situ hybridization indicated that the four treatments all increased markedly the expression of PI3Kp85αand Akt with the IOD increased significantly.RO and SW group improved the PI3Kp85αand Akt expression most among the four groups.
4.Correlation
Resistin levels and the expression of resistin mRNA were obviously correlated negatively with NO and eNOSmRNA expression and positively with ET、PAI、vWF and ETmRNA expression.Resistin levels and the expression of resistin mRNA were correlated negatively with the expression PI3Kp85αand Akt.Even after correction for weight,Lee index,insulin,ISI and lipids, resistin and the expression of resistin mRNA still correlated significantly with endothelial secretion markers,PI3K and Akt.
Conclusions
1.There were obvious endothelium dysfunction with PI3K、Akt mRNA and protein expression decreased after injection of resistin.
2.High fat diet induced high resistin rats and normal resistin rats at the same time.The serum resistin and resistin mRNA expression in adipose tissue were higher markedly in high-resistinemia rats than those in normal resistin rats.There were obvious obesity,insulin resistance and abnormal lipid in both high resistin rats and normal resistin rats.
3.The endothelial dysfunction was worse in high fat diet induced hyper-resistinemia rats than that in high fat diet induced normal resistin rats.The expression of PI3K and Akt in hyper-resistinemia rats were decreased significantly compared with the normal resistin rats.
4.Resistin increased with the occurrence of endothelial dysfunction in obesity.The results indicate that resistin was an early marker for endothelium dysfunction.
5.After correction for weight,Lee index,insulin,ISI and lipids,the level of resistin and resistin mRNA expression were correlated with endothelial secretion markers and the expression of PI3K and Akt.The result indicated that PI3K/Akt signaling pathway might contribute to the development of endothelial secretion dysfunction by resistin.
6.Rosiglitazone,swimming,diet-control and resistin antibody can improve endothelial dysfunction separately with increased PI3K、Akt expression.Swimming with diet-control and rosiglitazone with diet-control were most effective treatment methods.
Innovations and meanings:
1.This study demonstrated in vivo the effect of resistin on endothelium function.The endothelium functions were different in the high fat diet rats with different resistin level. The result certified that resistin was the independent risk factor of endothelium dysfunction after correction for interferences.It was crucial to detect and treat resistin for the prevention of endothelium dysfunction.
2.This study demonstrated in vivo the effect of resistin on PI3K/ Akt signaling pathway for the first time.There were different expressions of PI3K and Akt in individuals with different resistin.The result indicated the relationship among resistin,endothelium function and PI3K/Akt signaling pathway. The result indicated for the first time that PI3K/Akt signaling pathway might contribute to the development of endothelial secretion dysfunction by resistin.
3.This study first contrasted and demonstrated the effects in different treatment methods on endothelium function.Swimming and diet control were two effective methods for child to decrease the expression of resistin and improve endothelium function.
动脉粥样硬化(Atherosclerosis,AS)是严重威胁人类健康和生命的心血管疾病。目前被普遍接受的Ross“损伤反应”学说认为,血管内皮功能障碍(endothelial dysfuction,ED)是动脉粥样硬化形成的早期始动环节,并且贯穿于动脉粥样硬化发生发展的全过程。研究证实,内皮功能障碍的程度还是预测急性心血管事件的重要标志。因此早期检测内皮功能障碍的危险因子及其引发内皮功能障碍的可能机制,对于预防动脉粥样硬化的发生发展有重要意义。
血管内皮细胞具有十分活跃的代谢与内分泌功能,能感受生理刺激并作出调节性反应,以维持血管内环境的平衡。在各种内外因素的作用下,内皮细胞合成和释放多种血管活性物质,细胞因子之间的平衡被打乱,导致其调节血管运动功能、抗血小板和白细胞黏附功能以及抗凝血功能等失调,造成内皮功能障碍。既往研究较多且证实关系密切的内皮功能障碍危险因子有血脂异常、高血压、糖尿病、肥胖、炎症与感染、高同型半胱氨酸血症、吸烟等。新近报道抵抗素(resistin)亦与早期血管内皮细胞功能失调相关。抵抗素系近年发现的一种由脂肪细胞特异分泌的蛋白质,因其具有抵抗胰岛素的作用,故被命名为抵抗素。目前关于抵抗素与血管内皮功能的研究主要集中在体外细胞培养和离体器官培养等方面。研究认为抵抗素在动脉粥样硬化发生前对内皮细胞就有激活作用,经抵抗素孵育的猪冠状动脉环发生了内皮依赖性血管舒张功能障碍,并伴有氧化应激增强和内皮型一氧化氮合酶表达下降。用抵抗素孵育人隐静脉内皮细胞的研究证实,抵抗素能够提高内皮素—1(endothelin—1,ET—1)编码基因启动子的活性,使ET—1释放增多,使血管细胞粘附分子—1(vascularcellular adhesion molecule—1,VCAM—1)与单核细胞趋化蛋白—1(monocyte chemotactic protein—1,MCP—1)的表达分泌增多,促进白细胞的趋化。
但是抵抗素在体内是否依然对血管内皮功能有影响?抵抗素对血管内皮功能作用的可能途经、机制是什么?调节抵抗素表达后血管内皮分泌功能有何变化?这些均是抵抗素研究中有待解决的关键问题,但迄今罕见研究报道。本实验分别观察了外源性、内源性高抵抗素对在体环境中大鼠血管内皮功能的作用,检测血管内皮细胞活化通路中磷脂酰激醇3激酶/蛋白激酶B(PI3K/AKT)信号蛋白表达的改变,比较研究不同干预方法对抵抗素、内皮功能、PI3K/AKT信号通路的影响,探讨抵抗素作用于内皮细胞的可能机制,并试图寻求能够调节抵抗素、保护内皮功能的安全有效之干预方法。
研究方法:
1、外源性高抵抗素血症模型建立,观察外源性高抵抗素对血管内皮功能的影响。4周龄SD大鼠16只,随机分为抵抗素(RE)组与生理盐水对照(NS)组,各8只。抵抗素组大鼠在禁食10小时后给予尾静脉注入重组大鼠抵抗素(Phoenix Pharmaceuticals Ltd,美国)10μg,2小时后第二次给予10μg抵抗素,作为短期高抵抗素血症模型。生理盐水对照组在相同时间给予等容量生理盐水尾静脉注射。于第二次注药2小时后腹腔麻醉,留取血液及组织标本。比较两组抵抗素、胰岛素、血管内皮分泌因子、血脂等血液指标以及主动脉内皮eNOS、ET、PI3K、AKT表达差异。
