【作者】 吴西玲；

【导师】 杜立中；

【作者基本信息】 浙江大学 ， 儿科学， 2009， 博士

【摘要】 新生儿持续性肺动脉高压（Persistent Pulmonary Hypertension of the Newborn,PPHN）是新生儿危重症之一,缺氧是PPHN发生和病情恶化的重要危险因素之一。目前调节血管张力的三条主要途径为:一氧化氮（nitric oxide,NO）/环鸟甘单磷酸（cyclic guanosine monophosphate,cGMP）通路、前列环素/环腺甘单磷酸（cyclicadenosine monophosphate,cAMP）通路和内皮素通路,尤其是NO/cGMP途径是最近PPHN研究的一个热点。作为一种重要的内皮源性舒张因子,它除了激活鸟苷酸环化酶生成第二信使cGMP发挥生物学作用外,现在研究较多的是NO可通过蛋白质巯基硝基化来执行其生理功能。蛋白质巯基硝基化是一种氧化还原依赖的蛋白质翻译后的可逆修饰。许多含有巯基的蛋白可以通过硝基化和去硝基化调节蛋白的生物学活性,从而调节细胞信号的传递。亚硝基硫醇（nitrosothiols,RSNO）是含有硫醇的蛋白和小分子物质硝基化的产物,是体内NO的主要贮存体和载体。亚硝基硫醇浓度的变化和一系列生理病理的改变有关。最近发现乙醇脱氢酶Ⅲ,能特异性分解亚硝基谷胱甘肽（nitrosoglutathione,GSNO）,是目前所知的唯一一种能分解GSNO而不伴随NO释放的蛋白,故又把它命名为亚硝基谷胱甘肽还原酶（GSNO reductase,GSNOR）。GSNO是该蛋白的最佳底物。由于GSNO和其他亚硝基硫醇通过转硝基反应处于动态平衡中,所以GSNOR可以直接控制GSNO的水平,间接控制RSNO水平。三类一氧化氮合酶（nitric oxidesynthase,NOS）是NO和RSNO的主要来源。最近提出一种假说:内源性NO/GSNO/SNO的平衡受NOS-GSNOR双向调控。低氧性肺动脉高压与体内反应性氧化物过度增加、氧化还原平衡破坏和NO/RSNO的缺乏有关。烟酰胺腺嘌呤二核苷酸磷酸氧化酶（NADPH oxidase,NOX）尤其是NOX2,4是血管内生成活性氧簇(（reactive oxygen species,ROS）的最主要酶体,在氧化还原信号的传递中起重要的调节作用。硫氢化钠（NaHS）可以补充体内的硫化氢（hydrogen sulfide,H₂S）舒张血管,同时也是一种重要的抗氧化剂,干预后可以改变机体的氧化还原状态。RSNO的分解异常与肺动脉高压的研究报道很少,其机制尚不清楚。本课题以野生型和eNOS基因敲除的C57BL/6J小鼠作为研究对象,建立常压低氧的慢性肺动脉高压模型,探讨GSNOR在低氧性肺动脉高压中的变化,同时观察NOX2,4及ROS变化,以及NaHS干预等手段,对其调控机制进行初步的探讨,为肺动脉高压的治疗探索一种新的途径。第一部分低氧性肺动脉高压与亚硝基谷胱甘肽还原酶的表达目的:1.在低氧性肺高压形成过程中,GSNOR在mRNA和蛋白水平表达的变化及相应的酶活性变化。2.GSNOR的表达变化和内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS）,诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）之间的关系。3.通过缺氧过程中氧化还原指标还原型谷胱甘肽和氧化型谷胱甘肽的比值（glutathione/glutathione disulfide,GSH/GSSG）和肺组织NO含量的变化,探讨体内氧化还原平衡与肺动脉高压的关系。方法:1.模型建立和分组缺氧组:将C57BL/6J小鼠置于密封有机玻璃箱中,调整氧气和氮气的比例,使箱内氧浓度维持在10%左右。对照组:吸入FiO₂为0.21（即空气）,具体方法及实验控制因素同缺氧组。2.评价模型是否成功观察随缺氧时间的延长,外周血pH值和红细胞压积的变化。测量缺氧21天后右心室收缩压的变化,并用α-SMA免疫组化染色观察肺血管的肌化程度,测量右心室与左心室加室间隔的比值[ratio of right ventricle to left ventricle plus septum,RV/（LV+S）]评价右心室肥厚情况。3.荧光定量RT-PCR检测对照组和缺氧1,3,7,14,21天小鼠肺组织GSNOR,eNOS和iNOS在mRNA水平的变化。免疫组化染色观察GSNOR,eNOS和iNOS蛋白在小鼠肺组织内的定位情况。Western blot检测对照组和缺氧1,3,7,14,21天小鼠肺组织GSNOR和eNOS蛋白水平。用生物化学方法,检测肺组织GSNOR活性的变化,GSH/GSSG比值及NO含量的变化。结果:1.缺氧21天后右心室收缩压显著上升（对照组:21.41±1.92 mmHg;缺氧组:32.64±3.56mmHg,P＜0.01）。随着缺氧时间的延长,血pH值下降（P＜0.01）,红细胞压积逐渐上升（P＜0.01）。小动脉的血管壁增厚,非肌型血管出现了不同程度的肌化。RV/（LV+S）的比值显著增加（P＜0.01）。2.GSNOR变化:GSNOR mRNA在缺氧第1天下降（P＜0.05）,缺氧第3天和常氧组无显著差异,缺氧第7天其表达水平为对照组的1.36倍（P＜0.01）,随后逐渐下降。GSNOR蛋白在缺氧第7天显著上升（P＜0.05）,14天达高峰,随后下降。GSNOR酶活性在缺氧7天后显著增加（P＜0.01）。3.NOS变化:eNOS基因在缺氧3天后表达显著增加（P＜0.01）,14天达高峰,随后下降。eNOS蛋白在缺氧3天后表达显著上升。iNOS基因表达的变化和内皮型一氧化氮合酶基因表达变化相一致。4.GSH/GSSG比值及NO含量的变化:缺氧后肺组织GSH/GSSG比值和NO含量均较基础状态出现了显著的下降（分别为P＜0.05和P＜0.01）结论:1.缺氧诱导了GSNOR酶的活性持续升高,是缺氧后肺组织亚硝基硫醇缺乏的原因之一。2.缺氧后eNOS和iNOS的表达增加,而肺组织硝酸根和亚硝酸根浓度下降提示缺氧会导致体内NO缺乏。3.氧化还原平衡破坏参与了低氧性肺动脉高压的发病。第二部分低氧性肺动脉高压时亚硝基谷胱甘肽还原酶表达变化及调控机制研究目的:1.通过对eNOS基因敲除小鼠和野生型小鼠缺氧后及硫氢化钠（NaHS）干预后肺小动脉的重塑和右心室肥厚情况观察,探讨eNOS在肺小动脉的重塑中的作用。2.比较eNOS基因敲除小鼠和野生型小鼠缺氧及干预后GSNOR,iNOS,NOX2,NOX4在mRNA水平的差异。3.通过对eNOS基因敲除小鼠和野生型小鼠缺氧后及干预后肺组织反应性氧化物的变化,进一步证明氧化还原平衡与低氧性肺动脉高压的关系。方法:1.模型建立和分组常氧组:同实验第一部分。缺氧组:将野生型和eNOS基因敲除的C57BL/6J小鼠置于密封有机玻璃箱中,通入氧气和氮气,使箱内氧浓度维持在10%左右。干预组:低氧暴露方法同缺氧组,在缺氧前将硫氢化钠（NaHS）用生理盐水稀释后,以14μmol/kg的剂量腹腔注射,随后一天一次,持续21天。