伐地那非通过改善氧化应激水平治疗肺动脉高压的临床与基础研究

发布时间：2018-06-04 02:11

本文选题：肺动脉高压 + 伐地那非　；参考：《山东大学》2014年博士论文

【摘要】：肺动脉高压(pulmonary arterial hypertension, PAH)是一类多病因、进展性的疾病,其定义为静息状态右心导管(RHC)测定平均肺动脉压大于等于25mmHg,最终进展为右心衰竭,直至死亡。肺动脉高压是多种生理途径和细胞类型引起的多因素的病理生理改变,包括肺血管收缩、增殖,血管阻塞性重构,炎性浸润及原位血栓形成等机制。越来越多的研究表明,氧化应激参与了肺动脉高压的发病过程。氧化应激被定义为氧化物的增加(如过氧化氢、超氧阴离子等)伴或不伴有抗氧化物或抗氧化酶(如超氧化物歧化酶)的降低。增加的氧化物不但能够通过氧化特定的生物分子,直接损伤组织器官,包括脂质过氧化和DNA氧化,而且可以与一氧化氮(NO)结合,导致毒性分子过氧亚硝酸的产生和内皮一氧化氮合成酶(endothelial nitric oxide synthase, eNOS)的脱偶联。在过去几年里,肺动脉高压的药物治疗有了很大进展,文献报导显示使用靶向药物治疗的肺动脉高压患者比安慰剂组的死亡率下降了43%,住院率下降了61%31。目前应用于肺动脉高压治疗的靶向药物有以下3类：1)前列环素类似物；2)内皮素受体拮抗剂；3)5-磷酸二酯酶抑制剂。其中5-磷酸二酯酶抑制剂包括西地那非、伐地那非和他达拉非,5-磷酸二酯酶抑制剂在肺血管系统中大量表达。5-磷酸二酯酶抑制剂通过NO/cGMP通路起到扩张血管和抗增殖作用。5型磷酸二酯酶(phosphodiesterase-type5, PDE5)是在肺组织中广泛表达的的磷酸二酯酶异构酶,它能够在鸟苷酸环化酶(guanylate cyclase)的作用下降解环磷酸鸟苷(cyclic guanosine monophosphate, cGMP),引起皿官收缩。PDE5抑制剂,包括西地那非(sildenafil)和他达拉非(tadalafil),目前已经被批准用于肺动脉高压的临床治疗。伐地那非(vadenafil),属于新型PDE5抑制剂。与传统的PDE5抑制剂相比,它完全溶于水和乙醇,对PDE5的特异性作用更强。2011年,通过一项临床双盲、随机、安慰剂实验,研究者发现伐地那非能够有效的改善PAH患者的运动能力、血流动力学指数和心功能,但机制尚不明确。在勃起功能障碍的基础和临床研究中,研究人员发现除了PDE5抑制功能,PDE5抑制剂还能够通过抑制氧自由基形成,降低NADPH氧化酶起到强大的抗氧化作用,从而缓解内皮功能障碍和血管重构,但其他PAH靶向药物,如依前列醇(epoprostenol)并没有此功能。Ghofrani等报道给予PAH患者短期伐地那非治疗后,显示虽然伐地那非能引起肺血管收缩,但其对肺循环血管缺乏特异性,另外,伐地那非中长期治疗也有个案报道,显示其对PAH的治疗并没有特殊效果。因此,伐地那非治疗PAH的有效性及耐受性仍值得进一步商榷。 2009年,荆志成教授及其课题组,通过对45名PAH患者的研究,显示1年的伐地那非治疗能够有效地治疗PAH。2011年,该课题组继续进行了一项随机、双盲、安慰剂-对照及多中心的研究。结果显示与西地那非和他达拉非不同,伐地那非单药治疗即能够有效的延缓PAH患者的临床恶化程度,改善其血流动力学变化。然而伐地那非治疗肺动脉高压的具体机制仍不明确。鉴于目前伐地那非治疗PAH功能研究的缺乏,本课题收入15名特发性PAH患者,同时以野百合碱诱导的PAH大鼠为体内模型,以H202诱导的氧化应激内皮细胞为体外模型,研究伐地那非治疗PAH的机制研究和可能涉及的信号通路,为PAH的治疗提供了依据。研究分以下三部分：第一部分伐地那非改善氧化应激并治疗特发性肺动脉高压的临床研究本研究选择特发性肺动脉高压患者作为研究对象,分析了伐地那非对特发性肺动脉高压患者的治疗作用,及其对患者体内氧化应激水平的改善作用。1)我们选取了EVALUATION研究中的15名特发性肺动脉高压的患者,并选择了年龄及性别与之相匹配的健康人群作为对照。入选患者给予伐地那非每次5mg,每日1次口服1个月,若患者未出现严重不良反应,则加量至每次5mg,每日2次继续口服2个月。 2)我们于基线及3个月后评估完成以下内容：标准病史与体格检查,6分钟步行距离,WHO肺动脉高压功能分级,血生化指标,心电图,肺功能检查,和完整的右心导管检查所测血流动力学资料。结果显示3个月的伐地那非治疗能显著改善特发性肺动脉高压患者WHO功能分级、六分钟步行距离、心指数、肺血管阻力等指标。尽管跟用药前相比较,患者平均肺动脉压力、肺毛细血管楔压、平均右房压力较前有降低,但并没有统计学差异。以上结果说明伐地那非能够改善肺动脉高压患者临床症状及血流动力学变化。 3)随后我们采用商品化试剂盒测量了肺动脉高压患者及健康对照组血液中一氧化氮、丙二醛、8-异前列素F2α、3-硝基酪氨酸、超氧化物歧化酶等氧化应激及氮化应激相关产物的水平。发现肺动脉高压患者血浆中氧化损伤产物丙二醛、8-异前列素F2α、3-硝基酪氨酸明显高于正常人,氧化应激保护性产物一氧化氮、超氧化物歧化酶明显低于正常对照者。