【作者】 孟然；

【导师】 王亚平； 朱大龙；

【作者基本信息】 南京大学 ， 内科学（专业学位）， 2011， 博士

【摘要】 随着社会经济的发展,糖尿病(diabetes)正成为全球范围流行的疾病。2型糖尿病(type2diabetes)是糖尿病中最主要的类型,多由于机体产生胰岛素相对不足或由于机体细胞对胰岛素反应性下降,且2型糖尿病常见于肥胖(obesity)个体。近年来,肥胖的发生率越来越高,而肥胖的一个重要特征是胰岛素抵抗(insulin resistance),后者恰是2型糖尿病的主要发生机理。胰岛素抵抗是指机体需要较之正常状态更多的胰岛素才能产生与正常状态同等生物学效应的一种病理状态。越来越多来自动物以及临床研究的证据表明,炎症(inflammation)反应与胰岛素抵抗有着密切联系。在啮齿类动物的肥胖模型中已经证明,肿瘤坏死因子(tumor necrosis factor, TNF)-α这一前炎症因子在肥胖导致的胰岛素抵抗中起着极其重要的作用。现在的观点认为,脂肪是产生炎症相关细胞因子及其他生物活性物质的场所。除TNF-a之外,这些细胞因子还包括白介素(interleukin, IL)-6,瘦素(leptin),血清淀粉样蛋白A (serum amyloid A, SAA),抵抗素(resistin),单核细胞趋化蛋白(monocyte chemoattractant protein, MCP)-1,血浆纤溶酶原激活物抑制剂(plasminogen activator inhibitor)-1,血管紧张肽原(angiotensinogen),内脂素(visfatin),视黄醇结合蛋白(retinol-binding protein)-4等等。此外,随着细胞内炎症信号通路方面的研究,对于胰岛素抵抗产生机制的认识也越来越清楚。现有的研究表明,c-JNK的N末端激酶(c-JUN N-terminal kinase, JNK)以及核因子-κ B (nuclear factor-κ B, NF-κ B)信号通路在胰岛素抵抗发生中起着极其重要的作用。除了炎症,活性氧自由基(reactive oxygen species, ROS)水平的上升是胰岛素抵抗发生诸多因素中的重要因素之一,主要的实验证据有：1)在体外培养的3T3-L1脂肪细胞中,高剂量的过氧化氢或者其他可导致ROS产生的物质处理后可引起胰岛素抵抗；2)在肥胖个体或实验动物研究中发现氧化应激(oxidative stress)的指标均明显升高。研究还发现,在高脂饮食(high-fat diet, HFD)喂养的小鼠中,ROS产生相关的基因在胰岛素抵抗或者肥胖出现之前就已经开始上调。此外,多项临床研究还发现胰岛素抵抗或糖尿病患者经抗氧化剂治疗后,机体的胰岛素敏感性增加。Apocynin (4-hydroxy-3-methoxy-acetophenone)是药用植物Himalayan herb Picrorhiza kurrooa Royle提取物的组成成分,具有烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate, NADPH)氧化酶的特异性抑制作用,因而作为一种抗氧化剂被广泛用于多项研究之中。除了抗氧化作用之外,apocynin还被发现具有抗炎的作用,它可抑制自发性高血压大鼠中NF-κB的激活,下调TNF-α在冠状动脉中的表达,在静脉内皮细胞中降低TNF-α所致的血管细胞间粘附分子(vascular cell adhesion molecule, VCAM)-1的表达,抑制多形核粒细胞(polymorphonuclear granulocyte)的趋化,减少小鼠气管内过氧亚硝基阴离子,减轻人关节软骨中的炎症介导的软骨破坏,并且降低人单核细胞中环加氧酶(cyclooxygenase, COX)-2的表达。但是,apocynin在胰岛素抵抗中的治疗作用及其机制还没有被系统的研究。本研究主要目的之一是观察apocynin对高脂诱导胰岛素抵抗小鼠的治疗效果,并探讨其与炎症抑制和氧化损伤拮抗相关的治疗机制。促红细胞生成素(Erythropoietin, EPO)是肾脏产生的一种造血生长因子,上世纪被用于临床疾病的治疗。后续的临床应用研究发现,EPO除了具有促进机体造血功能之外,还呈现抗炎、抗氧化、抗凋亡等作用。临床上发现,在伴有或不伴有糖尿病终末期肾衰(end-stage renal disease, ESRD)的透析治疗患者,采用EPO治疗后胰岛素抵抗有所减轻,但相关机制尚未阐明。本研究在观察分析Apocynin、EPO胰岛素抵抗治疗作用的同时,将深入探讨这两种药物针对胰岛素抵抗的治疗机制。第一部分Apocynin的抗氧化作用对高脂小鼠胰岛素抵抗的影响这部分主要研究apocynin对高脂饮食导致的胰岛素抵抗小鼠的氧化应激和抗氧化酶的影响。C57BL/6J小鼠经12周高脂饮食喂养后,呈现明显的胰岛素抵抗特征。再此基础上给予apocynin (2.4g/L,饮水中)治疗5周。结果显示apocynin治疗可明显改善高脂小鼠的高血糖、高胰岛素血症以及高脂血症。同时腹腔注射糖耐量实验(intraperitoneal glucose tolerance test, IPGTT)以及胰岛素抵抗的动态模型评估(homeostasis model assessment of insulin resistance, HOMA-IR)均显示高脂小鼠经apocynin治疗后其胰岛素敏感性增高。与未治疗组相比,apocynin治疗组的血清丙二醛(malondialdehyde, MDA)明显降低而血清过氧化物歧化酶(superoxide dismutase, SOD)明显增高,提示apocynin可抑制系统的氧化应激。同样,在肝脏中MDA的水平也在apocynin治疗后明显降低,而与抗氧化系统(antioxidative defense system)相关的SOD、谷胱甘肽过氧化物酶(glutathione-peroxidase, GSHpx)均升高。与此对应,肝脏内谷胱甘肽(glutathione, GSH)含量则减少。此外,我们还意外的发现肝脏过氧化氢酶(catalase, CAT)在apocynin治疗后明显降低,这可能是apocynin治疗后肝脏内过氧化氢以及氧化应激减少的佐证。这些结果揭示了apocynin可以在高脂小鼠中通过减轻机体细胞的氧化应激来缓解胰岛素抵抗。第二部分Apocynin在高脂小鼠中通过抑制炎症而改善胰岛素抵抗这一部分主要研究apocynin对高脂小鼠导致的胰岛素抵抗模型中炎症方面的影响。经12周高脂饮食喂养后,有明显胰岛素抵抗表征的C57BL/6J小鼠进行apocynin治疗实验(2.4g/L,加入饮用水中,持续治疗5周)。结果显示空腹血糖、胰岛素耐量以及糖耐量实验均显示了高脂小鼠胰岛素敏感性的改善。血清内炎症因子TNF-α、IL-6以及Leptin的水平在apocynin治疗组均明显降低。肝脏TNF-α、IL-6、MCP-1以及脂肪组织内TNF-α、IL-6、MCP-1和瘦素的基因表达在apocynin治疗后也明显降低。更重要的是,肝脏内转录因子NF-κB的活性在apocymn治疗后被明显抑制。上述结果表明,apocynin在高脂小鼠导致的胰岛素抵抗模型中可减少循环血液、肝脏以及脂肪组织内的炎症因子,可能与apocynin减轻高脂小鼠胰岛素抵抗的机制有关。第三部分EPO的抗炎和抗氧化作用对高脂小鼠胰岛素抵抗的影响越来越多的研究发现EPO除了具有促进红细胞生成的作用之外,还有其他多种生物学功能。