普罗布考保护血管舒张功能及其对平滑肌细胞表型的影响

发布时间：2018-05-06 07:16

本文选题：普罗布考 + 血管功能　；参考：《中南大学》2014年博士论文

【摘要】：第一部分Probucol通过促进eNOS活化保护H202诱导的内皮依赖性血管舒张功能的降低背景普罗布考(Probucol, PB,又名丙丁酚)是一种人工合成的抗氧化剂。它作为一种双酚类化合物,具有降低胆固醇、抗氧化、抗炎及抗动脉粥样硬化等多重作用,最初是作为降脂药物应用于临床。临床试验、动物及体外实验发现probucol对某些病理改变下血管收缩舒张功能有直接作用,并证实其对血管功能的作用独立于其降脂效果。氧化应激是高血压、动脉粥样硬化等心血管疾病的发病机制,氧化应激通过作用于血管细胞,从而影响血管的收缩和舒张活性。而probucol的抗氧化性不仅表现在抑制脂质(如低密度脂蛋白LDL)的氧化,也可以清除血液和组织中自由基,这提示probucol的抗氧化作用是否是其影响氧化应激下血管功能改变的机制,以及是否对于心血管疾病的血管舒缩功能有保护作用。血管内皮是维持血管稳态的主要调节者。已知内皮细胞是血管收缩和舒张活性的重要组成部分,而内皮功能障碍可以导致血管收缩及舒张功能平衡紊乱,血压调节异常；此外内皮细胞受损还可以导致血液细胞黏附增加,炎症细胞浸润血管以及促-抗凝血平衡失调等。临床试验证实内皮细胞功能障碍是心血管事件的危险因素之一。而氧化应激对血管性疾病的发生发展有促进作用,活性氧族(reactive oxygen species, ROS)首先破坏内皮细胞功能,影响内皮的调压、抗凝及内分泌合成功能,这可能是动脉粥样硬化、高血压、糖尿病等血管病变的原因之一。目的 Probucol多效性作用机制尚不明确,而且probucol对血管舒缩功能的作用仍有争议。本实验为了探讨probucol对血管收缩及舒张功能的作用；并根据probucol的抗氧化特性进一步探讨,在H2O2诱导氧化损伤基础下probucol对血管舒张功能的影响及作用机制。方法使用大鼠离体胸主动脉血管环构建H2O2损伤内皮依赖性血管舒张功能的模型。(1)使用myograph技术研究离体血管环的收缩及舒张功能,使用高钾(KCl)、去氧肾上腺素(PE)分别诱导血管收缩反应,乙酰胆碱(ACh)、硝普钠(SNP)诱导血管舒张反应(内皮依赖性和非内皮依赖性)。观察血管在H2O2的持续作用下,血管基础功能的变化情况。(2)在H2O2作用的基础上,myograph检测PB干预处理对血管功能的影响,并且使用western blot技术检测干预后血管环内皮特异性一氧化氮合酶(eNOS)蛋白的表达情况,探讨PB对血管环舒张功能的作用是否是通过影响内皮细胞eNOS的产生来实现的。(3)使用血管环组织培养技术进一步探讨PB对不良应激下内皮依赖性舒张功能的作用,myograph检测培养后血管环活性的改变。建立apoE-/-动脉粥样硬化小鼠模型,在体实验研究在动脉粥样硬化疾病血管损伤基础上PB对血管舒张功能的影响,及其对血脂代谢的作用与血管功能改变的相关性培养原代人脐静脉内皮细胞(HUVECs),同样在体外实验中建立H202损伤血管内皮功能的模型,主要进行以下实验：(1) HUVECs的培养及鉴定；(2)实时荧光定量RT-qPCR及western blot检测PB对内皮细胞eNOS mRNA、蛋白表达水平的影响,对eNOS活化程度的影响；(2)在H2O2损伤情况下,PB对内皮细胞eNOS表达水平的作用,探讨PB是否通过影响eNOS表达来保护内皮细胞功能；(3)免疫荧光显微镜观察以及荧光定量检测HUVEC在H2O2诱导氧化损伤时NO的生成,以及PB对NO产生的作用,探讨PB是否能激活eNOS-NO生成途径达到保护内皮介导舒张功能的作用。结果 1.H2O2在一定浓度及作用时间下可以诱导氧化损伤,选择性抑制大鼠离体胸主动脉血管环ACh诱导的内皮依赖性舒张功能,对血管收缩功能无明显作用。高浓度或长时间H2O2作用可以造成不可逆的血管收缩及舒张功能的丧失。 