罗格列酮对博莱霉素所致肺动脉高压大鼠肺动脉iNOS表达和iNOS作用的影响
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摘要
目的:博莱霉素(bleomycin, BLM)是抗肿瘤的化疗药物,其主要副作用是引起肺泡炎和肺纤维化。气管内一次性滴注BLM复制肺纤维化模型已得到国内外公认。肺损伤是引起肺动脉高压的常见原因。目前对由BLM诱导肺纤维化过程中肺血管的变化所知甚少。本课题组以往研究结果显示:气管内滴注BLM后第14天,大鼠出现肺动脉高压,肺动脉内皮依赖性舒张反应受损;罗格列酮(Rosiglitazone, RSG)—过氧化物酶体增殖活化受体γ(peroxisome prolifertor-actived receptor-γ, PPAR-γ)激动剂能有效地防治BLM诱导的肺动脉高压。现已知,诱导型一氧化氮合酶( inducible nitro oxide synthase, iNOS)参与肺动脉高压的形成,但iNOS在BLM所致的肺动脉高压形成机制中的作用,尚不清楚。本实验通过观察模型大鼠肺动脉壁iNOS表达、iNOS在肺动脉血管反应性异常中的作用以及RSG对上述变化的影响,为阐明BLM诱导肺动脉高压的机制和RSG的防治机制提供资料。
第一部分罗格列酮对博莱霉素所致肺动脉高压大鼠肺动脉iNOS表达的影响
方法:选用免疫组织化学方法,观察气管内滴注BLM后第14天,大鼠各级肺动脉iNOS表达和分布的变化及RSG整体治疗以及离体孵育对肺动脉iNOS表达的影响。
健康雄性SD大鼠36只,体重(160~180g),随机分为四组:①NS+NS组大鼠(n=6) :一次性气管内滴入生理盐水(normal saline,NS,0.5ml/kg),NS (2ml/d)灌胃14天;②BLM+NS组(n=18) :一次性气管内滴入BLM (5mg/0.5ml/kg),NS (2ml/d)灌胃14天;③BLM+RSG组(n=6):一次性气管内滴入BLM (5mg/0.5ml/kg),RSG (3mg/kg·d溶于NS2ml/d)灌胃14天;④NS+RSG组(n=6) :一次性气管内滴入NS(0.5ml/kg),RSG (3mg/kg·d溶于NS2ml/d)灌胃14天。
各组大鼠均在气管给药后第14天取材。大鼠在10%水合氯醛(3ml/kg,ip.)麻醉后,放血处死。将肺动脉及左肺置于新鲜配置的4%多聚甲醛溶液中固定。BLM+NS组有6只大鼠的肺动脉立即被置于4%多聚甲醛固定,另12只大鼠的肺动脉在体外孵育(DMEM培养液,37℃,5% CO2 )24小时后被置于4%多聚甲醛中固定。体外孵育分两组:单纯培养液组(DMEM)和RSG孵育组(DMEM中含RSG10μmmol/L),每组6只大鼠。
固定后的组织经石蜡包埋、切片,用于iNOS免疫组织化学染色。JEDA-801D形态学图像分析系统计算iNOS免疫染色阳性的平均光密度值及阳性面积百分比。整体给药各组肺动脉行HE染色进行形态学观察。
结果:1注BLM大鼠肺动脉主干iNOS的表达及RSG的影响NS+NS组肺动脉壁(平滑肌层和内皮)有少量iNOS表达(平均光密度0.29±0.03,阳性面积6.80±1.80);NS+RSG组肺动脉壁(内皮和平滑肌层)iNOS表达(平均光密度0.30±0.01,阳性面积5.90±1.17)与NS+NS组相比,其差异无统计学意义(P>0.05)。BLM+NS组肺动脉壁(内皮和平滑肌层)iNOS表达(平均光密度0.39±0.02,阳性面积16.82±2.42)明显强于NS+NS组(P<0.01)。BLM+RSG组肺动脉壁(内皮和平滑肌层)iNOS表达(平均光密度0.33±0.02,阳性面积11.03±2.06)弱于BLM+NS组(P<0.05),但仍强于NS+NS组、NS+RSG组(P<0.05)。离体水平RSG孵育组(DMEM+RSG10μmmol/L)肺动脉壁iNOS表达(平均光密度0.32±0.02,阳性面积11.09±1.58)弱于单纯DMEM孵育组(平均光密度0.39±0.02,阳性面积16.60±2.75)(P<0.05)。
2注BLM大鼠肺内动脉iNOS的表达及RSG的影响肺内中等动脉(直径>100μm): NS+NS组肺内中动脉有少量iNOS表达(平均光密度0.30±0.06,阳性面积4.96±1.00)。与NS+NS组相比,NS+RSG组肺动脉iNOS表达无明显变化(平均光密度0.30±0.01,阳性面积5.02±1.40)(P>0.05)。BLM+NS组肺动脉iNOS表达(平均光密度0.37±0.02,阳性面积16.71±5.00)强于NS+NS组(P<0.05)。BLM+RSG组肺内中动脉iNOS表达(平均光密度0.31±0.01,阳性面积6.19±2.65)弱于BLM+NS组(P<0.05),并且与NS+NS组、NS+RSG组相比,其差异无统计学意义(P>0.05)。
肺内小动脉(直径<100μm): NS+NS组肺内小动脉有少量iNOS表达(平均光密度0.30±0.05,阳性面积5.27±0.44)。与NS+NS组相比,NS+RSG组肺动脉iNOS表达无明显变化(平均光密度0.30±0.01,阳性面积4.41±0.72)(P>0.05)。BLM+NS组肺内小动脉iNOS表达(平均光密度0.36±0.01,阳性面积17.91±3.07)强于NS+NS组(P<0.01)。BLM+RSG组肺内小动脉iNOS表达(平均光密度0.33±0.01,阳性面积10.32±3.55)弱于BLM+NS组(P<0.05),但仍强于NS+NS组、NS+RSG组(P<0.05)。
3 RSG对BLM所致的肺动脉高压大鼠肺动脉形态学改变的影响光镜下可见NS+NS组、NS+RSG组血管内皮细胞完整,内膜连续,内皮下弹力纤维完整,平滑肌层结构整齐;BLM+NS组肺动脉血管内皮细胞有脱落,内膜不连续,内皮下弹力纤维结构尚可;BLM+RSG组肺动脉内皮细胞损伤较轻,内膜基本完整,内皮下弹力纤维完整。
4小结:气管内滴入BLM第14天,大鼠肺动脉iNOS表达明显增加,肺动脉内皮细胞损伤;RSG阻止肺动脉iNOS高表达,减轻肺动脉内皮细胞损伤。