最近研究发现EPO可以减轻胰岛素抵抗,但是其确切机制尚未阐明。在本部分研究中,高脂导致的胰岛素抵抗小鼠根据不同的EPO治疗剂量以及时程分为4个大组。结果发现,EPO减轻胰岛素抵抗的作用可在2周、5周、以及7周治疗组中体现。在对其治疗机制的研究中,我们分析了EPO对胰岛素抵抗小鼠的胰岛素通路、脂质代谢、氧化应激以及炎症等方面的影响。结果发现：1)EPO可明显升高肝脏AKT的活性,而AKT是胰岛素通路中的关键因子；2)EPO可降低肝脏固醇调节元件结合蛋白-1c (sterol regulatory element binding protein isoform, SREBP1-c)、脂肪酸合酶(fatty acid synthase, FAS)的表达,降低脂肪组织内SREBP1-c的表达,增加脂肪组织脂蛋白酶(lipoprotein lipase, LPL)的表达；3)EPO可明显增高肝脏SOD. Cu-Zn SOD. GSH. GPx的水平,降低MDA;4) EPO可降低肝脏以及脂肪组织内炎症相关基因的表达,同时降低血清TNF-α、IL-6以及leptin的水平。EPO还抑制了肝脏内JNK1/2以及NF-κB的活性。我们的工作还发现,低剂量、长程的EPO治疗并不引起血红细胞的升高。总之,本研究在动物模型中证明了EPO可以通过改善胰岛素通路、脂质代谢、氧化应激,降低炎症水平等生物学效应,从而改善胰岛素抵抗。因此,EPO是胰岛素抵抗乃至糖尿病治疗有潜在应用前景的药物之一。更多还原

【Abstract】 The prevalence of diabetes is reaching epidemic expansion worldwide. Type2diabetes is the most common form of diabetes. In type2diabetes, either the body does not produce enough insulin or the cells ignore the insulin. Obesity is commonly associated with type2diabetes. The prevalence of obesity has increased dramatically in recent years. Insulin resistance is a key feature of obesity and is a main mechanism of type2diabetes. Insulin resistance is defined as a state that requires more insulin to obtain the biological effects achieved by a lower amount of insulin in the normal state.Increasing evidence from human studies and animal researches has established correlative as well as causative links between inflammation and insulin resistance. The proinflammatory cytokine tumor necrosisi factor (TNF)-a has been demonstrated to mediate insulin resistance as a result of obesity in many rodent obesity models. The concept of fat as a site for the production of cytokines and other bioactive substances quickly extended beyond TNF-a to include IL (interleukin)-6, leptin, serum amyloid A (SAA), resistin, monocyte chemoattractant protein-1(MCP-1), plasminogen activator inhibitor-1(PAI-1), angiotensinogen, visfatin, retinol-binding protein-4, and others. The investigations that focused on intracellular pathways activated by inflammation, instead of individual cytokines, have helped to restructure the framework for thinking about insulin resistance. It has been demonstrated that c-JUN N-terminal kinase (JNK) and nuclear factor-KB (NF-κB) pathway play the crucial roles in the insulin resistance.Besides inflammation, reactive oxygen species (ROS) production is one of many factors that have been suggested to play a role in the development of insulin resistance, based on the following evidence:(1) high doses of hydrogen peroxide and reagents that accumulate ROS can induce insulin resistance in3T3-L1adipocytes, and (2) increased markers of oxidative stress were observed in obese humans and rodents. It has been also demonstrated that the up-regulation of genes responsible for ROS production occurs in both the liver and adipose tissue before the onset of insulin resistance and obesity in mice fed with high-fat diet (HFD). Otherwise, several clinical trials have demonstrated improvement of insulin sensitivity in insulin-resistant and diabetic patients treated with antioxidants.Apocynin (4-hydroxy-3-methoxy-acetophenone) is a constituent of the Himalayan herb Picrorhiza kurrooa