【作者】 张丽君；

【导师】 裴建明； 李娟；

【作者基本信息】 第四军医大学 ， 生理学， 2013， 博士

【摘要】 背景：低氧性肺动脉高压（hypoxic pulmonary hypertension，HPH）是由于各种呼吸系统疾病引起的低氧血症导致的肺动脉高压。慢性阻塞性肺病（chronic obstructivepulmonary disease，COPD）以及长期处于高原环境均可导致HPH的发生。低氧引起的肺动脉收缩（hypoxic pulmonary vasoconstriction，HPV）和肺血管增殖重构（pulmonary artery remodeling，PAR）是HPH的两个重要病理生理环节。首先，低氧引起的肺动脉收缩本是机体的一种代偿反应，可促进肺泡的血流从低氧区流向高氧区，改善肺的通气/血流比值。然而，长期的低氧环境可引起肺动脉的持续不可逆收缩，导致肺动脉高压的形成。其次，低氧早期可伴有舒张因子（一氧化氮、前列腺素等）和收缩因子（内皮素、血管紧张素Ⅱ等）的失衡，引起内皮功能的失调，并逐渐引起肺动脉平滑肌细胞的增殖与肺动脉血管的重构，最终导致肺动脉高压和右心室肥厚。因此，舒张肺动脉、预防内皮功能失调和抑制平滑肌细胞的增殖是防治HPH的潜在策略。研究发现，内源性阿片肽（endogenous opioid peptide，EOP）及其受体在心肺血管系统发挥了重要的调节作用，κ-阿片受体（Kappa-opioid-receptor，κ-OR）是其中之一。本室前期研究发现，心肌缺血/再灌注后给予κ-阿片受体激动剂U50,488H可对抗心律失常的发生，减少心肌细胞坏死与凋亡，抑制再灌注后的炎症反应，对心肌发挥直接的保护作用，其机制与κ-OR介导激活PI3K-Akt-eNOS信号通路、增加NO生成有关。Akt-eNOS信号通路障碍是内皮功能失调的重要机制之一，然而，在HPH时，κ-OR能否通过介导激活Akt-eNOS信号通路，恢复肺动脉内皮功能，目前尚不清楚。另外，HPV和PAR是肺动脉高压形成的基本环节，前期研究发现，U50,488H对肺动脉具有明确的血管舒张效应，对肺动脉平滑肌细胞的增殖具有明确的抑制作用，但是这些作用在HPH是否存在？是否由κ-OR所介导也不清楚。本研究拟采用大鼠HPH模型，研究激活κ-OR能否对抗HPH，并进一步从改善内皮功能失调、舒张肺动脉血管和抑制肺动脉血管增殖等方面阐明κ-OR在抗HPH中的作用及可能机制。研究结果可为HPH的预防和治疗提供新的受体靶点，也为临床应用阿片类物质预防和治疗HPH提供实验依据，因而具有一定的理论和临床应用意义。目的：1.研究激活κ-OR在抗HPH中的作用。2.探讨激活κ-OR对HPH大鼠肺血管的舒张效应。3.探讨激活κ-OR对低氧引起的肺动脉平滑肌细胞增殖的影响4.探讨激活κ-OR对HPH大鼠内皮功能失调的影响方法：1.模拟海拔5000～5500米气压环境（大气压50kPa,氧浓度约10%），通过低压低氧动物仓，构建HPH动物模型。在整体动物上观察κ-OR激动剂U50,488H与κ-OR阻断剂nor-BNI对HPH形成的影响。2.自大鼠右侧颈外静脉插入直径为1mm的聚乙烯塑料导管，沿上腔静脉进入右心房、再通过三尖瓣口进入右心室，最后到达肺动脉干。运用RM-6280多道智能生理信号采集和记录系统，测量大鼠平均肺动脉压（mPAP）和右心室压力（RVP）等肺血流动力学指标，并通过该系统分析以上指标。3.剖胸取出大鼠心脏，沿室间隔边缘分离出右心室（RV）和左心室以及室间隔（LV＋S），称重计算RV、LV+S，以RV/（LV+S）比值和RV/BW（体重）比值来反映右心室肥厚程度。Western blot法检测肺动脉上κ-OR的蛋白表达情况。ELISA试剂盒检测内源性的κ-OR激动剂强啡肽（DynorphinA）的变化。4.分离HPH大鼠肺动脉，采用离体血管灌流技术，观察κ-OR激动剂U50,488H与内源性的κ-OR激动剂强啡肽（Dynorphin A）对HPH大鼠肺动脉血管环的舒张作用。5.用3H-TdR掺入实验和细胞MTT活性检测，观察κ-OR激动剂U50,488H对低氧下培养的肺动脉平滑肌细胞（PASMCs）增殖的影响。6.采用离体血管灌流技术检测HPH大鼠肺动脉对内皮依赖性舒血管物质ACh的反应及U50,488H腹腔注射对该反应的影响，7.用硝酸还原酶法检测血清NO水平的变化，并观察κ-OR阻断剂nor-BNI、PI3K抑制剂wortmannin、选择性Akt抑制剂AI以及非选择性NOS抑制剂L-NAME对U50,488H刺激NO生成作用的影响。8.建立肺微血管内皮细胞（PMVECs）的原代和传代培养。采用TUNEL法检测U50,488H对低氧时PMVECs凋亡情况的影响，同时应用Western blot法检测低氧环境下给予U50,488H后PMVECs中Akt和eNOS的磷酸化水平。结果：1.大鼠慢性低氧2W后形成稳定的HPH。血流动力学指标mPAP和RVP明显升高。隔天、低氧前10分钟腹腔注射κ-OR激动剂U50,488H（1.5mg/kg）、连续2周，可显著降低低氧两周大鼠的mPAP、RVP以及右心室肥厚指标（RV/（LV+S））和RV/BW，这些效应可被κ-OR阻断剂nor-BNI所阻断。2.慢性低氧可以上调肺动脉上κ-OR的表达，U50,488H可以进一步上调慢性低氧时肺动脉上κ-OR的表达，该作用可被κ-OR阻断剂nor-BNI所阻断。3.低氧1W和低氧2W均可上调大鼠血清中内源性κ-OR激动剂强啡肽（DynorphinA）的水平，而低氧4W后强啡肽（Dynorphin A）的水平回复至基线水平以下。4. U50,488H可呈浓度依赖性地舒张HPH大鼠的离体肺动脉，该作用可以被κ-OR阻断剂nor-BNI和非选择性NOS抑制剂L-NAME所阻断。强啡肽（DynorphinA）对HPH大鼠离体肺动脉亦具有显著的舒张作用，该作用可被κ-OR阻断剂nor-BNI所完全阻断。5. PASMCs在低氧条件下3H-TdR掺入量和MTT的OD值明显升高，而U50,488H可以明显降低低氧条件下培养的大鼠PASMCs的3H-TdR掺入量和MTT的OD值，该效应呈明显的浓度依赖性（10~100μmol/L），且该作用可被κ-OR阻断剂nor-BNI所阻断。6. U50,488H可改善HPH大鼠对内皮依赖性血管舒张剂乙酰胆碱（ACh）的舒张效应。7. U50,488H可明显增加慢性低氧大鼠血清中NO的生成，该作用可被κ-OR阻断剂nor-BNI、PI3K抑制剂wortmannin、选择性Akt抑制剂AI以及非选择性NOS抑制剂L-NAME所阻断。8. U50,488H具有抗内皮细胞凋亡的作用，该作用可被κ-OR阻断剂nor-BNI、PI3K抑制剂wortmannin、选择性Akt抑制剂AI以及非选择性NOS抑制剂L-NAME所阻断。9. U50,488H可明显增加PMVECs中Akt和eNOS的磷酸化水平，该效应可被κ-OR阻断剂nor-BNI和PI3K抑制剂wortmannin所阻断。结论：1.激活κ-OR具有抗HPH的作用，低氧早期可刺激大鼠内源性阿片肽Dynorphin的释放，同时上调κ-OR的表达，这可能是机体的一种代偿反应，但尚需进一步确认。2. κ-OR介导的抗HPH的作用机制可能涉及舒张大鼠肺动脉、改善HPH大鼠肺动脉内皮细胞功能以及抑制低氧诱导的PASMCs过度增殖等。3.外源性给予U50,488H可进一步激活κ-OR，其一方面通过NOS途径发挥对HPH大鼠肺血管的舒张效应；另一方面，通过PI3K-Akt-eNOS信号通路改善HPH大鼠肺动脉内皮细胞功能。更多还原

