【作者】 甘可欣；

【导师】 王富军；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:2型糖尿病(Type 2 diabetes mellitus,T2DM)大血管并发症患病率是非糖尿病人群的3～5倍,是2型糖尿病患者的主要并发症和致死的主要原因。而大血管病变的主要病理改变是动脉粥样硬化。糖尿病与动脉粥样硬化关系密切,表现为糖尿病患者出现动脉粥样硬化的时间早、程度重和预后差,控制血糖可以明显减少慢性微血管并发症,但不能显著降低动脉粥样硬化所致的大血管并发症。基质金属蛋白酶(matrix metalloproteinases, MMPs)是一大类能降解细胞外基质(extracellular matrix,ECM)和基底膜成分的锌依赖性肽链内切酶家族,与结缔组织的降解和重建有关。金属蛋白酶抑制剂(tissue inhibitor of matrix metalloproteinases, TIMPs )为其特异性抑制剂。二者在体内通过特定的途径调节,若调节发生紊乱,将导致各种疾病的发生。最近研究发现MMPs、TIMPs及二者的平衡失调与T2DM合并动脉粥样硬化(atherosclerosis,AS)的发生、发展有关。过氧化物酶体增殖蛋白激活性受体γ(Peroxisome Proliferator-activated receptor-γ,PPARγ)是细胞核内受体大家族成员,在脂质和脂蛋白新陈代谢、葡萄糖代谢中发挥关键作用。噻唑烷二酮(thiazolidinediones,TZDs)类药物是PPARγ的激动剂,可有效改善IR,降低血糖,纠正脂代谢紊乱,已日益广泛应用于2型糖尿病的治疗中。最近研究表明:PPARγ参与了动脉血管炎症反应和AS的形成和发展, PPARγ的活化可直接作用于血管壁,对血管壁具有保护作用。PPARγ激动剂对血管炎症的抑制作用可能对慢性炎症疾病,尤其是糖尿病大血管并发症的治疗具有重要意义。罗格列酮和吡格列酮是目前最常用的TZDs类药物,除降低高血糖、改善脂质代谢紊乱,还有降低血压、减少蛋白尿及抗动脉粥样硬化等作用。胶原酶-1(MMP-1)主要裂解间质Ⅰ型、Ⅲ型胶原,引起胶原变性,使其变得对明胶酶的水解作用敏感,在ECM的降解过程中具有重要作用,作用于AS发生、发展过程中的所有关键环节。关于MMP-1与斑块的稳定性的研究较多,在动脉粥样硬化斑块中表达水平较高,过度表达使斑块破裂的危险增加。目前对MMP-1在糖尿病大血管病变早期病变中的作用的研究较少。本试验研究目的在于观察链脲佐菌素(Streptozotocin,STZ)诱导的高脂高糖喂养的糖尿病大鼠模型中大动脉壁MMP-1、TIMP-1的表达变化,并应用吡格列酮进行干预,观察其对MMP-1、TIMP-1表达及血管内皮功能的影响,探讨吡格列酮在糖尿病大血管病变中的作用机制,并为预防和延缓糖尿病大血管病变提供有力理论依据和有效治疗方法。方法:健康雄性SD大鼠45只,8周龄,体重216±15g,由河北省实验动物中心提供。随机分为正常对照组(NC组)15只和实验组30只,NC组喂以标准大鼠饲料,实验组喂以高糖高脂高热量饲料,4周后实验组给予一次性腹腔注射链脲佐菌素30mg/kg,NC组大鼠给予同等剂量柠檬酸钠缓冲液腹腔注射,各组以原饲料继续喂养2周后,内眦静脉取血测空腹血糖≥7.8mmol/L者为糖尿病大鼠。再将糖尿病大鼠随机分为糖尿病组(DM组)和吡格列酮干预组(PIO组)。PIO组每日给予吡格列酮11mg /(Kg·d )灌胃治疗, NC组及DM组每日给予同PIO组等体积的生理盐水灌胃。实验末NC组剩余大鼠15只,DM组12只,PIO组13只。每组大鼠于实验开始、注射STZ前及注射STZ两周后和实验结束称重、取血。大鼠自采血前一日20:00起禁食,实验日8:00分别从内眦静脉采血3ml,分离血清,-20℃保存,用于各指标的检测。于第14周末(即造模成功后8周)处死动物,留取胸主动脉4%中性多聚甲醛固定,待组织学检查。1血糖、胰岛素测定血糖采用葡萄糖氧化酶法测定。空腹胰岛素(FINS)采用放射免疫法测定。应用稳态模式评估法胰岛素抵抗指数(HOMA-IR)及空腹胰岛素敏感性指数(ISI)评价胰岛素抵抗的程度。2血脂的测定甘油三酯(TG)、总胆固醇(TC)采用比色法测定(以mmol/L计)。FFAs用Cu~+比色法测定。3血管内皮功能生化指标的测定血浆内皮素(ET)采用放射免疫分析法测定,单位:pg/ml。血清NO测定采用硝酸还原酶法,单位:μmol/L。4动脉组织形态学检查常规方法制备大鼠动脉光镜组织切片,行苏木素-伊红(HE)染色,Masson染色,免疫组织化学染色。5统计学处理所有数据用SPSS13.0软件处理,计量资料以均数±标准差表示,组间差异用两样本均数的t检验进行比较,两组以上比较采用单因素方差分析。显著性用P值表示,P<0.05为差异有显著性,P<0.01为差异有极显著性。结果:1实验开始时大鼠随机分组,各组体重无统计学差别。高糖高脂喂养1月时即注射STZ前大鼠实验组体重明显高于NC组,差异有极显著性(P<0.01)。造模成功后大鼠出现明显的多饮、多食、多尿症状,体重明显下降,活动减弱,一般状态差。实验末PIO组和DM组体重明显高于NC组,有极显著性差异(P<0.01), PIO组较DM组体重下降,有显著性差异(P<0.05)。2 STZ造模前各组大鼠空腹血糖无差别。腹腔注射STZ后2周时,实验组血糖明显高于对照组,有极显著性差异(P<0.01)。实验末PIO组血糖低于DM组(P<0.05),但两组血糖仍高于NC组,有极显著性差异(P<0.01)。造模前实验组胰岛素水平明显高于NC组,有极显著性差异(P<0.01)。实验末DM组与PIO组胰岛素水平仍高于对照组,有显著性差异(P<0.05),PIO组胰岛素水平低于DM组,有显著性差异(P<0.05)。造模前实验组大鼠的TC、TG、FFA均明显高于NC组,有显著性差异(P<0.01)。实验末PIO组的TC、TG、FFA明显低于DM组(P<0.05),但仍高于NC组(P<0.05)。造模前实验组大鼠的ET均明显高于NC组,有显著性差异(P<0.01)。实验末PIO组的ET低于DM组(P<0.05),但仍高于NC组(P<0.05)。造模前实验组大鼠的NO均明显低于NC组,有显著性差异(P<0.01)。实验末PIO组的NO高于DM组(P<0.05),但仍低于NC组(P<0.05)。3组织学观察显示:实验末光镜下NC组大鼠动脉壁未见明显病理变化,糖尿病大鼠动脉壁出现局限性内膜增厚,但未见大量泡沫细胞等典型AS病理改变。主动脉横断面上可见较多的内膜增厚病灶,病灶处内皮细胞连续性断裂或缺失,内弹力板厚薄不均,有断裂分离现象,中膜浅层SMC轻度增生、排列紊乱。Masson染色:NC组动脉壁胶原含量较少,内膜和中膜仅见少量绿染区域;DM组和PIO组内膜增厚,内膜绿染区明显增加,红色的平滑肌细胞周围可见绿染;计算机图文分析染为绿色总面积占统计场总面积的百分比对胶原含量进行半定量,并做统计学分析显示:实验组与NC组比较均具有统计学意义(P<0.05),PIO组较DM组胶原含量减少,有显著性差异(P<0.05)。4免疫组化检测显示实验末NC组主动脉MMP-1、TIMP-1表达较少,仅内皮细胞胞浆少量表达。DM组MMP-1、TIMP-1表达明显增多,阳性细胞呈棕黄色,定位于细胞浆,内皮细胞和内膜下区域可见阳性表达,与NC组相比有统计学意义( P<0.05)。而PIO组表达水平低于DM组,高于NC组有显著性差异( P<0.05)。结论:1高糖高脂饮食加小剂量链脲佐菌素腹腔注射成功制备2型糖尿病大鼠模型,糖尿病大鼠模型8周时即出现动脉粥样硬化早期病理改变。2 2型糖尿病大鼠主动脉早期大血管病变中MMP-1与TIMP-1的表达和活性失调,导致血管壁胶原代谢紊乱,促进了动脉粥样硬化的发生发展。3吡格列酮通过激活PPARγ具有改善糖脂代谢、增加胰岛素敏感性、减少胰岛素抵抗的作用,还可通过调节MMP-1与TIMP-1的表达及改善血管内皮功能等,对糖尿病状态下的大血管起到了保护作用,减缓了糖尿病大血管病变的发展进程。更多还原