伐地那非治疗3个月后,氧化损伤产物8-异前列素F2α及3-硝基酪氨酸水平均较前降低,但仍高于正常人,而丙二醛的水平并没有明显改变。血管扩张产物一氧化氮较治疗前明显升高,基本恢复正常水平。抗氧化酶超氧化物歧化酶较治疗前升高,与正常人相比,仍处于较低水平。以上以上结果显示,肺动脉高压患者体内存在较高的氧化应激水平,抗氧化能力明显较正常人减弱。而伐地那非治疗能够降低患者的氧化应激水平,改善其抗氧化能力,起到保护性作用。第二部分伐地那非改善野百合碱诱导肺动脉高压大鼠氧化应激水平的研究本研究选择了34只雄性SPF级Sprague-Dawley (SD)大鼠,体重200-220g,随机分为正常对照组、野百合碱模型组以及伐地那非治疗组。给予50mg/kg野百合碱21天后,伐地那非治疗组大鼠予1mg/kg/d灌胃共21天。之后进行如下研究： 1)应用右心导管检查术测量大鼠的血流动力学指标,发现MCT组大鼠的mPAP、RVSP和PVR明显高于正常对照组,而CO则明显低于正常对照组。给予伐地那非治疗后,伐地那非组大鼠的mPAP、RVSP和PVR较MCT组分别降低41%、25%和26%。以上结果与我们临床试验结果相一致,进一步验证了伐地那非对血流动力学的改善作用。 2)为研究伐地那非对血流动力学改善的原因,我们对大鼠肺组织进行了组织学染色并应用相关软件对血管组织进行测量。与正常对照组相比,野百合碱组大鼠的%WT和%WA明显增高。而给予伐地那非治疗后,伐地那非组的%WT和%WA明显降低。以上结果在50μm-100μm、100-200μm直径的肺血管中均有所体现。之后我们通过a-SMA抗体染色,检测大鼠的血管肌化程度。跟正常对照组相比,野百合碱组血管肌化程度显著增加,镜下观察平滑肌细胞明显肥大增生。给予伐地那非治疗后,伐地那非组肌化程度较MCT组降低了39%,平滑肌细胞增生肥大有所改善。最后我们对肺血管平滑肌细胞进行了PCNA及TUNEL染色,发现伐地那非对血管厚度的改善是通过诱导大鼠肺动脉平滑肌细胞的增值,同时促进其凋亡而实现的。我们应用werstern blot的方法及商品化试剂盒,研究了伐地那非治疗肺动脉高压的相关机制。与正常对照组相比,野百合碱组大鼠血浆及肺组织内NO的浓度降低,.给予伐地那非治疗后,跟野百合碱组相比,伐地那非组血浆及肺组织内NO水平均较前恢复。原因是通过伐地那非上调eNOS蛋白的表达而实现的。之后我们发现伐地那非能够改善大鼠体内氧化应激水平,与我们临床试验结果相一致。为了进一步研究可能的机制,我们选择了氧化应激过程中主要的酶,NADPH氧化酶和超氧化物歧化酶,进行下一步研究。结果显示与正常对照组相比,野百合碱大鼠大鼠体内NADPH氧化酶催化亚基,NOX2和NOX4,的水平明显上调,但激活亚基RAC1水平并没有明显变化。伐地那非治疗能显著改善NOX2和NOX4的表达水平,却同样不影响RAC1亚基的表达水平。相反,MCT组大鼠体内SOD活性较正常对照组相比明显降低,而伐地那非治疗后,SOD活性得到了显著改善。以上结果提示,伐地那非对氧化应激水平的改善,其中一部分是通过改善NADPH氧化酶和超氧化物歧化酶而实现的。第三部分伐地那非通过BMP信号通路改善内皮细胞氧化损伤的机制研究通过第一部分和第二部分的体内研究,我们明确了伐地那非能改善氧化应激同时治疗肺动脉高压,但具体机制仍不清楚。因此第三部分,我们应用CRL-1730内皮细胞系为研究模型,给予不同剂量外源性H202造成其氧化应激损伤,进一步研究了伐地那非对氧化应激损伤保护性作用的具体机制。 1)通过MTT及TUNEL的方法,我们发现与对照组比较,H202组细胞活力显著降低,伐地那非各剂量组与H202组比较,细胞活力显著升高,且具有明显的剂量依赖性。与对照组比较,H202组细胞凋亡率显著增高,给予伐地那非处理后,细胞凋亡率较前明显改善,且具有剂量依赖性。以上结果于体内试验结果相互印证：伐地那非对内皮细胞起到了强烈的保护性作用,且该作用呈剂量依赖性。 2)之后我们检测了骨形态发生蛋白(bone morphogenetic proteins,BMPs)信号通路在各组细胞中的表达,BMP信号通路广泛参与了肺动脉高压、动脉粥样硬化等血管疾病。结果显示,氧化应激随时能够上调BMP受体1A、BMP受体1B、BMP受体2,同时上调胞浆内p-smad水平,最后进入细胞核内,上调转录因子MSX2的表达。而伐地那非能够改善上述通路,起到保护性作用。 3)最后我们应用BMP信号通路的抑制剂,Dorsomorphin干预细胞,并通过TUNEL检测细胞凋亡情况。我们发现,与对照组比较,H202组细胞内细胞凋亡率显著增高,伐地那非组与H202组比较,细胞凋亡率显著下降,而与伐地那非组比较,Dorsomorphin干预组细胞凋亡率明显上升。说明Dorsomorphin能抑制伐地那非对内皮细胞的保护性作用,进一步说明BMP信号通路参与了伐地那非对内皮细胞氧化应激损伤的改善。综上所述,我们通过临床研究、动物实验及体外细胞培养,证明了伐地那非对PAH的治疗作用以及对PAH患者体内氧化应激水平的改善作用,及其相关的机制。