第二部分罗格列酮对博莱霉素所致肺动脉高压大鼠肺动脉血管反应性的影响及iNOS的作用
方法:采用离体肺动脉血管环张力测定方法和硝基酪氨酸(nitrotyrosine,NT)免疫组化的方法,观察罗格列酮对气管内滴注BLM后第14天大鼠肺动脉血管反应性的影响及iNOS的作用。
健康雄性SD大鼠67只,体重(160~180g),随机分为六组:
①有内皮NS+NS组(n=9) :一次性气管内滴入生理盐水(normal saline,NS,0.5ml/kg) ,NS (2ml/d)灌胃14天;
②无内皮NS+NS组(n=9) :一次性气管滴入NS 0.5ml/kg,NS(2ml/d)灌胃14天,去除肺动脉内皮;
③有内皮BLM+NS组(n=7) :一次性气管滴入BLM5mg/0.5ml/kg , NS (2ml/d)灌胃14天④无内皮BLM+NS组(n=7):一次性气管滴入BLM5mg/0.5ml/kg,NS (2ml/d)灌胃14天,去除肺动脉内皮;
⑤有内皮BLM+RSG组(n=9) :一次性气管滴入BLM5mg/0.5ml/kg,RSG (3mg/kg/d溶于NS2ml/d)中灌胃14天;
⑥无内皮BLM+RSG组(n=8) :一次性气管滴入BLM5mg/0.5ml/kg,RSG (3mg/kg/d溶于NS2ml/d)中灌胃14天,去除肺动脉内皮。
各组大鼠均在气管给药后第14天取材。腹腔注射10%水合氯醛3ml/kg麻醉动物,固定,放血处死动物,迅速摘取心肺,置于4℃新鲜配置的Krebs液中,并通入含95%O2—5%CO2的混合气体。仔细分离左、右侧肺动脉,剪成2-3mm长的肺动脉环(pulmonary artery rings,PARs)。注意避免损伤肺动脉内皮。另外去内皮组在分离出肺动脉后用打磨的玻璃电极轻轻穿入肺动脉腔小心转动去除内皮。
将分离后的血管环垂直悬挂于盛有10ml Krebs液的浴槽中,用两个不锈钢针轻轻穿过血管环,一端固定于浴槽底部,另一端连接张力传感器,与3066平台记录仪相连。温度保持在37℃左右,并持续通入上述混合气体。PARs在2g张力下平衡1h,期间每隔15分钟换液一次,用10-6mol/L苯肾上腺素(Phenylephrine, PE)检测PARs的反应性,待收缩反应曲线至平台后冲洗,基础张力稳定后,对有内皮组肺动脉测定:⑴对10-6mol/L PE的收缩反应;⑵对10-6mol/L Ach的舒张反应;⑶选择性iNOS抑制剂氨基胍(Aminoguandine,AG) 10-4mol/L孵育20分钟后,各组PARs对10-6mol/L PE的收缩反应;⑷非选择性NOS抑制剂N (omega) -nitro-L-arginine methly ester (L-NAME) 10-4mol/L孵育20分钟后,各组PARs对10-6mol/L PE的收缩反应。在去内皮组,用10-6mol/L PE预收缩PARs至反应平台后,肺动脉对10-6mol/L Ach的舒张反应消失,说明内皮已去除干净,然后对PARs进行血管张力测定(方法同有内皮组PARs)。实验结束后将PARs烘烤至重量不再变化,记录干重。收缩反应结果用每毫克血管环干重的克张力(g/mg·d·w)表示,舒张反应结果以占PE (10-6mol/L)收缩值的百分比表示。
NS+NS组、BLM+NS组、BLM+RSG组各取6只大鼠的肺动脉主干及部分肺组织置于4%多聚甲醛中,石蜡包埋切片用于免疫组织化学。
结果:1 RSG对PARs收缩反应变化的影响有内皮BLM+NS组PARs对PE收缩反应(0.67±0.15 g/mg.d.w)较有内皮NS+NS组(0.99±0.28g/mg.d.w)明显减弱(P<0.05);有内皮BLM+RSG组PARs对PE的收缩反应(1.12±0.36 g/mg.d.w)明显强于有内皮BLM+NS组(P<0.05),与有内皮NS+NS组相比,其差异无显著性(P>0.05)。无内皮BLM+NS组PARs对PE的收缩反应(0.77±0.22 g/mg.d.w)较无内皮NS+NS组PARs (1.19±0.34 g/mg.d.w)明显降低(P<0.05);无内皮BLM+RSG组PARs对PE的收缩反应(1.18±0.33 g/mg.d.w)较无内皮BLM+NS组显著增强(P<0.05),与无内皮NS+NS组相比其差异无显著性(P>0.05)。提示BLM气管滴入导致肺动脉对PE的收缩反应减弱,RSG对肺动脉平滑肌有保护作用,可恢复肺动脉对PE的收缩反应。
2 RSG对PARs舒张反应的影响有内皮BLM+NS组PARs对Ach的舒张反应(0.43±0.14)较有内皮NS+NS组PARs (0.93±0.08)显著降低(P<0.01)。有内皮BLM+RSG组PARs对Ach的舒张反应(0.64±0.20)明显高于有内皮BLM+NS组( P<0.05 ),但仍低于有内皮NS+NS组(P<0.05)。提示RSG可以部分改善气管内滴入BLM所致的PARs对Ach的舒张反应降低。
3 AG (10-4mol/L)孵育对PARs反应性的影响无内皮NS+NS组、无内皮BLM+NS组、无内皮BLM+RSG组PARs在AG孵育前后对PE收缩反应性无明显变化,提示离体情况下肺动脉平滑肌层iNOS-NO对PARs的收缩反应无明显影响。有内皮NS+NS组、有内皮BLM+NS组、有内皮BLM+RSG组PARs在AG孵育前后对PE的收缩反应无明显变化,提示在离体情况下肺动脉内皮iNOS-NO对PARs的收缩反应无明显影响。
4 L-NAME (10-4mol/L)孵育对PARs反应性的影响有内皮NS+NS组PARs在L-NAME孵育后对PE的收缩反应与孵育前相比明显增强(1.64±0.53 g/mg.d.w vs 1.08±0.37 g/mg.d.w)(P<0.01) ;有内皮BLM+NS组PARs经L-NAME孵育后对PE的收缩反应与孵育前相比无明显变化(P>0.05);有内皮BLM+RSG组PARs在L-NAME孵育后对PE的收缩反应与孵育前相比明显增强(1.75±0.39g/mg.d.w vs 1.27±0.40g/mg.d.w , P<0.05)。无内皮NS+NS组、无内皮BLM+NS组、无内皮BLM+RSG组PARs在L-NAME孵育前后对PE的收缩反应性均无明显改变。这表明,BLM+NS组肺动脉内皮损伤严重,RSG可部分恢复BLM所致的PARs内皮依赖性舒张反应降低。提示,RSG可能通过保护肺动脉内皮细胞,维持eNOS的正常功能,而降低肺动脉压。