【Abstract】 Objective: The morbidity rate of macroangiopathy in diabetic complication is 3 to 5 times of non-diabetic. It is the major complication of diabetic and main reason for the diabetic’s mutilation and early death. The abnomal metabolism of the diabetics cause the dysfunction of arteries. The main modules such as long-term hyperglycemia, hypertension, dyslipidemia, insulin resistence, lead to diabetic complicating with macroangiopathy. But atherosclerosis(AS) is the chief process of macroangiopathy. The closely relationship between diabetic and atherosclerosis show that the diabetics occurs AS earlier and more seriously, and they have bad prognosis. The recent study shows that matix metalloproteinases(MMPs) is related to the development of diabetic complicating with AS. Peroxisome Proliferator-activated receptor-γ(PPARγ) , a member of the nuclear hormone receptor superfamily, play a critical role in lipid and lipoprotein metabolism and glucose metabolism. Thiazolidinediones (TZDs), a kind of high affinity ligand of PPARγ, through activating PPARγ, effectively improving insulin resistence, cutting down the blood glucose, retrieving the lipid metabolic disturbance, is generally utilized in the treatment of T2DM. The research recently show that PPARγhas involved in arteriovascular inflammatory reaction and the development of AS. The activation of PPARγcan protect the vessel wall directly. As a PPARγagonist may be significance to the treatment of chronic inflammation disease,especially to diabetic macroangiopathy because of its inhibitory action to blood vessel inflammation. Pioglitazone is a kind of new drug belonging toTZDs. Pioglitazone has the founctions that not only lowering the hyperglycemia, improving lipid metabolic disturbance, but also depressing the hypertension, reducing albuminuria, and anti-atherosclerosis. In the diabetic macroangiopathy, the effect of Pioglitazone on expression of MMP-1 and TIMP-1 on the vessel wall is not report yet. In the present study,we investigate the observation that the expression of MMP-1,TIMP-1 in rat of diabetic macroangiopathy model induced by long-term high-fat feeding accompanied with intraperitoneal injection of streptozotocin (STZ), and also research the effect of Pioglitazone on expression of MMP-1 and TIMP-1,changes of the collogen content in the vascular, and alteration of the functions of endothelium, explored the mechanisms of diabetic macroangiopathy treatment by PPARγagonist.Methods:Male Sprague–Dawley rats (Hebei Medical University) weighing about 216±15g (about 8 weeks old), were used. The rats were housed under 12 h light/12 h dark conditions with adlibitum access to water and food. After breeded adaptively for 1 weeks,a total of 45 healthy male SD rats were divided randomly into 2 groups: normal control group (Group NC, n=15) received a regular diet and experimental model group (n =30) was fed for 1 months. When the high-fat diet animals appeared the insulin resistance evaluated by Li Guangwei HOMA index formula , STZ was administered