Royle (Scrophulariaceae) which is regarded as an inhibitor of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) and was widely used as an antioxidant in research. In addition, apocynin also exhibits anti-inflammatory effects in many previous studies. Apocynin has been demonstrated to suppress NF-κB activation in spontaneously hypertensive rats, down-regulate TNF-a protein expression in coronary arteries, decrease vascular cell adhesion molecule-1(VCAM-1) induction by TNF-a in vein endothelial cells, reduce polymorphonuclear granulocyte chemotaxis, inhibited peroxynitrite in airway lumen in mice, attenuated inflammation-mediated cartilage destructionm activity in human articular cartilage, and reduced cyclooxygenase (COX)-2expression in human monocytes. The therapeutic effect of apocynin on insulin resistance has not been explored. Hence, one main goal of this study is to determine that if apocynin could ameliate insulin resistance in HFD mice through its effects on the oxidative stress and inflammation.Erythropoietin (EPO), a kind of hematopoietic growth factor from kidney, was first used for clinic treatment in last century. In the latter utility and research, EPO was found to have many other effects like inhibiting inflammation and oxidative stress, anti-apoptosis. Interestingly, it was found that EPO could attenuate insulin resistance in diabetic or non-diabetic end-stage renal disease (ESRD) patients who were taking haemodialysis therapy, while the underlying mechanisms are still unclear. The third part of this thesis is aimed to explore the effects of EPO treatment on insulin resistance in mice. Part Ⅰ Anti-oxidative effect of apocynin on insulin resistance in HFD miceThis part examined the effects of apocynin on oxidative stress and antioxidant enzymes in HFD induced obese mice. After12weeks on HFD, the C57BL/6J mice that clearly exhibited insulin resistance received apocynin (2.4g/L) in their drinking water for5weeks. The results show that apocynin treatment significantly ameliorated hyperglycemia, hyperinsulemia and dyslipidemia in HFD mice. Also, the intraperitoneal glucose tolerance test (IPGTT) and homeostasis model assessment of insulin resistance (HOMA-IR) indicate significant improvement of insulin sensitivity in HFD fed mice after apocynin treatment. Compared to the HFD control mice, serum malondialdehyde (MDA) was significantly lower and serum superoxide dismutase (SOD) was significantly higher in apocynin treated HFD mice, which revealed apocynin suppressed systemic oxidative stress. In the liver, apocynin was found to significantly reduce the level of MDA. Accordingly, apocynin treatment strengthened the antioxidative defense system with an increased activity of SOD, glutathione-peroxidase (GSHpx) and content of reduced glutathione (GSH). We also found that hepatic catalase (CAT).activity significantly decreased after apocynin treatment which may reflect apocynin significantly reduced hydrogen peroxide and oxidative stress in the liver. These results suggest that apocynin may ameliorate insulin resistance through reducing systemic and hepatic oxidative stress in HFD fed mice.Part Ⅱ Apocynin improves insulin