雌激素减轻大鼠低氧性肺动脉高压的作用及机制研究

发布时间：2018-06-04 12:09

本文选题：雌激素 + 内源性雌激素　；参考：《第四军医大学》2014年博士论文

【摘要】：研究背景：低氧性肺动脉高压（Hypoxic pulmonary hypertension, HPH）是肺动脉高压（Pulmonary hypertension, PH）中的一个亚型，常见于罹患慢性阻塞性肺疾病与久居高原的人群。HPH以血管壁的增厚与重建为主要特征，其病理生理学基础包括低氧性肺血管收缩（Hypoxic pulmonary vasoconstriction, HPV），肺动脉平滑肌细胞的肥大增殖，以及后期的肺动脉血管的重构（Pulmonary vascular remodeling, PVR）和肺动脉压力升高。肺动脉血管的结构重构和晚期右心室功能衰竭是PH患者病情恶化及导致死亡的主要原因。小动脉壁的肌层增厚与无肌层小动脉肌化是HPH病理学改变的主要特征。研究发现HPH的发病存在有明显的性别差异，高原低氧环境中女性发病率远低于男性，且女性患者临床表现也相对较轻；后来研究发现雌激素在其中发挥了舒张血管、降低肺动脉压力等重要作用。因此，进一步揭示雌激素对HPH的保护作用及机制，寻找新的安全有效的外源性雌激素，将为早期干预和治疗PH提供新的作用靶点及手段。本实验拟从动物整体、器官与细胞水平观察内源性雌激素、外源性雌激素和植物性雌激素对HPH的作用并深入探讨其作用机制。实验目的：从整体、血管和细胞水平三个层次探讨内源性、外源性及植物雌激素对HPH的防治作用，进一步从细胞周期、炎症、氧化应激等方面揭示雌激素在防治HPH作用中的机制：（1）探讨内源性雌激素在HPH发生过程中的作用与机制。（2）研究AKT/Skp2/P27kip1途径在雌激素防治HPH中的作用。（3）探究植物性雌激素在防治HPH中的作用及其作用机制。实验方法：一、观察并探讨内源性雌激素在HPH中的作用及机制我们分别在动物、肺动脉血管环和肺动脉平滑肌细胞水平进行研究。首先将成年雌性、雄性、怀孕及卵巢切除的SD大鼠（Sprague-Dawley rats）随机分为常氧组和低氧组等不同的8个组。常氧组置于自然条件（西安地区大气压力约为718mmHg，pO2为150.6mmHg），低氧组置于低氧舱中减压至380mmHg（相应的pO2减至79.6mmHg，相当于海拔5000米高度的氧分压），连续21天复制HPH的大鼠模型，观察对比各组大鼠的血流动力学指标，如右心室收缩压（Right ventricular systolic pressure,RVSP）、平均肺动脉压（mean pulmonary arterial pressure, mPAP）及肺动脉的病理结构变化等指标。其次，将雄性和不同动情周期的雌性大鼠麻醉后取肺动脉第3级血管（血管外径300μm），将血管条浸置于37℃的Krebs-Henseleit液中并通有混合气（95%O2:5%CO2）条件下60分钟，再更换为低氧混合气（95%N2:5%O2），分别用雌激素受体调节剂雷洛昔芬和MAPK抑制剂U0126（MAPK为雌激素膜受体GPR30的下游通路）进行干预，并用放射性免疫法测定各组大鼠的内源性雌激素的水平，观察内源性雌激素的生理性变化在急性低氧条件下对肺动脉血管收缩的作用。最后，采用取自不同性别和动情周期的大鼠肺动脉，原代培养平滑肌细胞，并于2%低氧条件下培养，分别用雷洛昔芬和U0126进行干预，[3H]-胸腺嘧啶脱氧核苷掺入法测定低氧引起的肺动脉平滑肌细胞增殖，进而观察内源性雌激素水平与低氧肺动脉平滑肌细胞增殖的关系。二、观察外源性雌激素在HPH中的作用及探讨P27kip1是否参与其内在保护机制我们在复制雄性大鼠HPH模型的基础上，同时给予外源性雌激素β-雌二醇干预，观察大鼠HPH相关的血流动力学参数与肺小动脉的病理形态学改变，通过免疫印迹实验、逆转录PCR方法测定了肺组织与肺动脉平滑肌细胞中与细胞周期相关的AKT/Skp2/P27kip1等关键分子，并结合β-雌二醇对低氧导致的肺动脉平滑肌细胞增殖的影响，在整体和细胞水平探讨外源性雌激素是否通过调节细胞周期而发挥对HPH的保护作用。三、观察并初步阐释植物性雌激素白藜芦醇对HPH的保护作用与机制为了寻找防治效果好，副作用小的化合物，，我们进一步观察植物雌激素白藜芦醇对HPH的防治作用。