via intraperitoneal injection at 30mg/kg to make model. Then the modeling rats were further divided randomly into 2 groups: non-intervention of T2DM group (Group DM, n=15) was fed the high-fat diet continuously for 2 months, and Pioglitazone -treated group (Group PIO, n=15) was fed a high-fat diet meanwhile Pioglitazone was administered by oral gavage (11mg/kg) once daily. The experiment lasted 14 weeks. Except for accidental death and abortive sample, there remain 15 rats in Group NC,12 rats in Group DM and 13 rats in Group PIO at the end of experiment.All the rats were weighed and their blood was taken at the beginning, before injecting STZ and end of the experiment. The rats were fasted at 20:00 in the day before experiment, blood was taken from the angular vein at 8:00 of experimental day, and the blood serum was separated and stored at -20℃for all the indexes’detection (the mensuration of the indexes were performed with the same batch of kits). At the end of experiment, the vascular tissues were taken and fixed for histological analysis by neutral paraform. 1 Assays blood glucose and insulinWe measured fasting glucose by glucose oxidase method, and insulin by radioimmunoassay on serum samples. The level of insulin resistance was evaluated according to Homeoestasis Model Assessment (HOMA-IR) and Insulin Sensitivity Index (ISI).2 Assays of blood fatTriglyceride(TG), total cholesterol(TC) were detected. FFA was measured by Cu2+chromatometry.3 Assays of vascular parametersEndothelin(ET) was measured by radioimmunoassay on serum samples. NO measured by nitrate reductase method.4 Cell morphology examination of vessel wallThe tissue section was made by routine method for light microscope examination, and proceed with HE staining,masson staining, MMP-1 and TIMP-1 immunohisto-chemistry staining. Observed vessel wall microstructure with light microscopes. Measured vessel wall collagen content by picric acid - brilliant green staining. Detected the protein expression level and activity of MMP-1 and TIMP-1 in vessel wall by immunohistochemical staining. hese graphic results were analyzed with computer image-analysis system and the integral optical density (IOD) counts of MMP-1、TIMP-1 and collagen content were calculated.5 Data analysis and statistical evaluationAll data were treated with SPSS 13.0. Measurement data were expressed as mean values±standard deviation. The difference between means was compared by the Student’s t-test, and analysis of variance for one factors (ANOVA) was used to compare continuous variables among groups. The degree of significance was judged by P values when necessary. P<0.05 means the difference is significant , P<0.01 means the difference is highly significant.Results:1 Body weight: There was no statistically significant difference in weight among each group of the rats at the beginning of the experiment. At the end of the experiment, the weight of rats in diabetic rats and was significantly higher than that in normal control rats (P<0.01), and the weight of rats treated with Pioglitazone was lower than that diabetic rats(P<0.05).2 Blood glucose and insulin: There was no significant difference in fasting glucose among the rats of each group at the beginning of the