5注BLM大鼠肺动脉主干硝基酪氨酸(nitrotyrosine, NT)的表达及RSG的影响NS+NS组肺动脉壁(平滑肌层和内皮)有极少量NT表达(平均光密度0.31±0.01,阳性面积6.87±0.56)。BLM+NS组肺动脉壁(内皮和平滑肌层)NT表达(平均光密度0.39±0.02,阳性面积21.42±1.84)明显强于NS+NS组(P<0.01)。BLM+RSG组肺动脉壁(内皮和平滑肌层)NT表达(平均光密度0.34±0.01,阳性面积9.80±0.94)弱于BLM+NS组(P<0.05),但仍强于NS+NS组(P<0.05)。
6注BLM大鼠肺内动脉NT的表达及RSG的影响肺内中等动脉(直径>100μm): NS+NS组肺内中动脉有少量NT表达(平均光密度0.31±0.01,阳性面积5.52±0.83)。BLM+NS组肺动脉NT表达(平均光密度0.38±0.02,阳性面积16.47±1.82)强于NS+NS组(P<0.01)。BLM+RSG组肺内中动脉NT表达(平均光密度0.32±0.02,阳性面积6.23±1.11)弱于BLM+NS组(P<0.01),与NS+NS组相比,其差异无统计学意义(P>0.05)。
肺内小动脉(直径<100μm): NS+NS组肺内小动脉有少量NT表达(平均光密度0.31±0.01,阳性面积4.89±0.97)。BLM+NS组肺内小动脉NT表达(平均光密度0.40±0.02,阳性面积14.60±2.55)强于NS+NS组(P<0.01)。BLM+RSG组肺内小动脉NT表达(平均光密度0.31±0.01,阳性面积5.54±1.02)弱于BLM+NS组(P<0.05),与NS+NS组相比,其差异无统计学意义(P>0.05)。
小结:BLM气管内滴入可导致肺动脉的收缩反应降低,内皮依赖性舒张反应受损,肺动脉内皮细胞损伤,平滑肌功能异常,肺动脉NT显著增加,这可能是BLM诱导肺动脉高压的机制之一。RSG能够减轻肺动脉氧化损伤,恢复肺动脉的收缩反应,改善内皮依赖性舒张反应,保护肺动脉平滑肌、内皮,维持eNOS的正常功能,这可能是RSG降低BLM诱导的肺动脉高压的机制之一。
结论:
1气管滴入BLM第14天肺各级动脉iNOS、NT表达明显增强,肺动脉内皮细胞受损。上述异常可能在BLM诱导的肺动脉高压过程中发挥重要作用。
2 BLM诱导的肺动脉高压大鼠肺动脉收缩反应降低,内皮依赖性舒张反应受损,导致肺动脉血管反应性紊乱,上述异常可能参与了肺动脉高压的形成。
3 RSG降低肺动脉iNOS、NT表达,保护肺动脉内皮细胞,部分恢复eNOS功能,完全恢复肺动脉平滑肌功能,改善肺动脉血管反应性,这可能是RSG降低肺动脉压的机制之一。
Objective:Bleomycin is a kind of anti-tumor drug whose main side effect is to induce pulmonary injury and fibrosis. The modle of pulmonary fibrosis induced by a single intratracheal instillation of blemycin (BLM) is generally accepted. Pulmonary fibrosis can result in pulmonary hypertension. Up to now, the abnormal changs of pulmonary artery, during BLM-induced lung fibrosis, has not been clear yet. Our previous study has shown that pulmonary hypertension and the disorder of pulmonary artery in rats on d 14 after a single intratracheal instillation of blemycin (BLM), and that rosiglitazone (RSG), a potent synthetic agonist of peroxisome proliferator-gamma (PPAR-γ), can effectively prevent the BLM-induced pulmonary hypertension. It is reported that inducible nitro oxide synthase ( iNOS ) participated in the development of pulmonary hypertension, but the effect of iNOS on the pathogenises of BLM-induced pulmonary hypertension remains unclear. In the present study, the effects of RSG on the expression and action of iNOS in pulmonary artery of BLM-induced pulmonary hypertension rats were investigated.
PartⅠEffects of rosiglitazone on expression of iNOS in pulmonary artery of bleomycin-induced pulmonary artery hypertension rats.
