【作者】 刘峰伊；

【导师】 姜一农；

【作者基本信息】 大连医科大学 ， 内科学， 2014， 博士

【摘要】 动脉粥样硬化以及由其引发的心脑血管疾病严重危害着人类的健康,关于动脉粥样硬化的发生机制,有多种学说包括脂质浸润学说,血栓形成学说,平滑肌细胞克隆学说及炎症损伤反应学说,目前最为认可的是ROSS提出的炎症损伤反应学说,他认为在各种刺激因素的作用下,首先动脉内膜损伤,内皮功能被破坏,继而分泌各种炎性因子,通过炎性因子的作用,平滑肌细胞表现出异常的多度增殖及凋亡不足,形成动脉粥样斑块从而引发各种心血管疾病。肾素-血管紧张素系统(Renin-Angiotensin System,RAS)作为人体的一个重要的调节系统,参与调节人体血压,水分及电解质以保持人体内环境的稳定。RAS的过度激活可导致多种心血管疾病的发生,例如高血压,动脉粥样硬化,心肌肥厚,心力衰竭等。血管紧张素Ⅱ(Angiotensin-Ⅱ,AngⅡ)是RAS的主要活性物质,AngⅡ与其受体结合后可发挥一系列促进增殖,促纤维化和促炎性反应,继而导致靶器官的损害,其在平滑肌细胞的增殖迁移及动脉粥样硬化的形成中发挥重要作用。肾素前体主要由肾脏分泌,并且在肾脏被激活转换为有活性的肾素。肾素前体本身一直被认为是没有活性的,但在上世纪80年代有报道显示在糖尿病患者中血浆肾素前体水平增多,随后的研究显示糖尿病患者中肾素前体的增多与微血管并发症有关联,并且早于微量白蛋白尿的出现,肾素前体水平连同糖化血红蛋白可以用来预测微量白蛋白尿的出现。这些足以说明肾素前体是具有功能的,提示机体可能处于一种病理状态,甚至可以作为预测某些疾病的一个指标。直到2002年Nguyen等提出有功能的肾素(前体)受体[(pro)renin receptor,PRR],并证实了它存在于心脏、大脑、胎盘、肾脏和肝脏,发现肾素前体可与PRR在纳摩尔水平结合,除了通过传统的AngⅡ途径发挥作用,还可以不依赖于AngⅡ的方式发挥作用,激活信号通路,引起促纤维化促炎症等一系列反应,导致靶器官的损伤,这使人们对RAS有了新的认识。阻断RAS系统被认为具有心血管获益并能减低心血管疾病的风险,但却并不能完全抑制心血管疾病的发生,其原因可能存在其他机制导致心血管疾病的发生,肾素前体及PRR产生的独立于AngⅡ却类似于AngⅡ的作用可能为其中的一种机制。我们将以此为基础,研究肾素前体及PRR在不依赖于AngⅡ的作用下对人脐动脉平滑肌细胞增殖及凋亡蛋白表达的影响。动脉粥样硬化的发生发展被认为是一种慢性炎症过程,而炎症的一个重要标志就是活性氧(reactive oxygen species,ROS)的产生。氧化应激是一种ROS过量生成损害了细胞内抗氧化系统的病理状态。大量的研究证实,ROS的产生在心血管疾病的病理生理过程中发挥重要作用。由炎症细胞及血管壁上各型细胞产的ROS能引起细胞的增殖、凋亡、迁移及促炎性基因的表达增多,ROS引起的各型细胞的损伤及功能上的变化形成了动脉粥样硬化的病理基础。NADPH氧化酶系统是血管壁上ROS的主要来源,AngⅡ能够激活NADPH氧化酶生成ROS,进一步损伤内皮功能,加速降解NO,使内皮收缩功能降低,同时促进平滑肌细胞的表型的转换,引起异常的增殖迁移,基质蛋白生成增多。对于PRR作用的分子机制尚不完全明确,PRR能否像AngⅡ一样通过氧化应激引起平滑肌细胞发生各种促进动脉粥样硬化形成的改变?我们将以此为研究对象,研究肾素前体及PRR对人脐动脉平滑肌细胞(human umbilical artery smooth muscle cells,HUASMCs)氧化应激的影响。促分裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)是细胞内信号转导的重要蛋白酶,包括ERK、JNK、p38MAPK及ERK5四个亚族,其中细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK1/2)信号转导通路与细胞的增殖分化密切相关。各种刺激因素通过激活ERK1/2信号通路,使其磷酸化水平增多而发挥促进增殖作用,促炎性因子的表达等。研究证实AngⅡ可通过氧化应激产生ROS进一步激活ERK1/2信号通路,从而促进平滑肌细胞的增殖。因此,我们将以此为基础,探讨肾素前体及PRR能否通过激活HUASMCs中ERK1/2信号通路进一步影响细胞增殖作用,并探讨ERK1/2信号通路及其与氧化应激的关系。目的：观察PRR在人脐动脉平滑肌细胞中的分布,观察肾素前体及其受体对HUASMCs增殖的影响及对凋亡蛋白Bcl-2,Bax表达的影响,研究ROS产生和ERK1/2信号通路在其中的作用。方法：1.用免疫荧光法观察PRR在HUASMCs中的分布。2.在缬沙坦及PD123319阻断AngⅡ的作用下,分别以浓度为O.1nM,1nM,10nM的肾素前体干预HUASMCs24-48h,用CCK-8检测细胞的增殖水平。3.在缬沙坦及PD123319阻断AngⅡ的作用下,分别以浓度为0.1nM,1nM,10nM的肾素前体干预HUASMCs24h,各组用real-time PCR2及western blot方法检测Bcl-2,Bax蛋白mRNA及蛋白表达水平。4.用siRNA PRR转染HUASMCs24,48及72小时后,用real-time PCR及western blot方法检测PRR的mRNA及蛋白表达,确定转染效率最强时间点。5.用siRNA的方法使PRR表达下调后,再用缬沙坦及PD123319预处理30min后,以浓度为10nM的肾素前体干预HUASMCs24h,分别用CCK-8检测细胞的增殖水平,用real-time PCR及western blot方法检测Bcl-2,Bax蛋白mRNA及蛋白表达水平。6.在缬沙坦及PD123319阻断AngⅡ的作用下,分别以浓度为0.1nM,1nM,10nM的肾素前体干预HUASMCs24h,然后收集细胞,在流式细胞仪上检测ROS水平。7.用siRNA的方法使PRR表达下调后,再用缬沙坦及PD123319预处理30min后,以浓度为10nM的肾素前体干预HUASMCs24h,在流式细胞仪上检测ROS水平。8.在缬沙坦及PD123319阻断AngⅡ的作用下,以浓度为10nM的肾素前体干预HUASMCs0-60min,用western blot方法检测ERK1/2磷酸化水平。9.用DPI(NADPH氧化酶抑制剂)预处理HUASMCs30min后,再以浓度为10nM的肾素前体干预HUASMCs,用western blot方法检测ERK1/2磷酸化水平。10.分别用PD98059(ERK1/2信号通路阻断剂)和DPI预处理HUASMCs30min后,再以浓度为10nM的肾素前体干预HUASMCs,用CCK-8检测细胞的增殖,用real-time PCR及western blot方法检测Bcl-2,Bax蛋白mRNA及蛋白表达水平。结果：1.HUASMCs存在PRR的表达,细胞整个胞质中均有表达,在细胞核周围表达更明显。2.不同浓度肾素前体可在不依赖于AngⅡ的作用下促进HUASMCs的增殖,并且浓度在10nM时最强。3.用10nM浓度的肾素前体干预HUASMCs24-48h,随着时间延长,增殖作用明显增强。4.肾素前体可在不依赖于AngⅡ的作用下上调Bcl-2蛋白的表达,下调Bax蛋白的表达,抑制凋亡。5.siRNA PRR转染后,能减弱肾素前体引起的HUASMCs增殖作用,并且减弱肾素前体调节HUASMCs凋亡蛋白表达的作用。6.肾素前体在不依赖于AngⅡ的作用下,能够促进HUASMCs ROS的生成,并且在肾素前体10nM浓度条件下的促增殖作用最明显,而siRNAPRR转染后能够减少肾素前体引起的HUASMCs中ROS的生成。7.肾素前体可在不依赖于AngⅡ的作用下将ERK1/2信号通路激活,并且磷酸化程度在15min-30min时作用最强。8.DPI及PD98059能抑制肾素前体引起的ERK1/2信号通路激活。9.DPI及PD98059可使肾素前体引起的HUASMCs的增殖作用明显减弱.10.DPI及PD98059可明显减弱肾素前体引起的HUASMCs的抗凋亡蛋白Bcl-2mRNA及蛋白表达的上调作用。11.DPI及PD98059可明显减弱肾素前体引起的HUASMCs的促凋亡蛋白Bax mRNA及蛋白表达的下调作用。结论：1.PRR存在于HUASMCs中.2.肾素前体可通过PRR在不依赖于AngⅡ的作用下,促进HUASMCs的增殖,并上调抗凋亡蛋白Bcl-2的表达,下调促凋亡蛋白Bax的表达。3.肾素前体可在不依赖于AngⅡ的作用下,促进HUASMCs ROS产生增多,并且这种作用是通过PRR介导的。4.肾素前体可在不依赖于AngⅡ的作用下,激活HUASMCs ERK1/2信号通路,并且这种作用通过ROS产生介导。5.阻断ROS产生和ERK1/2信号通路能减弱肾素前体及受体引起的HUASMCs的增殖作用,并且减弱对凋亡蛋白Bcl-2,Bax表达的调节作用。更多还原