[Abstract]:Pulmonary arterial hypertension (PAH) is a multi cause, progressive disease defined as resting right cardiac catheterization (RHC) for the determination of the mean pulmonary artery pressure greater than equal to 25mmHg, and eventually to right heart failure until death. Pulmonary hypertension is a multifactor pathogeny caused by multiple physiological pathways and cell types. A growing number of studies have shown that oxidative stress is involved in the pathogenesis of pulmonary arterial hypertension. Oxidative stress is defined as an increase in oxide (such as hydrogen peroxide, superoxide anion, etc.) with or without antioxidants or oxygen. The reduction of chemical enzymes (such as superoxide dismutase). The increased oxide can not only directly damage the tissues and organs by oxidation of specific biomolecules, including lipid peroxidation and DNA oxidation, but also can be combined with nitric oxide (NO), resulting in the production of peroxynitrite and the endothelial nitric oxid (endothelium nitric oxide synthase). Decoupling of E synthase, eNOS).
In the past few years, there has been great progress in the treatment of pulmonary hypertension. The literature has reported that the mortality of pulmonary hypertension patients using targeted drug therapy has decreased by 43% than that in the placebo group, and the hospitalization rate has decreased by 61%31., the target drugs used for the treatment of pulmonary hypertension are 3 categories: 1) prostacyclin analogues; 2) endothelium. 3) 5- phosphodiesterase inhibitors, in which 5- phosphodiesterase inhibitors, including sildenafil, dedionafil and tatabafil, 5- phosphodiesterase inhibitors in the pulmonary vascular system, expressed a large number of.5- phosphodiesterase inhibitors through the NO/cGMP pathway to expand the blood tube and anti proliferative.5 phosphodiesterase (phosphod Iesterase-type5, PDE5) is a phosphodiesterase isomerase, widely expressed in lung tissue, which can decrease cyclic guanosine monophosphate (cGMP) in the role of guanosine cyclase (guanylate cyclase), causing Petri dish officers to constriction.PDE5 inhibitors, including sildenafil (sildenafil) and tarafil (tadalafil). It has been approved for the clinical treatment of pulmonary hypertension.