Methods:Thirty-six male Sprague-Dawley rats were randomly divided into four groups:①NS+NS group (n=6) : NS (0.5ml/kg) was administrated by single intratracheal instillation and then NS (2ml/d, i.g) for 14 days;②BLM+NS group (n=18): BLM (5mg·kg-1·0.5ml) was administrated by single intratracheal instillation and then NS (2ml/d, i.g) for 14 days;③BLM+RSG group (n=6): BLM (5mg·kg-1·0.5ml) was administrated by single intratracheal instillation and then RSG (3mg·kg-1per rat, dissolved in 2ml NS, i.g) for 14 days;④NS+RSG group (n=6): NS (0.5ml/kg) was administrated by single intratracheal instillation and then RSG (3mg·kg-1per rat, dissolved in 2ml NS, i.g) for 14 days.On day 14 after intratracheal instillation, All rats were killed, and then pulmonary tissue and pulmonary artery were collected for immunohistochemistry. Twelve pulmonary arteries of BLM+NS 14 day group were incubated in DMEM for 24 hours present or absent rosiglitazone (10um/L) under the condition of 37℃, and then were collected for immunohistochemistry.
Data were analyzed using SPSS software. Group mean values and standard deviations were calculated. After homogeneitic analysis, homogeneous data were analyzed with one-way analysis of variance and a post hoc test of least significant difference (LSD). Data of incubated pulmonary arteries were analyzed using independent-samples T test. The statistical significance level was set at P<0.05.
Results: 1 RSG reduces the expression of iNOS in pulmonary artery induced by BLM. In NS+NS group, the expression of iNOS in pulmonary artery was slight and localized to the smooth muscle layer and endothelium (average optical density: 0.29±0.03, percentage of positive areas:6.80±1.80), and the pulmonary artery expression of iNOS in NS+RSG group was similar to that in NS+NS group. Compared with NS+NS、NS+RSG group, the expression of iNOS in pulmonary artery of BLM+NS group was significantly increased (average optical density: 0.39±0.02, percentage of positive areas: 16.82±2.42) (P<0.01). Compared with NS+NS、NS+RSG group, the expression of iNOS in pulmonary artery of BLM+RSG group (average optical density: 0.33±0.02, percentage of positive areas: 11.03±2.06) were significiantly increased (P<0.05). Comparied with BLM+NS, the expression of iNOS protein in pulmonary artery of BLM+RSG group were singnificiantly reduced (P<0.05). In addition, sections showed that vascular endothelium was intact and continuous in NS+NS group; but vascular endothelial cells apeared losting in BLM+NS group; vascular endothelial cells were protected in BLM+RSG group. In incubation with DMEM present rosiglitazone group, the expression of iNOS was significantly reduced (average optical density: 0.32± 0.02,percentage of positive areas:11.09±1.58)compared with DMEM absent rosiglitazone (average optical density: 0.39±0.02,percentage of positive areas: 16.60±2.75) (p<0.05).
2 Rosiglitazone reduce the expression of iNOS in pulmonary artery in lungs induced by bleomycin. Small arteries in lung (diamerter<100um): In NS+NS group, the expression of iNOS in pulmonary artery was slight (average optical density: 0.30±0.05, percentage of positive areas: 5.27±0.44). The expression of iNOS in pulmonary arteries in NS+RSG group was similar with that in NS+NS group (P>0.05). Compared with NS+NS、NS+RSG group, the expression of iNOS in BLM+NS group increased significantly (average optical density: 0.36±0.01, percentage of positive areas:17.91±3.07) (P<0.01). Compared with NS+NS、NS+RSG group, the expression of iNOS in pulmonary artery (average optical density: 0.33±0.01, percentage of positive areas: 10.32±3.55) in BLM+RSG group were singnificantly increased (P<0.05). Compared with BLM+NS group, the expression of iNOS in pulmonary artery in BLM+RSG group was singnificiantly reduced (P<0.05). Larger arteries in Lung (diamerter>100um) : In NS+NS group, the expression of iNOS in pulmonary artery was slight (average optical density: 0.30±0.06, percentage of positive areas: 4.96±1.00). The expression of iNOS in pulmonary artery in NS+RSG group was similar to that in NS+NS group (P>0.05). Compared with NS+NS、NS+RSG group, the expression of iNOS in BLM+NS group increased significantly (average optical density: 0.37±0.02, percentage of positive areas: 16.71±5.00) (P<0.05 ) . Compared with BLM+NS group, the expression of iNOS in BLM+RSG group was markedly reduced (P<0.05). The expression of iNOS in BLM+RSG group was similar to that in NS+NS、NS+RSG group (P>0.05)
The results suggested that strong expression of iNOS and injury of endothelium in pulmonary arteries of rats on day 14 after intratracheal instillation of BLM, and rosiglitazone could significantly reduce the above changes.
PartⅡThe effects of RSG on reactivities of PARs in BLM-induced pulmonary hypertension rats and the roles of iNOS
Methods: 67 male Sprague-Dawley rats were randomly divided into six groups:①NS+NS with endothelium group: NS (0.5ml/kg) was administrated by single intratracheal instillation and NS (2ml/d, i.g) for 14 days;②NS+NS without endothelium group: NS (0.5ml/kg) was administrated by single intratracheal instillation and NS (2ml/d, i.g) for 14 days. The endothelium of pulmonary arteries was removed;③BLM+NS with endothelium group: BLM (5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and NS (2ml/d, i.g) for 14 days;④BLM+NS without endothelium group: BLM (5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and then NS (2ml/d, i.g) for 14 days. The endothelium of pulmonary artery was removed;⑤BLM+RSG with endothelium group: BLM (5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and then RSG (3mg·kg-1per rat,dissolved in 2ml NS, i.g.) for 14 days.⑥BLM+RSG without endothelium group: BLM (5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and then RSG (3mg·kg-1per rat,dissolved in 2ml NS, i.g.) for 14 days. The endothelium of pulmonary artery was removed. The rats were sacrificed on day 14 after single intratracheal instillation. Changs of vascular tension of pulmonary artery rings (PARs) were detected in vitro. The contraction responses to phenyleyphrine (PE 10-6mol/L) were then tested separatedly to observe the stability of the PARs reactivity.When the contraction responses had become stable, PARs of all groups were detected:①the contraction responses of PARs to PE (10-6mol/L) ;②the relaxation responses of PARs to acetylcholine (Ach 10-6mol/L);③the contraction responses of PARs to PE (10-6mol/L) after preincubation with aminoguanidine (AG 10-4mol/L) for 20 min;④the contraction responses of PARs to PE (10-6mol/L) after preincubation with N (omega) -nitro-L-arginine methyl ester (L-NAME 10-4mol/L) for 20 min.The PARs responses to PE were expressed as g/mg·d·w, and vascular relaxtion responses to Ach were expressed as percentage reduction of initial vascular tension induced by PE.