【Abstract】 Background:Hypoxic pulmonary hypertension (HPH) is a progressive disorder characterized byabnormally high blood pressure in the pulmonary artery caused by hypoxemia after allkinds of respiratory system diseases. Both chronic obstructive pulmonary disease andliving in the plateau for a long time can lead to HPH. Hypoxia-induced pulmonaryvasoconstriction and pulmonary vascular remodeling are two key pathophysiologicprocesses in HPH. First, hypoxia-induced pulmonary vasoconstriction is a compensatorymechanism, redirecting blood flow from alveoli with a lower oxygen content to alveoliwith a higher oxygen content and improving ventilation/perfusion ratio. However,long-term hypoxia can result in irreversible vasoconstriction in the pulmonary artery andfurther lead to HPH. Then, hypoxia upsets the balance between the vasoconstrictor (ET-1, Ang Ⅱ) and vasodilator (NO, PGI), triggering endothelial dysfunction and graduallyleading to vascular remodeling characterized by proliferation of pulmonary artery smoothmuscle cells. In the end, HPH and right ventricular hypertrophy come into being.Therefore, dilating pulmonary artery, preventing endothelial dysfunction and inhibitingsmooth muscle cell proliferation are potential strategies for the prevention and treatmentof HPH.It has been demonstrated that endogenous opioid peptide and its receptor playsignificant role in cardiovascular system, of which the main subtype is κ opioid receptor(κ-OR). Our previous study demonstrates that selective κ-OR agonist U50,488Hadministered after ischemia/reperfusion exhibits an anti-arrhythmic effect, decreasescardiomyocyte necrosis and apoptosis and inhibits inflammation after reperfusion. Theprotective effects of κ-OR are associated with the activation of PI3K-Akt-eNOS pathwaymediated by κ-OR, which increases NO synthesis. The impairment of Akt-eNOS pathwayis one of the important mechanisms of endothelial dysfunction. However, it remainsunclear whether κ-OR ameliorates endothelial function through the activation ofAkt-eNOS pathway. Moreover, hypoxia-induced pulmonary vasoconstriction andpulmonary vascular proliferation are two key processes in HPH. Our previous studydemonstrates that U50,488H has a definite dilating effect on pulmonary artery and adefinite inhibitive effect on pulmonary artery smooth muscle cell proliferation. However,whether those effects exist in HPH and whether those are mediated by κ-OR remainobscure.By utilizing rat HPH model, this study aims to determine whether κ-OR activationantagonizes HPH and further improves endothelial function and pulmonary artery dilation,and inhibits pulmonary artery smooth muscle proliferation and to elucidate the role ofκ-OR in antagonizing HPH and its underlying mechanism. The results may provide newtherapeutic