Vadenafil, a new type of PDE5 inhibitor. Compared with the traditional PDE5 inhibitor, it is completely soluble in water and ethanol and is more specific to PDE5 than.2011 years. Through a double blind, randomized, placebo experiment, researchers found that FD can effectively improve the exercise ability, hemodynamic index and heart of PAH patients. In the basic and clinical studies of erectile dysfunction, the researchers found that in addition to PDE5 inhibitory function, PDE5 inhibitors can also inhibit the formation of oxygen free radicals, reduce the potent antioxidant activity of NADPH oxidase, and thus alleviate endothelial dysfunction and vascular remodeling, but other PAH targeting drugs, such as Epoprostenol, which does not have the function of.Ghofrani and other reports, gives PAH patients a short term FV treatment, showing that although FD can cause pulmonary vasoconstriction, it is not specific to the pulmonary circulatory vessels, and the medium and long term treatment of vasopunal also has a case report, showing no special effects on the treatment of PAH. The efficacy and tolerability of FD in the treatment of PAH is still open to question.
In 2009, Professor Jing Zhicheng and his team, through a study of 45 PAH patients, showed that 1 years of FDA therapy could effectively treat PAH.2011 years. The group continued to conduct a randomized, double-blind, placebo-controlled and multicenter study. It can effectively delay the clinical deterioration of PAH patients and improve their hemodynamic changes. However, the specific mechanism of the treatment of pulmonary hypertension by FD is still not clear.
In view of the current lack of PAH functional study on FV, 15 patients with idiopathic PAH were collected, and PAH rats induced by monocrotaline were in vivo model, and the oxidative stress endothelial cells induced by H202 were used as models in vitro. The mechanism of PAH treatment and the possible signaling pathways were studied, which provided for the treatment of PAH. The three parts of the study are as follows:
The first part is the clinical study of the treatment of idiopathic pulmonary hypertension by using Vastatin to improve oxidative stress.
In this study, patients with idiopathic pulmonary hypertension were selected as research subjects, and the therapeutic effect of FD on patients with idiopathic pulmonary hypertension and the improvement of oxidative stress in patients with.1) were selected. We selected 15 patients with EVALUATION in the study of idiopathic pulmonary vein hypertension and selected age and sex. The matched healthy people were given a control. The patients were given 5mg for 1 months 1 times a day. If the patient had no serious adverse reactions, the patient was given a dose of 5mg each time and 2 times a day for 2 months.
2) we evaluated the following contents at baseline and 3 months later: standard medical history and physical examination, 6 minute walking distance, WHO pulmonary hypertension function classification, blood biochemical index, electrocardiogram, lung function examination, and complete right heart catheterization test. Results showed that 3 months of FDA could significantly improve the idiopathic effect. WHO functional classification, six minute walking distance, heart index, pulmonary vascular resistance, and other indicators. Although compared with pre medication, the average pulmonary artery pressure, pulmonary capillary wedge pressure, and the mean right atrial pressure were lower than before, but there was no statistical difference. The results showed that FD could improve the pulmonary hypertension. Clinical symptoms and hemodynamic changes.
3) then we measured the levels of nitric oxide, malondialdehyde (MDA), 8- isoprost F2 a, 3- nitrotyrosine, superoxide dismutase and other oxidative stress related products in the blood of patients with pulmonary hypertension and healthy control group. Prostacyclin F2 alpha and 3- nitro tyrosine were significantly higher than normal people. Oxidative stress protective product nitric oxide and superoxide dismutase were significantly lower than normal controls. After 3 months of treatment, the levels of 8- ISO Prost F2 A and 3- nitrotyrosine were lower than those in the front, but still higher than those of normal people, while the level of malondialdehyde was not. There was an obvious change. The nitric oxide of the vasodilator increased obviously and basically recovered to the normal level. The antioxidant enzyme superoxide dismutase was higher than that before the treatment, and it was still at a lower level compared with the normal person. Above results showed that the above results showed that there was a high level of oxidative stress in the patients with pulmonary hypertension, and the antioxidant capacity was significantly higher. Normal people were weakened, while treatment with fattening could reduce oxidative stress level and improve their antioxidant capacity.
The second part is the effect of FD on improving oxidative stress in rats with pulmonary hypertension induced by monocrotaline.