2.在H2O2诱导的氧化应激下,PB (5-20μM)保护大鼠及小鼠离体胸主动脉血管环ACh诱导的内皮依赖性血管舒张功能。 3.PB通过抑制H2O2诱导的离体血管环eNOS活性下降,保护内皮细胞功能,但对收缩功能无显著作用。 4.在组织培养过程中,PB对大鼠胸主动脉血管环的内皮依赖性舒张功能也有保护作用。 5.在apoE-/-动脉粥样硬化小鼠模型中,PB可以保护小鼠主动脉内皮依赖性舒张功能,且该作用与其降脂效果无关。 6.PB可以提高人脐静脉内皮细胞中p-eNOS/eNOS比值,促进eNOS的磷酸化程度(Ser-1177),激活eNOS氧化活性；H2O2抑制eNOS总蛋白表达水平,PB保护H2O2诱导氧化损伤情况下eNOS的磷酸化水平。 7.PB促进人脐静脉内皮细胞中eNOS活化的产物NO的产生。结论 PB对于H2O2诱导的内皮依赖性舒张功能障碍有保护作用,其机制可能是PB通过促进eNOS磷酸化,激活eNOS氧化酶活性,从而促进NO的生成有关。第二部分普罗布考对平滑肌细胞表型的作用及其与平滑肌细胞增殖迁移的关系背景血管平滑肌细胞VSMCs作为血管壁的重要构成部分,其功能的异常与心血管疾病有密切关系。VSMCs增殖及合成作用参与动脉粥样硬化斑块的形成,平滑肌细胞源性泡沫细胞是斑块的细胞成分之一。传统观点认为平滑肌细胞具有收缩型及合成型两种表型,而病理改变与合成/增殖型有关。而新观点认为病变血管周围或血液来源的干细胞才是斑块形成的主要细胞来源,它们拥有部分SMC表型标记,但不同于普通平滑肌细胞的功能,拥有更多干细胞的特性,因此提出这种多能干细胞性的平滑肌细胞是SMC参与血管病理改变的基础。因此SMC干细胞活性增加与血管病灶处SMC增殖或合成作用有关,主要以高分化平滑肌细胞收缩蛋白的降低(包括α-actin、SM22、calponin及SM-MHC (SM1、SM2))以及干细胞标志升高。动物实验发现probucol可以抑制家兔主动脉球囊扩张损伤后血管再狭窄,抑制平滑肌细胞增殖和迁移,抑制内膜增厚、管腔变窄,并且改善血管重构。因此我们从SMC对血管疾病的作用机理和表型变化出发,探讨PB的对心血管疾病的保护作用是否与SMC表型有关,及其对SMC增殖迁移的作用。目的探索probucol对平滑肌细胞增殖及迁移的作用以及该作用是否与平滑肌细胞的表型变化有关。方法 (1)使用组织块贴壁法提取并培养原代大鼠的血管平滑肌细胞,使用免疫荧光染色鉴定原代VSMCs。观察原代细胞提取和培养过程中细胞形态及增殖分化过程,使用PB干预血管组织块以观察细胞从组织块原位的迁出情况；并且在细胞接种过程中直接计数PB对细胞增殖数目的影响。 (2)使用Transwell细胞迁移实验检测PB及oxLDL对VSMCs的迁移影响 (3)采用RT-qPCR技术、western-blot技术研究VSMCs中表型相关蛋白α-actin、干细胞标志Sox-10的表达变化,以及平滑肌细胞转录激活因子myocardin的表达改变情况。结果(1) α-actin+SM22+Sox-10-是高分化VSMCs的表型特征； (2)PB单独作用抑制VSMCs干细胞标志Sox-10的表达,也抑制myocardin、SM22表达,但不影响收缩蛋白α-actin的表达。H202诱导氧化应激促进Sox-10表达并下调myocardin表达,而PB可以部分逆转该变化,即H2O2作用基础上PB抑制Sox-10,促进myocardin表达。 (3)PB抑制VSMCs从组织内迁移以及在体外培养过程中的迁移及增殖速率；结论在体外研究中发现PB可以抑制大鼠VSMCs的迁移及增殖,其机制可能与PB抑制平滑肌干细胞的分化及运动能力有关。
[Abstract]:The first part of Probucol protects H202 induced endothelium-dependent vasodilation by promoting eNOS activation.