【Abstract】 Atherosclerosis and its related cardiovascular diseases have seriously threatened human health. Concerning the pathogenesis of atherosclerosis, there are several theories including lipid infiltration theory, thrombosis theory, smooth muscle cell clone theory and inflammation theory. The most recognized theory is the inflammation theory proposed by ROSS, and he considered that in the stimulation of various factors, the injured arterial intima will secrete a variety of inflammatory factors which can further impaire endothelial function. And the inflammatory factors also cause the imbalance of proliferation and apoptosis of vascular smooth muscle cells which promote the formation of atherosclerotic plaques.Renin Angiotensin System (RAS) as an important part of the regulation of human body system, is involved in regulating blood pressure, water and electrolyte in order to keep the stability of the environment in the human body. The excessive activation of the RAS can lead to a variety of cardiovascular diseases, such as hypertension, atherosclerosis, myocardial hypertrophy and heart failure. Angiotensin Ⅱ (AngⅡ) is the main active component of RAS, and the binging to its receptor can elicit a series of effects including proliferation, fibrosis and proinflammatory response which cause the damage of target organs. AngⅡ also plays a key role in the formation of atherosclerosis because it can promote the proliferation and migration of vascular smooth muscle cells.Prorenin is mainly synthesized in the kidney in which prorenin is converted into the active renin. Proenin has been considered as the inactive renin precursor, but in the last century80’s, a report showed that plasma prorenin levels increased in the diabetic patients. Subsequent studies have shown increased plasma prorenin that occurred before the occurrence of microalbuminuria in diabetic patients is associated with microvascular complications. And increased prorenin levels along with glycated hemoglobin can be used to predict the onset of microalbuminuria. So the increased prorenin levels may be associated with various diseases, and even can be used as an indicator to predict some disease.In2002, Nguyen firstly proposed the functional (pro)renin receptor (PRR), and detected its distribution in the heart, brain, placenta, kidney and liver. Then researchers found that the binding of prorenin to PRR in the nanomolar level can active the signal pathway to elicit a series of profibrotic and inflammatory effects in both AngⅡ-dependent and AngⅡindependent way. The AngⅡ-independent role of PRR makes people have a new understanding of RAS. The blockade of the RAS is considered to be beneficial and to reduce cardiovascular risks, despite the fact that its inhibition cannot completely inhibit the development of these diseases. The answer to this question might reside in the AngⅡ-independent effects induced by the binding of prorenin with PRR. Based