2、内源性高抵抗素血症模型建立,观察内源性高抵抗素对血管内皮功能的影响。4周龄SD大鼠130只,随机分为高脂组122只,正常对照组(NC组)8只。高脂组大鼠饲高脂饲料,正常对照组饲基础饲料。高脂组给予高脂饲料饲养8周后,高脂组与正常对照组大鼠空腹颈静脉采血,测血清抵抗素水平。①高脂组中血清抵抗素大于正常对照组(?)+2s的大鼠共52只,从中随机选取大鼠40只并分为饮食诱导高抵抗素血症组(HRE组,n=8)及其他四种干预组(32只,详见研究方法3)。②高脂组中血清抵抗素低于正常对照组(?)+s的大鼠共24只,从中随机选取8只作为高脂饮食正常抵抗素组(NRE组,n=8)。
将筛选出的高抵抗素血症组(HRE组)、正常抵抗素组(NRE组)与正常对照组(NC组)各自按原喂养方法继续喂养8周。8周后禁食麻醉,留取血液及组织标本。比较三组间抵抗素、胰岛素、血管内皮分泌因子、血脂等血液指标以及主动脉内皮eNOS、ET、PI3K、AKT表达差异。
3、不同干预方法的效果比较。高脂组中(见研究方法2)血清抵抗素大于正常对照组(NC)(?)+2s的大鼠共52只,从中随机选取大鼠40只并分为饮食诱导高抵抗素血症组(HRE组,n=8)及四种干预组,分别为单纯调整饮食组(DC组,n=8)、运动+调整饮食组(SW组,n=8)、罗格列酮+调整饮食组(RO组,n=8)、抗体+调整饮食组(AB组,n=8)。正常对照组采用研究方法2中正常对照组(NC组,n=8)。
DC组将高脂饲料改为基础饲料,不采取其他措施;SW组采用无负重游泳方式,每周游泳5次,每次1小时,水温29±2℃,喂基础饲料;RO组给马来酸罗格列酮(Rosiglitazone)(葛兰素史克公司生产,文迪雅)2mg/(Kg·d),灌胃,喂基础饲料。AB组每日给予抵抗素抗体(Biovendor Laboratory Medicine,Inc.德国)500ug尾静脉注射,共3天,饮食改为基础饲料;高抵抗素血症组不采取干预措施,继续高脂饮食至试验结束。干预持续8周后,禁食麻醉,留取血液及组织标本待测。比较上述各组抵抗素、胰岛素、血管内皮分泌因子、血脂等血液指标以及主动脉内皮eNOS、ET、PI3K、AKT表达差异。
4、血液指标测定:
采用ELISA法测量外周血抵抗素、血浆纤溶酶原激活物抑制剂1(PAI—1)、von Willebrand因子(vWF);放免法测内皮素(ET)、胰岛素(INS);硝酸还原酶法测一氧化氮(NO);利用全自动生化分析仪测定血脂:胆固醇(CH)、甘油三酯(TG)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL),并计算动脉粥样硬化指数(AI):AI=(TC—HDL)/HDL。
5、逆转录—聚合酶链式反应(RT—PCR)方法测定胸主动脉血管内皮eNOS、ETmRNA表达;分别以eNOS、ET扩增条带吸光度比值(eNOS/actin、ET/actin)作为其表达强度。
6、免疫组化法检测胸主动脉血管内皮磷脂酰激醇3激酶(PI3K)p85a亚基、丝氨酸/苏氨酸激酶蛋白激酶B(Akt)—1蛋白表达。
7、原位杂交法检测胸主动脉血管内皮PI3Kp85a、Akt—1mRNA表达。
8、采用计算机显微图像分析系统计算原位杂交及免疫组化图像中血管内皮层阳性表达细胞积分光密度(IOD),作为PI3K与Akt的表达强度。
9、统计学处理:所有数据均以(?)±s表示,采用SPSS14.0软件分析;两组之间比较用t检验,多组数据比较用方差分析。试验指标均经正态性检验,符合正态分布的指标之间进行person相关分析与partial相关分析(控制混杂因素后进行相关分析)。P＜0.05认为有统计学意义。
研究结果:
1、外源性抵抗素对血管内皮功能的作用:
1.1注入外源性重组抵抗素后,抵抗素组(RE)大鼠血清抵抗素与胰岛素均明显高于生理盐水对照组(NS),胰岛素敏感指数在RE组降低。RE组大鼠血管内皮舒缩因子ET明显升高,NO降低;促凝因子PAI与vWF明显升高。两组体格测量指标与血脂无统计学差异。
1.2 RE组与NS组脂肪组织抵抗素表达无差异。RE组血管内皮eNOSmRNA表达降低,而ETmRNA表达升高。
1.3原位杂交与免疫组化均显示,RE组内皮细胞阳性着色颗粒少,着色浅,分布稀疏;RE组PI3K、Akt免疫组化及原位杂交图像分析积分光密度值均较NS组低,差异有统计学意义。
2、内源性抵抗素对血管内皮功能的作用:
2.1高脂喂养高抵抗素血症组(HRE组)与正常抵抗素组(NRE组)体重、Lee指数、TC、TG、LDL均显著高于正常对照组,HDL较正常对照绢(NC组)降低。
2.2 HRE组血清抵抗素显著高于NRE组和正常对照组。HRE组、NRE组比NC组胰岛素升高、胰岛素敏感指数下降,均有统计学差异。HRE组、NRE组均较NC组出现明显内皮功能紊乱,并且HRE组比NRE组改变更明显,表现为血管内皮因子NO显著降低,ET升高;促凝因子PAI与vWF明显升高。
2.3 HRE组脂肪组织抵抗素表达明显高于NC组与NRE组,而后两者抵抗素表达无统计学差异。在血管内皮eNOSmRNA表达方面,HRE组低于NRE组,NRE组低于正常对照组,差异均有统计学意义。内皮ETmRNA表达则在HRE组最高,其次为NRE组,NC组最低,三组间差异均有显著意义。