resistance through suppressing inflammation in HFD induced obese miceIn this part, we investigated the effects of apocynin on inflammation in HFD induced insulin resistance mice model. After12weeks of HFD fed, the C57BL/6J mice that exhibited insulin resistance then received5weeks of apocynin (2.4g/L, in water). Following apocynin treatment, fasting glucose, insulin and glucose tolerance test showed a significant improvement in insulin sensitivity in HFD fed mice. We demonstrated that serum levels of TNF-α, IL-6and leptin were remarkably reduced with apocynin treatment. We also found that mRNA expression of TNF-α, IL-6, MCP-1in the liver and mRNA expression of TNF-a, IL-6, MCP-1, leptin in adipose tissue were suppressed by apocynin. Furthermore, the activity of transcription factor NF-κB in the liver was significantly suppressed with apocynin treatment. These results suggest that apocynin may reduce inflammatory factors in the blood, liver and adipose tissue, resulting in amelioration of insulin resistance in HFD fed mice.Part III EPO attenuates insulin resistance through multiple protective functions in HFD fed miceIncreasing data indicated that erythropoietin (EPO) has multiple biological functions besides erythropoiesis. A few studies recently showed that EPO could ameliorate insulin resistance, but the related mechanisms are still obscure which were explored in the present study. The high-fat diet (HFD)-induced insulin resistance mice were divided into groups with different EPO dosages and treatment durations. The results showed that EPO exhibited its protective effects on insulin resistance in2-,5-and7-week EPO treatment groups. To demonstrating the undergoing mechanisms, the status of insulin signaling, lipid metabolism, oxidative stress and inflammation in the HFD mice with or without EPO treatment were assessed. Our data showed that:1) EPO could increase activity of AKT in the liver which is critical in insulin signaling;2) In the liver, EPO reduced the expression of sterol regulatory element binding protein isoform (SREBP)-1c and fatty acid synthase (FAS), which both related to the lipid metabolism. At also, EPO decreased SREBP-1c and increased lipoprotein lipase (LPL) expression levels in the adipose tissue;3) with regard to the effects of EPO on oxidative stress, increased superoxide dismutase (SOD), Cu-Zn SOD, glutathione (GSH), glutathione peroxidase(GPx), and decreased malondialdehyde (MDA) were detected in the liver;4) EPO decreased serum tumor necrosis factor(TNF)-a, interleukin(IL)-6and leptin. The gene expressions of inflammatory factors in the liver and adipose tissue were also reduced after EPO treatment. Moreover, EPO suppressed the activations of hepatic JNK1/2and nuclear factor-KB (NF-κB). In addition, our results demonstrated that EPO treatment with low-dose and long-term did not cause the side-effect of high hematocrit. In conclusion, our results found that EPO could ameliorate insulin resistance through its protective functions on effecting insulin signaling, lipid metabolism, oxidative stress and inflammation. Thus, EPO could be a promising treatment for insulin resistance, and even for type2diabetes.更多还原