background
Probucol (PB, also known proproopol) is a synthetic antioxidant. It is a kind of bisphenol compound, which has multiple effects on lowering cholesterol, antioxidation, anti-inflammatory and anti atherosclerosis. It was first used as a lipid lowering drug in clinical practice. In clinical trials, animal and in vitro experiments found probucol to some pathology. The function of vasoconstriction and vasodilatation has a direct effect, and it is confirmed that its effect on vascular function is independent of its lipid lowering effect. Oxidative stress is the pathogenesis of cardiovascular diseases such as hypertension and atherosclerosis. Oxidative stress acts on vascular cells and thus affects vasoconstrictor and diastolic activity of blood vessels. The antioxidant activity of probucol is not Only the inhibition of lipid (such as low density lipoprotein LDL) oxidation can also remove free radicals in the blood and tissue, which suggests that the antioxidant effect of probucol is the mechanism that affects the changes in vascular function under oxidative stress, and whether it has protective effects on vascular systolic function in cardiovascular disease.
Vascular endothelium is the main regulator for the maintenance of vascular homeostasis. Known endothelial cells are an important component of vasoconstriction and diastolic activity. Endothelial dysfunction can lead to a balance disorder of vasoconstriction and diastolic function, abnormal blood pressure regulation, and endothelial cell damage can also lead to increased blood cell adhesion and inflammatory cell infiltration. Blood vessels and anticoagulant balance disorders. Clinical trials have confirmed that endothelial dysfunction is one of the risk factors for cardiovascular events. Oxidative stress can promote the development of vascular diseases. Reactive oxygen species (ROS) first destroys endothelial cell function, affects endothelial pressure, anticoagulant and internal components. Secretory function, which may be one of the causes of atherosclerosis, hypertension, diabetes and other vascular diseases.
objective
The mechanism of Probucol's pleiotropic action is still unclear, and the effect of probucol on vasomotor function is still controversial. This experiment is to explore the effect of probucol on vasoconstriction and diastolic function, and further explore the effect of probucol on vasodilatation based on H2O2 induced oxidative damage and the effect of probucol on vasodilatation. Use the mechanism.
Method
A model of endothelium-dependent vasodilatation of H2O2 injury was constructed using the isolated thoracic aorta ring of rats. (1) the systolic and diastolic function of the isolated vascular rings was studied by myograph technique. The vasoconstrictor response was induced by high potassium (KCl), deoxyadrenaline (PE), acetylcholine (ACh), and sodium nitroprusside (SNP) induced vasodilatation. Skin dependence and non endothelium-dependent). Observe the changes of vascular basic function under the continuous action of H2O2. (2) on the basis of H2O2 action, myograph detected the effect of PB intervention on vascular function, and the expression of endothelial specific nitric oxide synthase (eNOS) protein expression after intervention using Western blot technique. To investigate whether the effect of PB on vascular ring diastolic function is achieved by affecting the production of eNOS in endothelial cells. (3) the effect of PB on endothelium dependent vasodilatation under adverse stress, and the changes of vascular ring activity after myograph were detected by vascular ring tissue culture.
The effect of PB on vasodilatation on the basis of vascular injury of atherosclerotic disease, and the correlation between the role of blood lipid metabolism and vascular function change in the experimental study of atherosclerotic apoE-/- mice model was established in vivo.
The primary human umbilical vein endothelial cells (HUVECs) were cultured and the models of vascular endothelial function damaged by H202 were also established in vitro. The following experiments were carried out: (1) the culture and identification of HUVECs; (2) the effect of real-time fluorescence quantitative RT-qPCR and Western blot on the level of eNOS mRNA and protein expression of endothelial cells, and the activation degree of eNOS. (2) (2) the effect of PB on the expression level of eNOS in endothelial cells and whether PB protects endothelial cell function by affecting the expression of eNOS; (3) immunofluorescence microscope observation and fluorescence quantitative detection of NO production in H2O2 induced oxidative damage, and the effect of PB on NO, and to explore whether PB can activate eNOS -NO production pathway can protect endothelial mediated diastolic function.
Result
1.H2O2 can induce oxidative damage in a certain concentration and time of action and selectively inhibit endothelium dependent vasodilatation induced by ACh in the isolated thoracic aorta ring of the rat, which has no obvious effect on the vasoconstrictor function. High concentration or long time H2O2 can cause irreversible loss of vasoconstriction and diastolic function.