target for the prevention and treatment of HPH and provide experimentalevidence for the clinical application of opioid in the prevention and treatment of HPH,which reveals significant theoretical and clinical implication. Aims:1. To investigate the effect of κ-OR activation on antagonizing HPH.2. To investigate the effect of κ-OR activation on dilating pulmonary artery in HPH rats.3. To clarify the effect of κ-OR activation on the pulmonary artery smooth muscle cellproliferation induced by hypoxia.4. To investigate the effect of κ-OR activation on endothelial dysfunction in HPH rats.Methods:1. Hypoxic condition was created for8hours every day with the exposure of the rats toboth low pressure and low oxygen (air pressure50kpa, oxygen concentration10%), sothe HPH rat model was established. The effects of κ-OR agonist U50,488H and κ-ORantagonist nor-BNI were studied in HPH rats in vivo.2. After hypoxia, rats were anesthetized via peritoneal injection with pentobarbitalsodium (60mg/kg, IP injected). A micro-catheter (diameter=1mm) was inserted intothe pulmonary artery through the right external jugular vein. Then the meanpulmonary arterial pressure (mPAP) and right ventricular pressure (RVP) weremeasured.3. The hearts and blood were then harvested. Each of the following was isolated in orderto calculate the right ventricular hypertrophy index (RVHI): body weight (BW), rightventricle (RV), left ventricle (LV), and septum (S). The RVHI itself was expressed asthe tissue weight ratio of RV/(LV+S) and RV/BW. Expression of κ-OR was determinedby Western-Blot, and concentration of endogenous dynorphin A was determined byELISA.4. The pulmonary artery was carefully isolated and cleaned of fat and connective tissue.In vitro vascular ring perfusion was utilized. U50,488H and dynorphin A were addedrespectively to determine its relaxation effect on the artery rings in HPH rats.5. The effect of U50,488H on the proliferation of pulmonary arterial smooth muscle cells (PASMCs) under hypoxic condition was measured by MTT and [3H]-thymidine(3H-TdR) incorporation assay.6. Isolated perfusion of pulmonary artery ring was used to determine the reaction of theartery ring to ACh, which is an endothelium-dependent vasodilator. Besides, the effectof U50,488H i.p. administration on the reaction of the artery ring to ACh.7. The serum NO was determined by measuring the concentration of nitrite, a stablemetabolite of nitric oxide, through a modified Griess reaction method. The underlyingmechanism was investigated by several inhibitors such as nor-BNI (a selective κ-ORantagonist), wortmannin (a selective PI3K antagonist), AI (a selective PI3Kantagonist), and L-NAME (a non-selective NOS inhibitor).8. The primary culture and subculture of pulmonary microvascular endothelial cells(PMVECs) was performed. The effect of U50,488H on PMVECs apoptosis induced byhypoxia was determined by TUNEL staining. The phosphorylation of Akt and eNOSin the PMVECs was detected by Western-Blot after U50,488H administration