2.4免疫组化与原位杂交结果均显示,NC组内皮细胞染色呈强阳性,细胞内有密集、强染、着棕黄色颗粒。NRE组内皮层着色明显,部分内皮细胞内有棕黄色阳性颗粒。HRE组内皮层着色较浅,只有少数内皮细胞内有阳性表达颗粒。PI3K、Akt图像分析积分光密度值HRE组最低,NRE组居中,NC组最高,差异有统计学意义。
3、不同方法干预效果的比较:
3.1单纯调整饮食组(DC)与运动+调整饮食组(SW)干预8周后,大鼠体重、Lee指数显著低于高抵抗素血症组(HRE),罗格列酮+调整饮食组(RO)、抵抗素抗体+调整饮食组(AB)则与HRE组无明显差异。SW组血脂改善最显著,DC组、RO组次之,AB组血脂各项指标与HRE组无显著差异。
3.2四种干预方法均使血清抵抗素、胰岛素显著低于高抵抗素血症对照组,胰岛素敏感指数显著升高。其中,RO组与SW组抵抗素、胰岛素下降明显,而DC组和AB组抵抗素、胰岛素虽已有统计学改变,但与正常对照组仍然有统计学差异。
3.3 RO组、SW组与AB组均使血清ET、PAI、vWF均较HRE组明显降低,NO显著升高;调整饮食组只有ET、PAI较HRE组显著降低,而vWF与NO的改变没有统计学意义。
3.4 SW组与RO组脂肪组织抵抗素表达下降最明显,DC组抵抗素表达有下降,但仍高于对照组。AB组抵抗素表达与HRE组无统计学差异。四个干预组均较HRE组内皮eNOSmRNA表达增加,ETmRNA表达降低,差异有统计学意义,其中RO组已经达到正常对照组水平。
3.5原位杂交与免疫组化显示,四种干预措施均使血管内皮PI3Kp85a、AktmRNA及蛋白表达升高,内皮层棕黄色阳性颗粒增多,染色加深,图像分析积分光密度值明显升高,有统计学意义。其中RO组与SW组改变程度较大。
4、相关分析:血清抵抗素、抵抗素mRNA与血清NO、内皮eNOSmRNA表达、内皮PI3Kp85a、AktmRNA及蛋白表达呈显著负相关,与血清ET、PAI、vWF、内皮ETmRNA表达显著正相关。控制体重、Lee指数、胰岛素、胰岛素敏感指数、血脂等混杂因素后,血清抵抗素、抵抗素mRNA仍然与内皮分泌因子、PI3K、AKT表达显著相关。
研究结论
1、外源性注射大鼠重组抵抗素后,试验大鼠出现明显血管内皮分泌功能紊乱,伴有血管内皮细胞活化通路信号PI3K、AktmRNA与蛋白表达的下降。
2、高脂饮食喂养大鼠可以诱导内源性高抵抗素血症模型及正常抵抗素大鼠。高抵抗素血症大鼠血清抵抗素水平与脂肪组织抵抗素表达均显著高于正常抵抗素大鼠。二者都有肥胖、血脂紊乱、动脉粥样硬化指数升高及不同程度的胰岛素抵抗现象。
3、高脂饮食诱导高抵抗素大鼠血管内皮分泌功能紊乱的程度显著高于高脂饮食正常抵抗素大鼠。高脂饮食诱导高抵抗素大鼠与高脂饮食正常抵抗素大鼠比较,血管内皮细胞PI3K、Akt的mRNA与蛋白表达均显著降低。
4、肥胖个体中抵抗素升高与血管内皮功能障碍同步发生,提示血清抵抗素检测可以作为肥胖个体血管内皮功能障碍的早期预测指标。
5、控制体重、Lee指数、胰岛素、血糖、血脂等因素的影响后,血清抵抗素、抵抗素mRNA与内皮分泌因子显著相关,与PI3K、Akt的表达显著负相关。提示抵抗素通过PI3K/Akt信号通路引起了血管内皮分泌功能紊乱。
6、调整饮食、运动、罗格列酮、抗体四种方法均可以使血管内皮分泌功能有不同程度改善,伴有血管内皮PI3K、Akt的mRNA与蛋白表达升高。其中运动+调整饮食或罗格列酮+调整饮食的干预效果最好。
创新和意义:
1、在体证实了抵抗素有引发血管内皮分泌功能障碍的作用。首次比较了高脂饮食状态下,不同抵抗素水平个体血管内皮分泌功能的差异;证实了在排除混杂因素后抵抗素是诱导血管内皮分泌功能障碍的独立危险因素。提出早期检测并干预抵抗素是预防血管内皮功能障碍的重要环节。
2、首次在体研究了抵抗素对内皮细胞活化通路PI3K/AKT蛋白的作用,比较了不同抵抗素水平个体PI3K/AKT表达的差异。排除混杂因素后,相关分析证实抵抗素、血管内皮分泌功能、PI3K/AKT三者有显著相关性;提示PI3K/AKT信号通路可能参与了抵抗素引起血管内皮分泌功能障碍的过程。
3、首次比较了不同干预措施对抵抗素表达及血管内皮功能的影响。提出运动与调整饮食结构是适用于高脂肥胖个体早期降低抵抗素表达、改善血管内皮功能的安全有效措施。
Background and objectives:
Atherosclerosis(AS),one of the cardiovascular diseases, is the most significant cause of mortality.Investigations in recent years have demonstrated that endothelial dysfunction is the initiating and early event in the development of atherosclerosis.Moreover,endothelial dysfunction exists in all periods of the development of AS and is a important marker for acute cardiovascular diseases.Therefore,it is crucial to detect the risk factor and mechanism of endothelial dysfunction earlier for the prevention of AS.