在整体水平，复制雄性大鼠HPH模型，同时给予植物雌激素白藜芦醇（40mg/kg/day，灌胃），同样观察大鼠HPH相关的血流动力学参数与肺小动脉的病理形态学改变，采用免疫组织化学染色方法观察大鼠肺动脉血管组织周围的炎症细胞浸润，Realtime-PCR检测肺组织中IL-6、TNF-α等炎症介质的表达，并采用免疫印迹实验等方法观察与炎症和氧化应激相关的HIF-1α、NF-κB、Nrf-2/Trx-1、H2O2、GSH、SOD等各关键分子的表达变化；在细胞水平低氧培养肺动脉平滑肌细胞的同时，给予不同浓度的植物雌激素白藜芦醇进行干预，同时给予雌激素受体抑制剂ICI182780阻断雌激素受体，采用WST-1实验观察植物雌激素白藜芦醇对低氧肺动脉平滑肌细胞增殖的影响，另外通过使用HIF-1α抑制剂KC7F2抑制HIF-1α，并分别用AKT和ERK抑制剂LY294002和PD98059抑制AKT通路与ERK通路，进一步从炎症和氧化应激两方面揭示植物雌激素白藜芦醇对大鼠HPH和低氧性肺动脉平滑肌增殖的影响及其机制。实验结果： 1）内源性雌激素的生理性变化可以影响大鼠HPH的发生发展，雌激素水平高的雌性低氧组大鼠比雄性低氧组大鼠表现出较轻的RVSP、mPAP等血流动力学指标的升高与肺小动脉壁增厚等表现，其中雌激素水平最高的孕鼠HPH参数变化最轻；而卵巢切除低氧组大鼠的雌激素水平接近于雄性大鼠，其RVSP、mPAP升高幅度及肺小动脉壁增厚等表现也接近于雄性低氧组。离体血管实验显示，与雄性大鼠和动情间期大鼠相比，取自雌激素水平较高的动情前期大鼠的肺动脉血管表现出较轻的HPV反应，MAPK抑制剂U0126增加了HPV反应。细胞水平实验进一步表明来自雌激素水平较高的动情前期大鼠的肺动脉平滑肌细胞表现出较轻的增殖反应，雷洛昔芬与U0126干预在一定程度上增强了低氧导致的肺动脉平滑肌细胞增殖，雷洛昔芬与U0126同时干预时表现出叠加效应，明显增加了低氧导致的肺动脉平滑肌细胞增殖。 2）低氧导致大鼠RVSP和右心室重构指数RVHI的明显升高、肺小动脉血管壁的显著增厚；并且伴随了肺组织中细胞周期抑制因子P27kip1的表达明显减少，以及P27kip1特异性降解酶S期激酶蛋白2（Skp-2）与磷酸化AKT蛋白水平表达的明显升高。外源性雌激素β-雌二醇有效减轻低氧导致的大鼠RVSP和右心室重构指数RVHI的升高，减轻了肺小动脉血管壁的增厚，在减少磷酸化AKT和Skp-2表达的同时升高细胞周期抑制因子P27kip1的表达；细胞实验进一步验证大体实验结果，并显示β-雌二醇能够显著降低低氧诱导的肺动脉平滑肌细胞的增殖。 3）植物性雌激素白藜芦醇明显抑制大鼠HPH的发生发展，减轻低氧导致的右心室压力升高与右心室重构，同时减少低氧引起的HIF-1α、NF-κB在肺组织中的表达增加，减轻了炎症细胞在肺动脉血管周围的浸润和肺组织中H2O2的生成，减少低氧引起的IL-6、TNF-α等炎症因子的mRNA水平的升高，增加了Nrf-2、Trx-1、GSH与SOD等抗氧化应激因子的表达；细胞实验表明白藜芦醇以浓度依赖的方式减少低氧导致的HIF-1α在肺动脉平滑肌细胞中的表达，并且抑制HIF-1α的表达的同时可以有效抑制低氧肺动脉平滑肌细胞增殖，雌激素受体抑制剂ICI182780不能阻断白藜芦醇抑制低氧肺血管平滑肌增殖的作用，白藜芦醇在抑制低氧导致的肺动脉平滑肌细胞增殖的同时也抑制磷酸化AKT和ERK的表达。结论： 1）内源性雌激素在大鼠HPH发生过程中发挥了拮抗性作用。可能通过非基因组作用途径（GPR30受体途径）降低HPV和基因组途径抑制PVR和PASMCs的增殖从而发挥其拮抗HPH的作用。 2）外源性雌激素β-雌二醇能有效改善大鼠HPH，通过调节AKT/Skp2/P27kip1途径抑制肺动脉平滑肌细胞的增殖是其发挥作用的重要机制之一。 3）植物雌激素白藜芦醇能减轻HPH，该作用与其抑制HIF-1α途径和降低炎症反应，以及激活Nrf-2/Trx-1通路，减少氧化应激反应有关，其作用可能为非雌激素受体依赖的方式，但与抑制AKT和ERK通路有关。
[Abstract]:Research background:
Hypoxic pulmonary hypertension (Hypoxic pulmonary hypertension, HPH) is a subtype of pulmonary hypertension (Pulmonary hypertension, PH). It is common in chronic obstructive pulmonary disease and people who live on high altitude..HPH is the main feature of vascular wall thickening and reconstruction, and its pathophysiological basis includes hypoxic pulmonary vasoconstriction (Hypox). IC pulmonary vasoconstriction, HPV), hypertrophy and proliferation of pulmonary artery smooth muscle cells, and later remodeling of pulmonary artery (Pulmonary vascular remodeling, PVR) and pulmonary artery pressure. Structural remodeling of pulmonary artery and late right ventricular failure are the main reasons for the deterioration of the patient's condition and the leading cause of death. Myometrial thickening and myocutaneous arterioles are the pathological changes of HPH.
Main features.
The study found that there was a significant gender difference in the incidence of HPH, the incidence of women in high altitude hypoxia was far lower than that of men, and the clinical manifestations of women were relatively light. Later, the study found that estrogen played an important role in diastolic blood vessels and decreased the pressure of pulmonary artery. Therefore, the protective effect of estrogen on HPH was further revealed. And the mechanism of finding new safe and effective exogenous estrogen will provide new targets and means for early intervention and treatment of PH. This experiment is to observe the role of endogenous estrogen, exogenous estrogen and phytoestrogen to HPH and explore the mechanism of its action from the animal whole, organ and cell level.
The effects of endogenous, exogenous and phytoestrogens on the prevention and control of HPH were discussed from three levels, including the whole, blood vessel and cell level, and the mechanisms of estrogen in the prevention and control of HPH were revealed from cell cycle, inflammation and oxidative stress.
(1) to explore the role and mechanism of endogenous estrogen in the pathogenesis of HPH.
(2) to study the role of AKT/Skp2/P27kip1 pathway in the prevention and treatment of HPH by estrogen.
(3) to explore the role and mechanism of plant estrogen in the prevention and treatment of HPH.
Experimental methods:
First, observe and explore the role and mechanism of endogenous estrogen in HPH.