对照组:低氧暴露方法同缺氧组,缺氧前用和干预组同剂量的生理盐水腹腔注射,随后一天一次,持续21天。2.实验指标检测比较野生型和eNOS基因敲除小鼠缺氧及NaHS干预21天后,肺小动脉重塑（观察media wall thickness,MT%）及右心室肥厚的变化。用RT-PCR方法检测GSNOR,eNOS,iNOS,NOX2和NOX4在mRNA水平的变化,用生物化学法检测肺组织反应性氧化物浓度的变化。结果:1.野生型小鼠缺氧21天后,肺组织NOX2,4mRNA水平显著上升（均为P＜0.01）,ROS的浓度显著下降（P＜0.01）。GSNOR的表达在缺氧第7天显著上升（P＜0.05）,随后下降。2.eNOS基因敲除小鼠缺氧21天后NOX2 mRNA的表达显著下降（P＜0.01）而NOX4 mRNA无明显变化。肺组织ROS的浓度无明显变化。iNOS的表达显著上升（P＜0.01）,GSNOR的表达显著下降（P＜0.05）。3.野生型小鼠NaHS干预21天后,MT%和RV/（LV+S）显著下降（分别为P＜0.05,P＜0.01）。野生型小鼠NaHS干预7天后,eNOS和iNOS的表达和缺氧7天组比较显著下降（均为P＜0.01）。干预21天后NOX2和NOX4的表达显著下降（均为P＜0.01）,肺组织ROS的浓度和缺氧组比较略有上升,但无统计学差异。GSNOR的表达也出现了显著下降（P＜0.05）。4.eNOS基因敲除小鼠NaHS干预21天后RV/（LV+S）显著下降（P＜0.05）而MT%变化不明显。NaHS干预21天后,NOX2,4和iNOS mRNA的表达和缺氧21天组比较无差异。肺组织ROS的浓度显著上升（P＜0.01）,GSNOR的表达显著增加（P＜0.01）。结论:1.通过缺氧21天,野生型和eNOS基因敲除小鼠均能成功建立肺动脉高压模型。两者对缺氧的反应程度一致,说明eNOS缺乏后会通过其他途径发生代偿。2.eNOS基因敲除的小鼠缺氧后NOX2和GSNOR的表达下降,NOX4无明显变化,提示eNOS对NOX和GSNOR的表达有诱导作用。3.NaHS对野生型小鼠肺血管重塑的逆转作用比eNOS基因敲除的小鼠明显,说明eNOS在NaHS舒张血管中起到了重要的作用。NaHS干预21天后,两种小鼠肺组织ROS上升,GSNOR的异常表达被部分纠正。GSNOR的过度表达与氧化还原失衡有关。更多还原

【Abstract】 Persistent pulmonary hypertension of the newborn（PPHN） is one of the critical diseases.Hypoxia is an important factor contributing to the formation and deterioration of PPHN.The disorders in the nitric oxide（NO）-cyclic guanosine monophosphate （cGMP）,prostacyclin-cyclic adenosine monophosphate（cAMP） and endothelin signaling pathways play an important role in the vascular abnormalities associated with PPHN.The NO-cGMP pathway has been a topic of particularly intense investigation over the past decade.Nitric oxide（NO） is an important endothelial relaxing factor.Recent studies have paid more attention to the nitrosylation of the proteins in the regulation of nitric oxide homeostasis,which is a cGMP-undependent signaling pathway.Nitrosylation of cysteine groups in proteins has now become established as the redox-based post-translational modification.Nitrosylation and denitrosylation can control the bioactivity of many proteins with cysteine groups.It can further change the signaling pathways and the function of some proteins.Nitrosothiols（RSNO） are the product of nitrosylation of protein and low molecular thiols and represent a means either for the storage or transport of NO.The change in nitrosothiols concentration is associated with a series of pathophysiology processes. Recent study has shown that classⅢalcohol dehydrogenase,also known as glutathione-dependent formaldehyde could selectively degrade nitrosoglutathione （GSNO）.The end product is ammonia not NO.It is also known as GSNO reductase （GSNOR）.GSNO is equilibrated with other RSNO through transnitrosation,so GSNOR can regulate the concentration of RSNO in vivo indirectly.Three kinds of nitric oxide synthesis take part in the synthesis of NO or RSNO.It is suggested that the concentration of NO/GSNO/RSNO is under the double gate control of nitric oxide synthase（NOS）-GSNOR.Hypoxic pulmonary hypertension was associated with reactive oxygen species overproduction,redox imbalance and NO/RSNO