Sections were stained with immunohistochemistry for the expression of NT .
Results: 1 Rosiglitazone improves PARs contraction responses to PE after BLM intracheal instillation Compared with the PARs in NS+NS group, the contraction reponses to PE of PARs in BLM+NS , either with endothelium or without endothelium, were significantly reduced (P<0.05). In BLM+RSG group, rosiglitazone administration reversed PARs contraction reponses to PE. The results suggested that PARs contraction responses to PE was damaged by BLM administratiom, and the damaged contraction reponses to PE was reversed by rosiglitazone.
2 Rosiglitazone protects endothelial cells of pulmonary artery and improves the endothelial cells-dependedent- relaxtion. Compared with PARs in NS+NS group with endothelium, the relaxation reponses to Ach of PARs in BLM+NS with endothelium group were significantly decreased (0.93±0.08 vs 0.43±0.14) (P<0.01) . In BLM+RSG group, the relaxtion responses to Ach of PARs were markedly improved compared with those in BLM+NS group (0.69±0.18 vs 0.43±0.14,P<0.05), which indicated that rosiglitazone impoves the function of pulmonary artery endothelial cells and reduce the damage to pulmonary artery endothelium induced by BLM, this may be one of mechanisms involved in rosiglitazone reversing the pulmonary artery hypertension induced by BLM.
3 Aminoguanidine (AG) has no effect on the PARs contraction reponses to PE in all groups. In all groups, the PARs contraction responses to PE were not changed after preincubation with AG(10-4mol/L), which indicated that in pulmonary artery hypertension induced by BLM NO derived by iNOS may not participate in the regulation of pulmoary artery vascular tension.
4 L-NAME preincubation enhance the PARs contraction responses to PE in groups with endothelium. After incubation with L-NAME, the contraction responses of PARs to PE in NS+NS group with endothelium significantly increased (1.20±0.50 g/mg·d·w vs 1.73±0.57 g/mg·d·w) (P<0.01) , while the contraction reponses of PARs in BLM+NS group with endothlium did not changed after preincubation with L-NAME. In BLM+RSG with endothelium group the contraction responses to PE markedly increased (1.31±0.38 vs 1.81±0.40) (P<0.05) . The contraction responses to PE in all groups without endothelium had no siginificant differences.The result indicated that rosiglitazone protect pulmonary artery vascular endothelial cells.
5 RSG reduces the expression of NT protein in pulmonary artery induced by BLM. In NS+NS group, the expression of NT in pulmonary artery was slight (average optical density: 0.31±0.01, percentage of positive areas:6.87±0.56). The expression of NT protein of pulmonary artery in BLM+NS group was significantly increased (average optical density: 0.39±0.02, percentage of positive areas: 21.42±1.84) compared with that in NS+NS group, (P<0.01). The expression of NT protein of pulmonary artery in BLM+RSG group (average optical density: 0.34±0.01, percentage of positive areas: 9.80±0.94) was lower than that in BLM+NS (P<0.05), and still higher than that in NS+NS group (P<0.05).
6 Rosiglitazone reduce the expression of NT in pulmonary artery in lungs induced by bleomycin. Small arteries in lung (diamerter<100um): In NS+NS group, the expression of NT in pulmonary artery was slight (average optical density: 0.31±0.01, percentage of positive areas: 4.89±1.00). Compared with NS+NS group, the expression of NT in BLM+NS group increased significantly (average optical density: 0.40±0.02, percentage of positive areas:14.60±2.55) (P<0.01). The expression of NT in pulmonary artery in BLM+RSG group (average optical density: 0.31±0.01, percentage of positive areas: 5.84±1.02) was lower than that in BLM+NS group (P<0.01) and was similar to that in NS+NS group(P>0.05). Larger arteries in Lung (diamerter>100um) : In NS+NS group, the expression of NT in pulmonary artery was slight (average optical density: 0.31±0.01, percentage of positive areas: 5.52±0.83).Compared with NS+NS group, the expression of NT in BLM+NS group increased significantly (average optical density: 0.38±0.02, percentage of positive areas: 16.47±1.82) (P<0.01). The expression of NT in BLM+RSG group was lower than that in BLM+NS group (P<0.01), and was similar to that in NS+NS group (P>0.05).
The results suggested that: (1) the contraction responses of PARs to PE and the endothlium-dependent relaxtion resposes of PARs to Ach were striking impaired by BLM, which led to PARs reactivties disorder; (2) the expression of NT in pulmonary arteries was markedly increased by BLM; (3) Rosiglitazone could reduce the expression of NT in pulmonary artery, partly protect the funtion of pulmonary artery endtholium, and reverse the abnormal funtion of smooth muscle induced by BLM.
Conclusions: 1 The expression of iNOS and NT in all kinds of pulmonary arteries was significantly increased and endothelium of pulmonary artery was injured by bleomycin, which maybe implicated in the bleomycin induced pulmonary artery hypertension.
2 Bleomycin induced the markedly impaired contraction to PE and the relaxtion to Ach of PARs, which had led to the disorder of pulmonary artery vascular reactivity. This might be one of the mechanisms of pulmonary hypertension in this model.
3 Rosiglitazone could significantly reduce the expression of iNOS and NT in all kinds of pulmonary arteries and reverse the impaired contraction responses of PARs. In addition, rosiglitazone could protect the function of pulmonary artery endothelial cells and correct the disorder of PARs induced by BLM. All above may be one of the mechanisms that rosiglitazone reverse the pulmonary artery hypertension induced by bleomycin.