We studied the level of the animal, pulmonary artery ring and pulmonary artery smooth muscle cells. First, the adult female, male, pregnant and ovariectomized SD rats (Sprague-Dawley rats) were randomly divided into 8 groups: the normal oxygen group and the hypoxic group. The atmospheric pressure in the normal oxygen group (the atmospheric pressure in Xi'an area was about 718mmHg, pO2 was 150.6m MHg), hypoxic group was placed in hypoxic chamber to decompress to 380mmHg (corresponding pO2 to 79.6mmHg, equivalent to oxygen partial pressure at altitude of 5000 m). The rat model of HPH was replicated for 21 days and the hemodynamic indexes of rats in each group were compared, such as right ventricular systolic pressure (Right ventricular systolic pressure, RVSP), mean pulmonary arterial pressure (mean pulmonar). Y arterial pressure, mPAP) and the pathological changes of the pulmonary artery. Secondly, the male and the female rats with different estrus cycles were taken to take the third stage of the pulmonary artery (300 m) of the pulmonary artery, and the vascular strips were immersed in the Krebs-Henseleit solution at 37 degrees C and 60 minutes under the condition of mixed gas (95%O2:5%CO2), and then replaced by the mixture of hypoxic oxygen. Gas (95%N2:5%O2), the estrogen receptor modulator raloxifene and the MAPK inhibitor U0126 (MAPK as the downstream pathway of the estrogen receptor GPR30) were intervened, and the endogenous estrogen levels were measured by radioimmunoassay, and the physiological changes of endogenous estrogen were observed in the pulmonary artery under the acute hypoxia condition. In the end, the rat pulmonary artery from different sex and estrus cycles was used to culture the smooth muscle cells in the primary culture, and cultured in 2% hypoxia conditions, using raloxifene and U0126 respectively. [3H]- thymidine deoxynucleoside incorporation was used to determine the proliferation of pulmonary artery smooth muscle cells induced by hypoxia, and then the endogenous female excitation was observed. Relationship between plasma level and proliferation of pulmonary artery smooth muscle cells in hypoxia.
Two, observe the role of exogenous estrogens in HPH and explore whether P27kip1 participates in its intrinsic protective mechanism.
On the basis of the replication of the HPH model of male rats, we also gave exogenous estrogen beta estradiol intervention to observe the hemodynamic parameters associated with HPH and the pathomorphological changes of the pulmonary arterioles. By immunoblotting experiments, the A related to cell cycle in pulmonary tissue and pulmonary artery smooth muscle cells was measured by reverse transcription PCR. KT/Skp2/P27kip1 and other key molecules, combined with the effect of beta estradiol on the proliferation of pulmonary artery smooth muscle cells induced by hypoxia, explore the protective effect of exogenous estrogen on HPH by regulating the cell cycle at whole and cell levels.
Three, we observed and preliminarily explained the protective effect and mechanism of phytoestrogen resveratrol on HPH.
In order to find a compound with good effect and small side effect, we further observe the effect of phytoestrogen resveratrol on the prevention and control of HPH. At the overall level, we replicate the HPH model of male rats, and give the phytoestrogen resveratrol (40mg/kg/day, gavage), and also observe the hemodynamic parameters related to HPH and the disease of the pulmonary arterioles in rats. Morphological changes, immunohistochemical staining method was used to observe the infiltration of inflammatory cells around the pulmonary artery tissue in rats. Realtime-PCR was used to detect the expression of IL-6, TNF- alpha and other inflammatory mediators in the lung tissue, and the immunoblot test was used to observe the HIF-1 alpha, NF- kappa B, Nrf-2/Trx-1, H2O2, GSH, SOD, and so on. The changes in the expression of the key molecules; at the same time in the cell level hypoxic culture of pulmonary artery smooth muscle cells, different concentrations of phytoestrogen resveratrol were given, and estrogen receptor inhibitor ICI182780 was given to block estrogen receptor. WST-1 experiment was used to observe the effect of resveratrol on hypoxic pulmonary artery smooth muscle. In addition to the effect of cell proliferation, the inhibition of HIF-1 alpha by using HIF-1 alpha inhibitor KC7F2 and the inhibition of AKT pathway and ERK pathway with AKT and ERK inhibitors LY294002 and PD98059 respectively, the effects and mechanisms of phytoestrogen resveratrol on the proliferation of HPH and hypoxic pulmonary artery smooth muscle in rats were further revealed from the two aspects of inflammation and oxidative stress.