deficiency. NADPH oxidase,especially for NOX2 and NOX4,was the main sourece of reactive oxygen species（ROS） in pulmonary vessels and important for redox signalling.NaHS injection could supplement the endogenous hydrogen sulfide（H₂S） and relaxed blood vessels.H₂S was proved to be an important antioxidant and could change the redox state in vivo.However,the role of GSNOR in regulating pulmonary vascular tone and remodeling has not yet been clearly addressed in hypoxic pulmonary hypertension.In this study,we select C57BL/6J wild-type（WT） and endothelial nitric oxide synthase （eNOS） knockout（KO） mice as study subjects to investegate the change of GSNOR in chronic hypoxic pulmonary hypertension.We also observed the change of NOX2,4 and ROS after NaHS administration to explore the regulatory mechanism of GSNOR in chronic hypoxia.The aim of this study was to explore a possible new therapy pathway for the treatment of neonatal pulmonary hypertension.PartⅠThe expression of GSNOR in hypoxic pulmonary hypertensionAim:1.To investigate the changes of GSNOR on mRNA and protein levels,and GSNOR enzymatic activities during the development of hypoxic pulmonary hypertension.2.To analyze the interactions among GSNOR,eNOS and inducible nitric oxide synthase（iNOS） in hypoxic pulmonary hypertension.3.To investigate the role of redox equilibration in the pathogenesis of hypoxic pulmonary hypertension by evaluating glutathione（GSH）/glutathione disulfide（GSSG） and nitrite/nitrate concentration.Methods:1.Pulmonary hypertension model and groupsHypoxic group:C57BL/6 mice were exposed to 10%oxygen in a forced-air environmental chamber for 21 days.Normoxic group:mice were exposed to 21%oxygen（room air） for 21 days.2.Assessment of pulmonary hypertensionThe right ventricular systolic pressure（RVSP） was detected in mice after 21 days’ hypoxic exposure.The change of blood pH and hematocrit was analyzed at different time point of hypoxic exposure.The degree of muscularization of pulmonary vessels was assessed by immunohistochemistry staining ofα-SMA.The ratio of the right ventricle（RV） to the left ventricle plus septum（LV+S） was used to evaluate the hypertrophy of right ventricle.3.Real-time PCR was used to analyze the GSNOR,eNOS and iNOSmRNA expression in mice lungs.Immunohistochemistry was used for the localization and expression of these proteins.Western blot analysis were used for the evaluation of GSNOR and eNOS protein levels in mice lungs.Biochemistry methods was used to assay the GSNOR enzymatic activity and the concentration of nitrite/nitrit in mice lungs.Results:1.RVSP increased significantly in mice after 21 days’ hypoxic exposure than that of normoxic group（N:21.41±1.92mmHg;H21:32.64±3.56mmHg,P＜0.01）.Blood pH and hematocrit decreased significantly with the duration of exposure to hypoxia （P＜0.01,respectively）.The number of muscular pulmonary vessels increased significantly in mice following 21 days’ hypoxic exposure than in normoxic group.The ratio of RV/（LV+S） was also