第一部分罗格列酮对博莱霉素所致肺动脉高压大鼠肺动脉iNOS表达的影响
方法:选用免疫组织化学方法,观察气管内滴注BLM后第14天,大鼠各级肺动脉iNOS表达和分布的变化及RSG整体治疗以及离体孵育对肺动脉iNOS表达的影响。
健康雄性SD大鼠36只,体重(160~180g),随机分为四组:①NS+NS组大鼠(n=6) :一次性气管内滴入生理盐水(normal saline,NS,0.5ml/kg),NS (2ml/d)灌胃14天;②BLM+NS组(n=18) :一次性气管内滴入BLM (5mg/0.5ml/kg),NS (2ml/d)灌胃14天;③BLM+RSG组(n=6):一次性气管内滴入BLM (5mg/0.5ml/kg),RSG (3mg/kg·d溶于NS2ml/d)灌胃14天;④NS+RSG组(n=6) :一次性气管内滴入NS(0.5ml/kg),RSG (3mg/kg·d溶于NS2ml/d)灌胃14天。
各组大鼠均在气管给药后第14天取材。大鼠在10%水合氯醛(3ml/kg,ip.)麻醉后,放血处死。将肺动脉及左肺置于新鲜配置的4%多聚甲醛溶液中固定。BLM+NS组有6只大鼠的肺动脉立即被置于4%多聚甲醛固定,另12只大鼠的肺动脉在体外孵育(DMEM培养液,37℃,5% CO2 )24小时后被置于4%多聚甲醛中固定。体外孵育分两组:单纯培养液组(DMEM)和RSG孵育组(DMEM中含RSG10μmmol/L),每组6只大鼠。
固定后的组织经石蜡包埋、切片,用于iNOS免疫组织化学染色。JEDA-801D形态学图像分析系统计算iNOS免疫染色阳性的平均光密度值及阳性面积百分比。整体给药各组肺动脉行HE染色进行形态学观察。
结果:1注BLM大鼠肺动脉主干iNOS的表达及RSG的影响NS+NS组肺动脉壁(平滑肌层和内皮)有少量iNOS表达(平均光密度0.29±0.03,阳性面积6.80±1.80);NS+RSG组肺动脉壁(内皮和平滑肌层)iNOS表达(平均光密度0.30±0.01,阳性面积5.90±1.17)与NS+NS组相比,其差异无统计学意义(P>0.05)。BLM+NS组肺动脉壁(内皮和平滑肌层)iNOS表达(平均光密度0.39±0.02,阳性面积16.82±2.42)明显强于NS+NS组(P<0.01)。BLM+RSG组肺动脉壁(内皮和平滑肌层)iNOS表达(平均光密度0.33±0.02,阳性面积11.03±2.06)弱于BLM+NS组(P<0.05),但仍强于NS+NS组、NS+RSG组(P<0.05)。离体水平RSG孵育组(DMEM+RSG10μmmol/L)肺动脉壁iNOS表达(平均光密度0.32±0.02,阳性面积11.09±1.58)弱于单纯DMEM孵育组(平均光密度0.39±0.02,阳性面积16.60±2.75)(P<0.05)。
2注BLM大鼠肺内动脉iNOS的表达及RSG的影响肺内中等动脉(直径>100μm): NS+NS组肺内中动脉有少量iNOS表达(平均光密度0.30±0.06,阳性面积4.96±1.00)。与NS+NS组相比,NS+RSG组肺动脉iNOS表达无明显变化(平均光密度0.30±0.01,阳性面积5.02±1.40)(P>0.05)。BLM+NS组肺动脉iNOS表达(平均光密度0.37±0.02,阳性面积16.71±5.00)强于NS+NS组(P<0.05)。BLM+RSG组肺内中动脉iNOS表达(平均光密度0.31±0.01,阳性面积6.19±2.65)弱于BLM+NS组(P<0.05),并且与NS+NS组、NS+RSG组相比,其差异无统计学意义(P>0.05)。
肺内小动脉(直径<100μm): NS+NS组肺内小动脉有少量iNOS表达(平均光密度0.30±0.05,阳性面积5.27±0.44)。与NS+NS组相比,NS+RSG组肺动脉iNOS表达无明显变化(平均光密度0.30±0.01,阳性面积4.41±0.72)(P>0.05)。BLM+NS组肺内小动脉iNOS表达(平均光密度0.36±0.01,阳性面积17.91±3.07)强于NS+NS组(P<0.01)。BLM+RSG组肺内小动脉iNOS表达(平均光密度0.33±0.01,阳性面积10.32±3.55)弱于BLM+NS组(P<0.05),但仍强于NS+NS组、NS+RSG组(P<0.05)。
3 RSG对BLM所致的肺动脉高压大鼠肺动脉形态学改变的影响光镜下可见NS+NS组、NS+RSG组血管内皮细胞完整,内膜连续,内皮下弹力纤维完整,平滑肌层结构整齐;BLM+NS组肺动脉血管内皮细胞有脱落,内膜不连续,内皮下弹力纤维结构尚可;BLM+RSG组肺动脉内皮细胞损伤较轻,内膜基本完整,内皮下弹力纤维完整。
4小结:气管内滴入BLM第14天,大鼠肺动脉iNOS表达明显增加,肺动脉内皮细胞损伤;RSG阻止肺动脉iNOS高表达,减轻肺动脉内皮细胞损伤。
第二部分罗格列酮对博莱霉素所致肺动脉高压大鼠肺动脉血管反应性的影响及iNOS的作用
方法:采用离体肺动脉血管环张力测定方法和硝基酪氨酸(nitrotyrosine,NT)免疫组化的方法,观察罗格列酮对气管内滴注BLM后第14天大鼠肺动脉血管反应性的影响及iNOS的作用。
健康雄性SD大鼠67只,体重(160~180g),随机分为六组:
①有内皮NS+NS组(n=9) :一次性气管内滴入生理盐水(normal saline,NS,0.5ml/kg) ,NS (2ml/d)灌胃14天;
②无内皮NS+NS组(n=9) :一次性气管滴入NS 0.5ml/kg,NS(2ml/d)灌胃14天,去除肺动脉内皮;
③有内皮BLM+NS组(n=7) :一次性气管滴入BLM5mg/0.5ml/kg , NS (2ml/d)灌胃14天④无内皮BLM+NS组(n=7):一次性气管滴入BLM5mg/0.5ml/kg,NS (2ml/d)灌胃14天,去除肺动脉内皮;
⑤有内皮BLM+RSG组(n=9) :一次性气管滴入BLM5mg/0.5ml/kg,RSG (3mg/kg/d溶于NS2ml/d)中灌胃14天;
⑥无内皮BLM+RSG组(n=8) :一次性气管滴入BLM5mg/0.5ml/kg,RSG (3mg/kg/d溶于NS2ml/d)中灌胃14天,去除肺动脉内皮。
各组大鼠均在气管给药后第14天取材。腹腔注射10%水合氯醛3ml/kg麻醉动物,固定,放血处死动物,迅速摘取心肺,置于4℃新鲜配置的Krebs液中,并通入含95%O2—5%CO2的混合气体。仔细分离左、右侧肺动脉,剪成2-3mm长的肺动脉环(pulmonary artery rings,PARs)。注意避免损伤肺动脉内皮。另外去内皮组在分离出肺动脉后用打磨的玻璃电极轻轻穿入肺动脉腔小心转动去除内皮。