Vascular endothelial cells play a central role in maintaining cardiovascular homeostasis in health.In addition to providing a physical barrier between the vessel wall and lumen, the endothelium secretes a number of mediators that regulate platelet aggregation,coagulation,fibrinolysis and vessel tone. The term endothelial dysfunction refers to an imbalance in the production of these mediators,which may promote vasospasm, thrombosis or vessel occlusion in experimental models and has been implicated in the pathogenesis of acute coronary syndromes and other cardiovascular disorders.The high risk factors for endothelial dysfunction include hyperlipidemia,hypertension, diabetes mellitus,inflammation,infection and smoking. Resistin was recently suggested to be involved in the development of endothelial dysfunction.Resistin is a recently described novel adipokine that has been suggested to play a role in the development of insulin resistance.Recent studies on the relationship between resistin and endothelial dysfunction have been predominantly in vitro.Some studies indicated resistin exerted direct effects to promote endothelial cell activation before the onset of overt AS.Incubation of porcine coronary rings with resistin impaired endothelium-dependent vasorelaxation,increased oxidative stress,and decreased eNOS expression.Endothelial cells(ECs)were activated by promoting ET-1 release,in part by inducing ET-1 promoter activity after incubated with human recombinant resistin.Furthermore, resistin upregulates adhesion molecules and monocyte chemotactic protein-1.
However,the researches on the relationship between resistin and endothelial dysfunction were inadequate.Invivo, there are few investations and reports on whether resistin have regulating effects on endothelial function.And little is known about the mechanisms of how resistin signals in the endothelium. These problems are to be solved in the research of resistin. Whether the endothelium secretion factors changed with the variation of resistin? In this study,we established hyper-resistin model in rats by high-fat diet and resistin injection to observe the effect of resistin on the endothelial function invivo.At the same time,the present study was designed to investigate the protein and gene expression of phosphatidylinositol 3 kinase in the hyper-resistinmodel.The effects of rosiglitazone,swimming,diet control and resistin antibody on the resistin,endothelial function and PI3K were contrasted.
Methods
1.The establishment of extrinsic hyper-resistin model
This study was conducted on 16 male 4 week old Sprague Dawley rats.The rats were divided into two groups randomly:resistin group(n=8 RS),normal saline group(n=8 NS).The rats in the resistin group received an infusion of rat recombinant resistin (Phoenix Pharmaceuticals Ltd,American)(10μg/rat)through the tail vein after 10 hours of fasting.A second infusion of rat recombinant resistin was given to the resistin group two hours after the first infusion.Sham operations were conducted on the NS group at the same time.Two hours after the second dose of resistin,the resistin group and the NS group were anesthetized and blood samples and thoracic arteries were collected for different detections.
2.The establishment of intrinsic hyper-resistin model
This study was conducted on 130 male 4 week old Sprague Dawley rats.The rats were divided into two groups randomly: high-fat diet group(n=122)and normal control group(NC n=8). All rats received high-fat chow except the rats in the normal control group,which received standard chow.After the high-fat diet group was fed for 8 weeks,blood samples of the two groups were collected from the right internal jugular vein for the measure of resistin.The rats with resistin levels 2 S.D.greater than the mean of resistin in the control group were defined as having diet-induced hyper-resistinemia model.There were 52 rats in the diet-induced hyper-resistinemia model.The 52 rats were divided randomly into hyper-resistinemia model group(HRE n=8) and other four groups(see method 3).The rats with resistin levels lower than X+1S.D.of resistin in the control group were defined as having diet-induced normal resistin model(24 rats). Eight rats were selected randomly from the diet-induced normal resistin model and defined as normal resistin goup(NRE n=8). Then the rats in the diet-induced hyper-resistinemia group(HRE), diet-induced normal resistin group(NRE)and normal control group(NC)were fed as before for 8 weeks.After 8 weeks,all rats in the three groups were anesthetized and blood samples and thoracic arteries were collected for different detections.