inhypoxia.Results:1. The HPH was established after exposing rats to chronic hypoxia for2weeks. ThemPAP and RVP of rats in hypoxia were significantly higher compared with both themPAP and RVP of normoxic rats. Compared with the hypoxia2w group, the hypoxia2w+U50,488H group showed a significant decrease in mPAP, RVP, RV/(LV+S) andRV/BW, and the effect of U50,488H was abolished by nor-BNI.2. Compared with the normoxia group, the expression of κ-OR in pulmonary artery wasincreased in the hypoxia group. U50,488H further up-regulated the expression of κ-ORin the hypoxia groups, which was abolished by nor-BNI.3. The level of dynorphin A was increased at1wk and2wks after hypoxia, whereas itreturned to below baseline level at4wks exposure to hypoxia.4. U50,488H exhibited both time-dependent and dose-dependent relaxation effect on HPH rat pulmonary artery rings. This vasorelaxing effect was abolished by nor-BNIand L-NAME. Dynorphin A also showed time-dependent relaxation effect and theeffect was completely abolished by nor-BNI.5. When PASMCs were exposed to hypoxia, the quantity of3H-TdR incorporation andOD optimum in MTT detection increased significantly compared to the normoxicgroup. U50,488H significantly inhibited the PASMCs proliferation in a dose dependentmanner (10~100μmol/L), which was also abolished by nor-BNI.6. Chronic hypoxia resulted in a significant endothelial dysfunction. This dysfunctionwas demonstrated through decreased vasorelaxation in response to ACh. U50,488Hadministration significantly improved the pulmonary artery relaxation response toACh.7. Hypoxia resulted in a significant decrease in serum NO compared with that in thenormoxic group. U50,488H pretreatment significantly restored serum NO content inHPH rats, which was abolished by nor-BNI, wortmannin (a PI3K inhibitor), AI (aselective Akt inhibitor) and L-NAME (a non-selective NOS inhibitor).8. U50,488H antagonized endothelial cell apoptosis, which was abolished by nor-BNI,wortmannin (a PI3K inhibitor), AI (a selective Akt inhibitor) and L-NAME (anon-selective NOS inhibitor).9. In the cultured PMVECs that were exposed to hypoxia, U50,488H treatment increasedAkt phosphorylation and endothelial nitric oxide synthase (eNOS) phosphorylationsignificantly, which were abolished by both nor-BNI and wortmannin.Conclusions:1. κ-opioid receptor activation exhibits definite effect against HPH. Hypoxia at an earlystage stimulates the release of dynorphin and upregulates κ-OR expression, which maybe a compensatory reaction but needs further investigation.2. The mechanism underlying the effect of antagonizing HPH mediated by κ-ORinvolves dilating pulmonary artery in HPH rats, improving pulmonary artery endothelial dysfunction in HPH rats and inhibiting excessive proliferation of PASMCsinduced by hypoxia, etc.3. U50,488H administration further activates κ-OR. For one thing, U50,488H dilatespulmonary artery through NOS pathway. For another, U50,488H improves endothelialdysfunction through PI3K-Akt-eNOS pathway.更多还原