将分离后的血管环垂直悬挂于盛有10ml Krebs液的浴槽中,用两个不锈钢针轻轻穿过血管环,一端固定于浴槽底部,另一端连接张力传感器,与3066平台记录仪相连。温度保持在37℃左右,并持续通入上述混合气体。PARs在2g张力下平衡1h,期间每隔15分钟换液一次,用10-6mol/L苯肾上腺素(Phenylephrine, PE)检测PARs的反应性,待收缩反应曲线至平台后冲洗,基础张力稳定后,对有内皮组肺动脉测定:⑴对10-6mol/L PE的收缩反应;⑵对10-6mol/L Ach的舒张反应;⑶选择性iNOS抑制剂氨基胍(Aminoguandine,AG) 10-4mol/L孵育20分钟后,各组PARs对10-6mol/L PE的收缩反应;⑷非选择性NOS抑制剂N (omega) -nitro-L-arginine methly ester (L-NAME) 10-4mol/L孵育20分钟后,各组PARs对10-6mol/L PE的收缩反应。在去内皮组,用10-6mol/L PE预收缩PARs至反应平台后,肺动脉对10-6mol/L Ach的舒张反应消失,说明内皮已去除干净,然后对PARs进行血管张力测定(方法同有内皮组PARs)。实验结束后将PARs烘烤至重量不再变化,记录干重。收缩反应结果用每毫克血管环干重的克张力(g/mg·d·w)表示,舒张反应结果以占PE (10-6mol/L)收缩值的百分比表示。
NS+NS组、BLM+NS组、BLM+RSG组各取6只大鼠的肺动脉主干及部分肺组织置于4%多聚甲醛中,石蜡包埋切片用于免疫组织化学。
结果:1 RSG对PARs收缩反应变化的影响有内皮BLM+NS组PARs对PE收缩反应(0.67±0.15 g/mg.d.w)较有内皮NS+NS组(0.99±0.28g/mg.d.w)明显减弱(P<0.05);有内皮BLM+RSG组PARs对PE的收缩反应(1.12±0.36 g/mg.d.w)明显强于有内皮BLM+NS组(P<0.05),与有内皮NS+NS组相比,其差异无显著性(P>0.05)。无内皮BLM+NS组PARs对PE的收缩反应(0.77±0.22 g/mg.d.w)较无内皮NS+NS组PARs (1.19±0.34 g/mg.d.w)明显降低(P<0.05);无内皮BLM+RSG组PARs对PE的收缩反应(1.18±0.33 g/mg.d.w)较无内皮BLM+NS组显著增强(P<0.05),与无内皮NS+NS组相比其差异无显著性(P>0.05)。提示BLM气管滴入导致肺动脉对PE的收缩反应减弱,RSG对肺动脉平滑肌有保护作用,可恢复肺动脉对PE的收缩反应。
2 RSG对PARs舒张反应的影响有内皮BLM+NS组PARs对Ach的舒张反应(0.43±0.14)较有内皮NS+NS组PARs (0.93±0.08)显著降低(P<0.01)。有内皮BLM+RSG组PARs对Ach的舒张反应(0.64±0.20)明显高于有内皮BLM+NS组( P<0.05 ),但仍低于有内皮NS+NS组(P<0.05)。提示RSG可以部分改善气管内滴入BLM所致的PARs对Ach的舒张反应降低。
3 AG (10-4mol/L)孵育对PARs反应性的影响无内皮NS+NS组、无内皮BLM+NS组、无内皮BLM+RSG组PARs在AG孵育前后对PE收缩反应性无明显变化,提示离体情况下肺动脉平滑肌层iNOS-NO对PARs的收缩反应无明显影响。有内皮NS+NS组、有内皮BLM+NS组、有内皮BLM+RSG组PARs在AG孵育前后对PE的收缩反应无明显变化,提示在离体情况下肺动脉内皮iNOS-NO对PARs的收缩反应无明显影响。
4 L-NAME (10-4mol/L)孵育对PARs反应性的影响有内皮NS+NS组PARs在L-NAME孵育后对PE的收缩反应与孵育前相比明显增强(1.64±0.53 g/mg.d.w vs 1.08±0.37 g/mg.d.w)(P<0.01) ;有内皮BLM+NS组PARs经L-NAME孵育后对PE的收缩反应与孵育前相比无明显变化(P>0.05);有内皮BLM+RSG组PARs在L-NAME孵育后对PE的收缩反应与孵育前相比明显增强(1.75±0.39g/mg.d.w vs 1.27±0.40g/mg.d.w , P<0.05)。无内皮NS+NS组、无内皮BLM+NS组、无内皮BLM+RSG组PARs在L-NAME孵育前后对PE的收缩反应性均无明显改变。这表明,BLM+NS组肺动脉内皮损伤严重,RSG可部分恢复BLM所致的PARs内皮依赖性舒张反应降低。提示,RSG可能通过保护肺动脉内皮细胞,维持eNOS的正常功能,而降低肺动脉压。
5注BLM大鼠肺动脉主干硝基酪氨酸(nitrotyrosine, NT)的表达及RSG的影响NS+NS组肺动脉壁(平滑肌层和内皮)有极少量NT表达(平均光密度0.31±0.01,阳性面积6.87±0.56)。BLM+NS组肺动脉壁(内皮和平滑肌层)NT表达(平均光密度0.39±0.02,阳性面积21.42±1.84)明显强于NS+NS组(P<0.01)。BLM+RSG组肺动脉壁(内皮和平滑肌层)NT表达(平均光密度0.34±0.01,阳性面积9.80±0.94)弱于BLM+NS组(P<0.05),但仍强于NS+NS组(P<0.05)。
6注BLM大鼠肺内动脉NT的表达及RSG的影响肺内中等动脉(直径>100μm): NS+NS组肺内中动脉有少量NT表达(平均光密度0.31±0.01,阳性面积5.52±0.83)。BLM+NS组肺动脉NT表达(平均光密度0.38±0.02,阳性面积16.47±1.82)强于NS+NS组(P<0.01)。BLM+RSG组肺内中动脉NT表达(平均光密度0.32±0.02,阳性面积6.23±1.11)弱于BLM+NS组(P<0.01),与NS+NS组相比,其差异无统计学意义(P>0.05)。
肺内小动脉(直径<100μm): NS+NS组肺内小动脉有少量NT表达(平均光密度0.31±0.01,阳性面积4.89±0.97)。BLM+NS组肺内小动脉NT表达(平均光密度0.40±0.02,阳性面积14.60±2.55)强于NS+NS组(P<0.01)。BLM+RSG组肺内小动脉NT表达(平均光密度0.31±0.01,阳性面积5.54±1.02)弱于BLM+NS组(P<0.05),与NS+NS组相比,其差异无统计学意义(P>0.05)。
小结:BLM气管内滴入可导致肺动脉的收缩反应降低,内皮依赖性舒张反应受损,肺动脉内皮细胞损伤,平滑肌功能异常,肺动脉NT显著增加,这可能是BLM诱导肺动脉高压的机制之一。RSG能够减轻肺动脉氧化损伤,恢复肺动脉的收缩反应,改善内皮依赖性舒张反应,保护肺动脉平滑肌、内皮,维持eNOS的正常功能,这可能是RSG降低BLM诱导的肺动脉高压的机制之一。
结论:
1气管滴入BLM第14天肺各级动脉iNOS、NT表达明显增强,肺动脉内皮细胞受损。上述异常可能在BLM诱导的肺动脉高压过程中发挥重要作用。
2 BLM诱导的肺动脉高压大鼠肺动脉收缩反应降低,内皮依赖性舒张反应受损,导致肺动脉血管反应性紊乱,上述异常可能参与了肺动脉高压的形成。
3 RSG降低肺动脉iNOS、NT表达,保护肺动脉内皮细胞,部分恢复eNOS功能,完全恢复肺动脉平滑肌功能,改善肺动脉血管反应性,这可能是RSG降低肺动脉压的机制之一。