3.The effects in different treatment methods
Forty rats were selected randomly from diet-induced hyper-resistinemia model(see method 2)and randomized into diet-control group(DC n=8),swimming with diet-control group (SW n=8),rosiglitazone with diet-control group(RO n=8) antibody with diet-control group(AB n=8)and hyper-resistinemia group(HRE n=8).The rats in DC group were given standard chow without other treatments.The rats in SW group swam one hour per day,five days per week.The rats in RO group were received rosiglitazone 2mg/(Kg·d)The antibody group received two injections of resistin antibody(Biovendor Laboratory Medicine, Inc.Germany)through the tail vein for 3 days.The rats in SW, RO and AB group were given standard chow and the rats in HRE group were given high-fat diet as before.The treatments in RO,SW and DC group were lasted for 8 weeks.After the treatments,all rats in NC,DC,SW,RO,AB and HRE groups were anesthetized and blood samples and thoracic arteries were collected for different detections.
4.Measurement of variables in serum
Serum levels of resistin,PAI-1,and vWF were measured by using ELISA kits.Insulin(INS)and endothelin(ET)were measured by radioimmunoassay.Serum nitric oxide(NO)production was detected by measuring the final stable equimolar degradation products nitrite and nitrate by using a standard colormetric assay.Total cholesterol(TC)and triglycerides(TG)were determined by enzymatic method and low-density lipoprotein(LDL) and high-density lipoprotein(HDL)cholesterol were determined by immuno—turbidimetry assay respectively.Atherosclerotic index(AI)was calculated from TC and HDL:AI=(TC—HDL)/HDL.
5.Determination of the mRNA expression of eNOS and ET by RT-PCR method:The relative expressing level was defined by optical density ratio of eNOS and ET to actin respectively(eNOS /actin、ET/actin).
6.Immunohistochemistry was employed to examine the expression of phosphatidylinositol 3kinase(PI3K)p85αsubunit and protein kinase B(Akt)in thoracic artery endothelium.
7.In situ hybridization was employed to examine the mRNA expression of PI3K p85αsubunit and Akt—1 in thoracic artery endothelium.
8.Quantification was performed through a microscope with a computer-assisted image analysis system(image-pro plus 5.0).
9.The data were indicated as mean±standard deviation((?)±s)and were analyzed by Student t test and one-way ANOVA using SPSS14.0 statistical software.The correlation between variables,which were considered to be normally distributed by normality plots,was analyzed using Person' s correlation and partial correlation.Value of P＜0.05 was considered significant.
Results
1.The effect of extrinsic resistin on endothelial function
1.1 After two injections of rat recombinant resistin,the levels of insulin and resistin in the resistin group were increased significantly,while the insulin sensitive indexs were lower in the resistin group than those in the NS group.In the resistin group,the levels of ET,PAI,and vWF were higher while the level of NO was lower than those in the NS group.There was no difference in body weight and lipids of the two groups.
1.2 There was no difference in resistin mRNA expression of RE and NS group.The eNOS mRNA expression in RE group was lower and ET mRNA higher than those in NS group.
1.3 The results in immunohistochemistry and In situ hybridization indicate that the expression levels of PI3K and Akt in the resistin group were much lower than that in the NS group,which had strong positive brown granules in the cytoplasm.
2.The effect of intrinsic resistin on endothelium function
2.1 The body weight,Lee index,TC、TG、LDL were much higher in high resistin group(HRE)and normal resistin group(NRE)than those in normal control group(NC),while the level of HDL in HRE and NRE group decreased.
2.2 The level of resistin in HRE group was the highest among HRE,NRE and NS group.The level of insulin increased and the level of ISI decreased in HRE and NRE group compared with normal control group.There was markedly endothelial dysfunction in HRE and NRE group.Moreover,the rats in HRE group had worse endothelial dysfunction than NRE group.The level of NO decreased significantly and the levels of ET,PAI and vWF increased in HRE group.
2.3 The resistin expression in HRE group was the highest among HRE,NRE and NC group.There was no difference between NRE and NC group in resistin expression.The eNOSmRNA expression in NRE group was higher than that in HRE group and lower than that in NC group.The ETmRNA expression in HRE group was the highest among HRE,NRE and NC group and the ETmRNA expression in NRE group was the second.