on this, we invetigated the Angll-independent role of prorenin and PRR on the proliferation and apoptosis in human umbilical artery smooth muscle cells (HUASMCs).Atherosclerosis is a chronic inflammatory process, and the important sign of inflammation is reactive oxygen species (ROS) production. Oxidative stress is a pathological condition in which excessive production of ROS damage the intracellular antioxidant system. A lot of studies have confirmed that ROS play an important role in the pathophysiology of cardiovascular disease. ROS produced by inflammatory cells and various types of cells in vascular wall can promote cells proliferation, apoptosis, migration and increase expression of proinflammatory genes. The damage and functional changes of cell induced by ROS constitute the pathological basis of atherosclerosis. Angiotensin Ⅱ can activate NADPH oxidase system that is the main source of the vessel wall ROS. The production of ROS can further cause the abnormal proliferation and migration of vascular smooth muscle cells by impairing endothelial function, accelerating the degradation of NO, reducing the endothelial contractile function, and promoting the phenotype transformation of smooth muscle cells. The molecular mechanism of PRR is still not completely clear, and whether the PRR can like AngⅡ through oxidative stress induce the changes which is responsible for atherosclerosis in smooth muscle cells is not defined. On basis of this, we invetigated the AngⅡ-independent role of prorenin and PRR on the production of ROS in HUASMCs.Mitogen activated protein kinase (MAPK) is an important enzyme in signal transduction, including ERK, JNK, p38MAPK and ERK5four subfamilies, and ERK1/2signal transduction pathway is closely related to the proliferation and differentiation of cells. ERK1/2activation by a variety of stimuli can promote cells proliferation and increase the inflammatory factor expression. The binding of AngⅡ to its receptor can promote the proliferation of smooth muscle cells through the phosphorylation of ERK1/2. In rat cardiomyocytes, AngⅡ can induce the phosphorylation of ERK1/2through oxidative stress. So, we explored the role of ERK1/2signal pathway in effects induced by prorenin and PRR, and study the relationship between ERK1/2and ROS.Ojective:To observe the distribution of PRR in HUASMCs. To observe the effect of prorenin on proliferation and apoptosis protein Bcl-2, Bax expression in HUASMCs. To explore the role of ERK1/2and ROS in prorenin-induced effects.Methods:1.We used fluorescence immunohistochemistry to determine the distribution of PRR in HUASMCs.2.In the presence of Valsartan and PD123319, we used prorenin of different concentration (0.1nM,1nM,10nM) to stimulate HUASMCs for24-48h and evaluated the proliferation by CCK-8.3.In the presence of Valsartan and PD123319, we used prorenin different concentration (0.1nM,1nM,10nM) to stimulate HUASMCs for24h and evaluated the expression of Bcl-2and Bax by western blot and real-time PCR.4.HUASMCs were transfected with PRR siRNA respectively24,48and72hours, and the mRNA and protein of PRR expression was detected by real-time PCR and western blot. 5.After transfection of PRR siRNA and preincubation with Valsartan and PD123319for30min, we used prorenin of concentration (10nM) to stimulate HUASMCs for24h and evaluated the proliferation by CCK-8and evaluated the