In this study, 34 male SPF grade Sprague-Dawley (SD) rats and weight 200-220g were selected and randomly divided into normal control group, monocrotaline model group and FD n non treatment group. After giving 50mg/kg monocrotaline for 21 days, the rats were given a total of 21 days with 1mg/kg/d. The following study was followed:
1) the hemodynamic indexes of rats were measured by right heart catheterization, and the mPAP, RVSP and PVR in group MCT rats were significantly higher than those of normal control group, while CO was significantly lower than that of normal control group. After the treatment, the mPAP, RVSP and PVR of FD group rats decreased by 41%, 25% and more of 26%. were compared with our clinical trial. The results were consistent, which further verified the improvement of Vernon on hemodynamics.
2) in order to study the reasons for the improvement of FD's hemodynamics, we stained the lung tissue of rats and measured the vascular tissue with relevant software. Compared with the normal control group, the%WT and%WA of the rats were significantly higher than those in the normal control group. The%WT and%WA decreased significantly after treatment with FD. The results were shown in the pulmonary vessels of 50 mu M-100 mu m and 100-200 m diameter. Then we detected the degree of vascular myosis in rats by a-SMA antibody staining. Compared with the normal control group, the degree of vascular myosis in the monocrotaline group increased significantly, and the smooth muscle cells were obviously hypertrophic and proliferated under the microscope. The degree of muscle myocytes in the group was 39% lower than that in the MCT group. The proliferation and hypertrophy of smooth muscle cells was improved. Finally, we performed PCNA and TUNEL staining on the pulmonary vascular smooth muscle cells. It was found that the improvement of vascular thickness by reducing the vascular thickness was achieved by inducing the proliferation of rat pulmonary artery smooth muscle cells and promoting its apoptosis.
We used werstern blot method and commercial kits to study the mechanism of pulmonary arterial hypertension treated with FD. Compared with the normal control group, the concentration of NO in the plasma and lung tissue of the rats in the monocrotaline group decreased. After the treatment, the plasma and the NO levels in the pulmonary tissue were compared with the monocrotaline group. We found that FDA was able to increase the expression of eNOS protein. After that, we found that FD could improve the level of oxidative stress in rats, consistent with our clinical trials. In order to further study the possible mechanisms, we chose the main enzymes, NADPH oxidase, and super enzymes in the oxidative stress process. The results showed that the level of NADPH oxidase catalyzed subunits, NOX2 and NOX4 in the rats of monocrotaline increased obviously compared with the normal control group, but the level of the activated subunit RAC1 was not significantly changed. The level of NOX2 and NOX4 expression could be significantly improved, but the RAC1 was not affected by RAC1. On the contrary, the activity of SOD in the MCT group was significantly lower than that in the normal control group, and the activity of SOD was significantly improved after the treatment. The results suggested that the level of oxidative stress was improved, some of which were achieved by improving the NADPH oxidase and superoxide dismutase.
The third part is the mechanism of improving endothelial cell oxidative damage by means of BMP signaling pathway.
In the first and second parts of the body, we have identified that FD can improve oxidative stress and treat pulmonary hypertension, but the specific mechanism is still unclear. So the third part, we applied the CRL-1730 endothelial cell line as the research model, and gave different doses of exogenous H202 to cause oxidative stress damage and further study. The protective mechanism of Vastatin on oxidative stress injury is discussed.
1) through the method of MTT and TUNEL, we found that compared with the control group, the cell viability of the H202 group decreased significantly. Compared with the H202 group, the cell viability was significantly increased, and the cell viability was significantly dependent on the control group. Compared with the control group, the apoptosis rate of the H202 group was significantly higher than that in the control group. The results were proved to be dose-dependent. The results of the above results showed that FD had a strong protective effect on endothelial cells, and the effect was dose-dependent.
2) after that, we detected the expression of bone morphogenetic proteins (BMPs) signaling pathway in each cell. BMP signaling pathway is widely involved in pulmonary arterial hypertension, atherosclerosis and other vascular diseases. The results show that oxidative stress can increase the BMP receptor 1A, BMP receptor 1B, BMP receptor 2, and up - up cytoplasmic p-. Smad level finally entered the nucleus and up-regulated the expression of transcription factor MSX2, while Vastatin could improve the above pathway and play a protective role.
3) at last, we used the inhibitor of BMP signaling pathway, Dorsomorphin interfered the cells, and detected the cell apoptosis by TUNEL. We found that compared with the control group, the H202 group was within the cell.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R725.4

【共引文献】