experiment. At 2 weeks after the injection of STZ, the blood glucose level of rats in Group DM and the rats in Group PIO significantly raised up from normal baseline (P<0.01). The blood glucose level of rats in Group PIO was lower than that in Group DM (P<0.05), but it was still hyperglycemia. The insulin level of rats in Group DM was markedly higher than that in Group NC (P<0.01), The insulin level of rats treated with Pioglitazone lower than that in Group DM and higher than that in Group NC(P<0.05).3 Blood lipid :The plasma levels of TG, TC, FFA in rats with T2DM were obviously higher than those in normal control rats (P<0.01). After treated with Pioglitazone,their plasma levels decreased to some extent, but they were still higher than those in normal control rats.4 vascular parameterThe plasma levels of ET in rats with T2DM were obviously higher than those in normal control rats(P<0.01). The plasma levels of ET in rats treated with Pioglitazone was lower than that in rats with T2DM(P<0.05), but still higher than that in normal control rats(P<0.05). And NO in rats with T2DM were obviously lower than those in normal control rats(P<0.01). The plasma levels of NO in rats treated with Pioglitazone was higher than that in rats with T2DM(P<0.05), but still lower than that in normal control rats(P<0.05).5 The pathological morphological change of the vascular tissuesHE-staining method: under light microscope, we observed that, compared with the Group NC,the vascular tissue of the diabetic rats show significantly thicken in local tunica intima,but can’t find the typical pathological change of AS, such as a great of foam cells.Masson-staining method: Under light microscope, the intima shows a little green in normal control rats, while the green area in Group DM and in Group PIO was bigger than that in Group NC (P<0.05),the green area in Group PIO was smaller than that in Group DM (P<0.05).6 Immunohistochemistry The MMP-1 and TIMP-1 expression of the rats treated with Pioglitazone was significantly higher than that of normal control rats (P<0.05), and lower than that of diabetic rats (P<0.05).Conclusions:1 Male SD rat was fed with a diet enriched with sucrose (20%),cholesterol(2.5%) and lard(15%) to induce insulin resistance, and after two month on the diet streptozotion(STZ,30mg/kg) was injected in abdominal cavity to induce hyperglycemia. The rat model was succeed in preparing for T2DM , with hyperglycemia, insulin resistance and dyslipidemia. It also shows that there exists early stages of atherosclerosis in the model of T2DM rats.2 The disturbance of MMP-1 and TIMP-1 activation and expression in diabetic rats constitutes the development of atherosclerosis,as one of the critical factors.3 Pioglitazone plays an important role in improving glucolipid metabolism, increasing the sensitivity of insulin and reducing the insulin resistance,by activating PPARγ. Moreover, it can regulate the expression of MMP-1 and TIMP-1 in vascular tissues, increase the activity of NO, reduce the activity of ET. Thus it exerts the action against inflammatory reaction and oxidative stress , improves the blood vessel endothelial function, protects vascular tissues in T2DM, and slows the process of diabetic atherosclerosis.更多还原