significantly increased after hypoxic exposure（P＜0.01）.2.The change of GSNOR:The mRNA expression of GSNOR decreased after exposure to hypoxia for 1 day（P＜0.05）,but increased significantly as high as 1.36-fold on day 7 compared to normoxic group（P＜0.01）.The protein expression of GSNOR and its bioactivities in the lung were also increased significantly after 7 days’ hypoxic exposure（P＜0.05,P＜0.01 respectively）.3.The change of NOS:The mRNA expression of eNOS increased significantly in mice after 3 day’ hypoxic exposure than in normoxic group（P＜0.01）.The peak expression was observed in day 14 of hypoxic exposure and decreased gradually thereafter.The protein expression of eNOS increased significantly in mice after 3 day’ hypoxic exposure than in normoxic group.The change of iNOS mRNA expression was correlated with that of eNOS.4.The ratio of GSH to GSSG and the concentration of nitrite/nitrate decreased significantly in mice lungs following hypoxic exposure than in normoxic group（P＜0.05, P＜0.01,respectively）.Conclusions:1.The increased expression of GSNOR is responsible for the depletion of RSNO in mice after hypoxic exposure.2.Increased eNOS or iNOS expression and decreased nitrite and nitrate concentration following hypoxic exposure suggest that hypoxia may lead to the deficiency of NO in vivo.3.The redox balance was impaired in mice after hypoxic exposure,which may be related to the pathogenesis pulmonary hypertension. PartⅡThe mechanism of hypoxia induced GSNOR overexpressionAim:1.To investigate the change of pulmonary vascular remodeling and right ventricle hypertrophy in wild-type or eNOS knockout mice after 21 days’ hypoxic exposure and the effect of NaHS administration.2.To explore the mRNA expressions of GSNOR,eNOS,iNOS and NADPH oxidase 2,4 in wild-type or eNOS knockout mice after hypoxic exposure and the effect of NaHS administration.3.To investigate the role of redox in the pathogenesis of hypoxic pulmonary hypertension by evaluating the change of reactive oxygen species（ROS） concentration in lung tissues in wild-type or eNOS knockout mice after hypoxic exposure and effect of NaHS administration.Methods:1.Pulmonary hypertension model and groupsNormoxic group:mice were exposed to 21%oxygen（room air） for 21 days.Hypoxic group:C57BL/6 mice were exposed to 10%oxygen in a forced-air environmental chamber for 21 days.Treatment group:NaHS,which was dissolved in normal saline at a dosage of 14μmol/kg,was administrated daily by intraperitoneal injection from day 0 to day 21.Control group:The same dosage of saline as treatment group was administrated daily by intraperitnoeal injection from day 0 to day 21.2.The morphological changes of the small pulmonary arteries were examined by media wall thickness（MT%）.The ratio of RV/（LV+S） was calculated to evaluate the hypertrophy of right ventricle.3.Real-time PCR was used to analyze the GSNOR,eNOS,iNOS and NOX2,4 mRNA expression in mice lungs after hypoxic exposure and the effect of NaHS administration.4.Biochemistry