Experimental results:
1) the physiological changes of endogenous estrogen could affect the development of HPH in rats. The female rats with high estrogen level showed a lighter RVSP, mPAP and other hemodynamic indexes, and the thickening of the pulmonary artery wall in the rats with the high level of estrogen, while the HPH parameters of the female rats with the highest efemale level were the lightest. The estrogen level of the ovariectomized hypoxic rats was close to that of the male rats. The elevation of RVSP, mPAP and the thickening of the pulmonary artery wall were also close to the male hypoxia group. In vitro, the blood vessel test showed that the pulmonary arteries of the higher estrus rats were compared with the male rats and the estrous interval rats. The light HPV reaction and the MAPK inhibitor U0126 increased the HPV reaction. The cell level experiment further showed that the pulmonary artery smooth muscle cells from the estrous proestrus rats showed a lighter proliferation response. The intervention of raloxifene and U0126 enhanced the proliferation of pulmonary artery smooth muscle cells induced by hypoxia to some extent, and Ralo. When Xifen and U0126 intervened at the same time, they showed a superposition effect, which significantly increased the proliferation of pulmonary artery smooth muscle cells induced by hypoxia.
2) hypoxia leads to a significant increase in RVSP and right ventricular remodeling index RVHI, significantly thickening of the vascular wall of the pulmonary arterioles, and a significant decrease in the expression of the cell cycle inhibitory factor P27kip1 in the lung tissue, as well as the apparent increase in the expression of the P27kip1 specific degrading enzyme S kinase protein 2 (Skp-2) and the phosphorylated AKT protein. Sexual estrogen beta estradiol effectively alleviates the increase of RVSP and right ventricular remodeling index RVHI in rats induced by hypoxia, reduces the thickening of the vascular wall of the pulmonary arterioles, increases the expression of the phosphorylated AKT and Skp-2, and increases the expression of the cell cycle inhibitory factor P27kip1; the cell experiment validates the general experimental results and shows the beta estradiol. It can significantly reduce the proliferation of pulmonary artery smooth muscle cells induced by hypoxia.
3) phytoestrogen resveratrol obviously inhibits the development of HPH in rats, reduces hypoxic induced right ventricular pressure and right ventricular remodeling, reduces HIF-1 a caused by hypoxia, increases the expression of NF- kappa B in lung tissue, reduces the infiltration of pulmonary artery circumference in the pulmonary artery and the formation of H2O2 in the lung tissue, and reduces hypoxia. The increase of mRNA levels of inflammatory factors, such as IL-6, TNF- a, increases the expression of antioxidant stress factors such as Nrf-2, Trx-1, GSH and SOD, and the cell test shows that veratrol reduces the expression of HIF-1 a induced by hypoxia in the pulmonary artery smooth muscle cells in a concentration dependent manner, and inhibits the expression of HIF-1 alpha effectively. The proliferation of hypoxic pulmonary artery smooth muscle cells and the inhibitory effect of resveratrol inhibitor ICI182780 on the proliferation of hypoxic pulmonary vascular smooth muscle can not be blocked by resveratrol inhibitor. Resveratrol inhibits the proliferation of pulmonary artery smooth muscle cells induced by hypoxia and inhibits the expression of phosphorylated AKT and ERK.
Conclusion:
1) endogenous estrogen plays an antagonistic role in the development of rat HPH. It may reduce the proliferation of PVR and PASMCs by reducing the HPV and genomic pathways through the non genomic pathway (GPR30 receptor pathway) and thus exerts its antagonistic effect on HPH.
2) exogenous estrogen beta estradiol can effectively improve the HPH of rat, and it is one of the important mechanisms of inhibiting the proliferation of pulmonary artery smooth muscle cells by regulating the AKT/Skp2/P27kip1 pathway.
3) phytoestrogen resveratrol alleviates HPH, which is related to the inhibition of HIF-1 alpha pathway and the reduction of inflammatory response, as well as the activation of the Nrf-2/Trx-1 pathway, and the reduction of oxidative stress. The effect may be a non estrogen receptor dependent manner, but it is related to the inhibition of the AKT and ERK pathways.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R544.1

【引证文献】