expression of Bcl-2and Bax by western blot and real-time PCR.6. In the presence of Valsartan and PD123319, we used prorenin of different concentration (0.1nM,1nM,10nM) to stimulate HUASMCs for24h and measured the production of ROS on a flow cytometer.7. After transfection of PRR siRNA and preincubation with Valsartan and PD123319for30min, we used prorenin of concentration (10nM) to stimulate HUASMCs for24h and measured the production of ROS on a flow cytometer.8. In the presence of Valsartan and PD123319, we used prorenin of concentration (lOnM) to stimulate HUASMCs and evaluated the phosphorylation of ERK1/2by western blot.9. After the preincubation with DPI for30min, we used prorenin of concentration (10nM) to stimulate HUASMCs for0-60min and evaluate the phosphorylation of ERK1/2by western blot.10. After the preincubation with PD98059and DPI respectively for30min, we used prorenin of concentration (10nM) to stimulate HUASMCs and evaluated the proliferation by CCK-8and evaluated the expression of Bcl-2and Bax by western blot and real-time PCR.Results:1.We determined the distribution of PRR in HUASMCs by fluorescence immunohistochemistry and detected the cytoplasmic expression of PRR.2. Different concentrations of prorenin can independently of AngⅡ promote the proliferation in HUASMCs and the effect of prorenin (10nM) was most significant.3. Prorenin (10nM) for24-48h, as stimulation time extended the effect of prolifertion in HUASMCs was more significant.4. Different concentrations of prorenin can independently of AngⅡ upregulate the expression of Bcl-2and downregulate the expression of Bax in HUASMCs and the effect of prorenin (10nM) was most significant.5.After transfection with a PRR siRNA, the effects that prorenin elicited on the proliferation of HUASMCs was significantly attenuated, and the upregulated expression of Bcl-2and the downregulated expression of Bax were also altered.6. Prorenin can independently of AngⅡ promote the production of ROS and the effect of prorenin (10nM) was most significant. Transfection with a PRR siRNA significantly decreased the production of ROS.7. Prorenin can independently of AngⅡ induce the phosphorylation of ERK1/2, and the effect at15min-30min was most significant.8. DPI (NADPH oxidase inhibitor) and PD98059(the ERK1/2inhibitor) respectively attenuated the phosphorylation of ERK1/2induced by prorenin.9. DPI and PD98059respectively attenuated the prorenin-induced proliferation of HUASMCs.10. DPI and PD98059respectively attenuated the prorenin-induced up-regulation of Bcl-2at mRNA and protein levels in HUASMCs.11. DPI and PD98059respectively attenuated the prorenin-induced down-regulation of Bax at mRNA and protein levels in HUASMCs.Conclusions:1. There were the expression of PRR in all the cytoplasm of HUASMCs.2. The binding of prorenin to PRR can promote the proliferation and upregulate the anti-apoptotic protein Bcl-2and downregulate the pro-apoptotic protein Bax independently of AngⅡ in HUASMCs.3. The binding of prorenin to PRR can independently of AngⅡ promote the production of ROS in HUASMCs.4. Prorenin can independently of AngⅡ induce the phosphorylation of ERK1/2through the production of ROS in HUASMCs.5. Inhibition of the production of ROS and the phosphorylation of ERK1/2respectively attenuated the prorenin-induced proliferation and regulation of apoptosis factors in HUASMCs.更多还原