methods were used to determine the concentration of ROS in mice lungs after hypoxic exposure and effect of NaHS administration.Results:1.The mRNA expression of NOX2,4 increased significantly in WT mice after 21 days’ hypoxic exposure than in normoxic group（P＜0.01,respectively） while the concentration of ROS decreased significantly（P＜0.01）.The expression of GSNOR increased significantly on day 7 in hypoxic exposure than in normoxic group（P＜0.05） and decreased gradually thereafter.2.The mRNA expression of NOX2 decreased significantly after 21 days’ hypoxic exposure in KO mice than in normoxic group（P＜0.01）.There was no significant change in the expression of NOX4 and the concentration of ROS in KO mice after 21 days’ hypoxic exposure.The expression of iNOS increased significantly（P＜0.01）.The expression of GSNOR decreased significantly in KO mice after 21 days’ hypoxic exposure than in normoxic group（P＜0.05）.3.The media width of small pulmonary vessels and the ratio of RV/（LV+S） were reduced significantly after NaHS administration in WT mice than in hypoxic group （P＜0.05,P＜0.01 respectively）.The mRNA expression of eNOS or iNOS decreased significantly in mice after NaHS administration for 7 days compared to in mice of 7 days’ hypoxic exposure（P＜0.01,respectively）.The expression of NOX2,4 also decreased significantly in mice after NaHS administration for 21 days compared to in mice of 21 days’ hypoxic exposure（P＜0.01,respectively）.The concentration of ROS had no change.The expression of GSNOR increased significantly after NaHS administration for 21 days compared to in mice of 21 days’ hypoxic exposure（P＜0.05）.4.The ratio of RV/（LV+S） decreased significantly（P＜0.05） while there was no significant change in MT%in KO mice after NaHS administration than in hypoxic group.The expression of iNOS,NOX2 and NOX4 had no significant change in KO mice after NaHS administration for 21 days than in mice of 21 days’ hypoxic exposure. The concentration of ROS and the expression of GSNOR increased significantly （P＜0.01,respectively）.Conclusion:1.Pulmonary hypertension model was successfully established in WT and KO mice after 21 days’ hypoxic exposure.There was no significant difference in response to hypoxia between the two kinds of mice.There may be other pathways to compensate for the deficiency of eNOS.2.The expression of NOX2 and GSNOR decreased significantly in KO mice after hypoxic exposure while there was no significant change in the expression of NOX4 compared to normoxic group.It suggests that eNOS could induce the expression of NOX and GSNOR.3.Remodeling of pulmonary vessels and right ventricle hypertrophy induced by hypoxia can be reversed after NaHS administration in WT mice.eNOS may take part in the process of vascular relaxation of NaHS.The concentration of ROS in lungs increased and the abnormal expression of GSNOR was corrected partly after NaHS administration in WT and KO mice.The expression of GSNOR may be associated with an imbalanced redox status.更多还原