2.4 The results in immunohistochemistry and In situ hybridization indicated that the expression levels of PI3K and Akt in the HRE group were much lower than that in the NRE group and NC group,which had strong positive brown granules in the cytoplasm.The IOD of PI3K and Akt in the HRE group were the lowest among HRE,NRE and NC group and those in NC group were the highest.
3.The effects in different treatment methods
3.1 After the treatments in RO and SW group lasted for 8 weeks, the body weight,Lee index were much lower in DC and SW group than those in HRE group.There was no difference in body weight, Lee index among RO,AB and HRE group.The lipids in SW group improved most and those in DC and RO group the second.There was no difference in lipids between HRE and NC group.
3.2 The four treatments all decreased the level of resistin and insulin and increased ISI markedly.The levels of resistin and insulin in RO and SW decreased most among the four treatment groups and those in DC and AB group decreased secondly.
3.3 The levels of ET、PAI、vWF in RO,SW and AB group were much lower and NO higher than those in HRE group.The levels of ET and PAI in De group decreased compared with the HRE group. There no difference in vWF and NO between DC and HRE group.
3.4 The resistin mRNA expression in SW and RO group were decreased mostly.There was no difference in resistin mRNA expression between AB and HRE group.The four treatments all increased markedly the expression of eNOSmRNA and decreased the expression of ETmRNA.There was no difference between HRE and NC group.
3.5 The results in immunohistochemistry and In situ hybridization indicated that the four treatments all increased markedly the expression of PI3Kp85αand Akt with the IOD increased significantly.RO and SW group improved the PI3Kp85αand Akt expression most among the four groups.
4.Correlation
Resistin levels and the expression of resistin mRNA were obviously correlated negatively with NO and eNOSmRNA expression and positively with ET、PAI、vWF and ETmRNA expression.Resistin levels and the expression of resistin mRNA were correlated negatively with the expression PI3Kp85αand Akt.Even after correction for weight,Lee index,insulin,ISI and lipids, resistin and the expression of resistin mRNA still correlated significantly with endothelial secretion markers,PI3K and Akt.
Conclusions
1.There were obvious endothelium dysfunction with PI3K、Akt mRNA and protein expression decreased after injection of resistin.
2.High fat diet induced high resistin rats and normal resistin rats at the same time.The serum resistin and resistin mRNA expression in adipose tissue were higher markedly in high-resistinemia rats than those in normal resistin rats.There were obvious obesity,insulin resistance and abnormal lipid in both high resistin rats and normal resistin rats.
3.The endothelial dysfunction was worse in high fat diet induced hyper-resistinemia rats than that in high fat diet induced normal resistin rats.The expression of PI3K and Akt in hyper-resistinemia rats were decreased significantly compared with the normal resistin rats.
4.Resistin increased with the occurrence of endothelial dysfunction in obesity.The results indicate that resistin was an early marker for endothelium dysfunction.
5.After correction for weight,Lee index,insulin,ISI and lipids,the level of resistin and resistin mRNA expression were correlated with endothelial secretion markers and the expression of PI3K and Akt.The result indicated that PI3K/Akt signaling pathway might contribute to the development of endothelial secretion dysfunction by resistin.
6.Rosiglitazone,swimming,diet-control and resistin antibody can improve endothelial dysfunction separately with increased PI3K、Akt expression.Swimming with diet-control and rosiglitazone with diet-control were most effective treatment methods.
Innovations and meanings:
1.This study demonstrated in vivo the effect of resistin on endothelium function.The endothelium functions were different in the high fat diet rats with different resistin level. The result certified that resistin was the independent risk factor of endothelium dysfunction after correction for interferences.It was crucial to detect and treat resistin for the prevention of endothelium dysfunction.
2.This study demonstrated in vivo the effect of resistin on PI3K/ Akt signaling pathway for the first time.There were different expressions of PI3K and Akt in individuals with different resistin.The result indicated the relationship among resistin,endothelium function and PI3K/Akt signaling pathway. The result indicated for the first time that PI3K/Akt signaling pathway might contribute to the development of endothelial secretion dysfunction by resistin.
3.This study first contrasted and demonstrated the effects in different treatment methods on endothelium function.Swimming and diet control were two effective methods for child to decrease the expression of resistin and improve endothelium function.
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