2. under the oxidative stress induced by H2O2, PB (5-20 M) protects ACh induced endothelium-dependent vasodilation function in isolated rat thoracic aorta rings.
3.PB inhibited endothelial function induced by H2O2 induced decrease of eNOS activity in isolated vascular rings, but had no significant effect on contractile function.
4. in tissue culture, PB also has protective effects on endothelium-dependent vasodilation of thoracic aorta rings in rats.
5. in the apoE-/- atherosclerosis mouse model, PB can protect the endothelium-dependent vasodilatation function of mice, and this effect has nothing to do with the lipid-lowering effect.
6.PB can increase the p-eNOS/eNOS ratio in human umbilical vein endothelial cells, promote the phosphorylation of eNOS (Ser-1177), activate eNOS oxidation activity, H2O2 to inhibit the expression level of eNOS total protein, PB protects the phosphorylation level of eNOS under the condition of H2O2 induced oxidative damage.
7.PB promotes the production of NO activated by eNOS in human umbilical vein endothelial cells.
conclusion
PB has a protective effect on H2O2 induced endothelium-dependent diastolic dysfunction. The mechanism may be that PB promotes the production of NO by promoting the phosphorylation of eNOS, activating the activity of eNOS oxidase, and thus promoting the formation of NO.
The second part of probucol on the phenotype of smooth muscle cells and its relationship with smooth muscle cell proliferation and migration
background
Vascular smooth muscle cell VSMCs is an important component of vascular wall. The abnormal function of vascular smooth muscle cells is closely related to cardiovascular disease..VSMCs proliferation and synthesis are involved in the formation of atherosclerotic plaques. Smooth muscle cell derived foam cells are one of the cellular components of plaque. Two phenotypes are formed, and pathological changes are associated with synthesis / proliferation. The new view is that the stem cells around the vessel or blood source are the main source of the plaque formation. They have some SMC phenotypic markers, but they are different from the function of ordinary smooth muscle cells and have more characteristics of stem cells. Therefore, this multipotent stem is proposed. Cellular smooth muscle cells are the basis of SMC's involvement in vascular pathological changes. Therefore, the increase of SMC stem cell activity is related to the proliferation or synthesis of SMC in vascular lesions, mainly with the decrease of contractile proteins of highly differentiated smooth muscle cells (including alpha -actin, SM22, calponin and SM-MHC (SM1, SM2)) and the rise of stem cell markers.
Animal experiments have found that probucol can inhibit vascular restenosis after balloon dilatation of rabbit aorta, inhibit the proliferation and migration of smooth muscle cells, inhibit intima thickening, narrowing of the lumen, and improve vascular remodeling. Therefore, we discuss the protective effect of PB on cardiovascular disease from the mechanism and phenotypic changes of SMC to vascular disease. Whether it is related to SMC phenotype and its effect on SMC proliferation and migration.
objective
To explore the effect of probucol on the proliferation and migration of smooth muscle cells and whether it is related to the phenotypic changes of smooth muscle cells.
Method
(1) the vascular smooth muscle cells of the primary rat were extracted and cultured with tissue block wall method, and the primary VSMCs. was detected by immunofluorescence staining to observe the cell morphology and proliferation and differentiation process during the extraction and culture of the primary cells. PB was used to intervene the vascular tissue in order to observe the migration of the cells from the group in situ; and the cells were inoculated in the cells. The effect of direct counting of PB on cell proliferation was studied.
(2) using Transwell cell migration assay to detect the effects of PB and oxLDL on VSMCs migration.
(3) RT-qPCR technology and Western-blot technique were used to study the phenotypic related protein alpha -actin in VSMCs, the expression of Sox-10 in stem cells, and the change of the expression of the transcriptional activator of smooth muscle cells (myocardin).
Results (1) alpha -actin+SM22+Sox-10- is a phenotypic characteristic of highly differentiated VSMCs.
(2) PB alone inhibits the expression of VSMCs stem cell marker Sox-10 and inhibits myocardin, SM22 expression, but does not affect the expression of Sox-10 induced by the expression of contractile protein alpha -actin to induce oxidative stress to promote Sox-10 expression and down regulation of myocardin expression, while PB can partly reverse the change.
(3) PB inhibited the migration of VSMCs from tissues and the migration and proliferation rate in vitro.
conclusion
In vitro studies showed that PB could inhibit the migration and proliferation of VSMCs in rats. The mechanism may be related to the inhibition of PB on the differentiation and motor ability of smooth muscle stem cells.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96

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