Objective:Bleomycin is a kind of anti-tumor drug whose main side effect is to induce pulmonary injury and fibrosis. The modle of pulmonary fibrosis induced by a single intratracheal instillation of blemycin (BLM) is generally accepted. Pulmonary fibrosis can result in pulmonary hypertension. Up to now, the abnormal changs of pulmonary artery, during BLM-induced lung fibrosis, has not been clear yet. Our previous study has shown that pulmonary hypertension and the disorder of pulmonary artery in rats on d 14 after a single intratracheal instillation of blemycin (BLM), and that rosiglitazone (RSG), a potent synthetic agonist of peroxisome proliferator-gamma (PPAR-γ), can effectively prevent the BLM-induced pulmonary hypertension. It is reported that inducible nitro oxide synthase ( iNOS ) participated in the development of pulmonary hypertension, but the effect of iNOS on the pathogenises of BLM-induced pulmonary hypertension remains unclear. In the present study, the effects of RSG on the expression and action of iNOS in pulmonary artery of BLM-induced pulmonary hypertension rats were investigated.
PartⅠEffects of rosiglitazone on expression of iNOS in pulmonary artery of bleomycin-induced pulmonary artery hypertension rats.
Methods:Thirty-six male Sprague-Dawley rats were randomly divided into four groups:①NS+NS group (n=6) : NS (0.5ml/kg) was administrated by single intratracheal instillation and then NS (2ml/d, i.g) for 14 days;②BLM+NS group (n=18): BLM (5mg·kg-1·0.5ml) was administrated by single intratracheal instillation and then NS (2ml/d, i.g) for 14 days;③BLM+RSG group (n=6): BLM (5mg·kg-1·0.5ml) was administrated by single intratracheal instillation and then RSG (3mg·kg-1per rat, dissolved in 2ml NS, i.g) for 14 days;④NS+RSG group (n=6): NS (0.5ml/kg) was administrated by single intratracheal instillation and then RSG (3mg·kg-1per rat, dissolved in 2ml NS, i.g) for 14 days.On day 14 after intratracheal instillation, All rats were killed, and then pulmonary tissue and pulmonary artery were collected for immunohistochemistry. Twelve pulmonary arteries of BLM+NS 14 day group were incubated in DMEM for 24 hours present or absent rosiglitazone (10um/L) under the condition of 37℃, and then were collected for immunohistochemistry.
Data were analyzed using SPSS software. Group mean values and standard deviations were calculated. After homogeneitic analysis, homogeneous data were analyzed with one-way analysis of variance and a post hoc test of least significant difference (LSD). Data of incubated pulmonary arteries were analyzed using independent-samples T test. The statistical significance level was set at P<0.05.
Results: 1 RSG reduces the expression of iNOS in pulmonary artery induced by BLM. In NS+NS group, the expression of iNOS in pulmonary artery was slight and localized to the smooth muscle layer and endothelium (average optical density: 0.29±0.03, percentage of positive areas:6.80±1.80), and the pulmonary artery expression of iNOS in NS+RSG group was similar to that in NS+NS group. Compared with NS+NS、NS+RSG group, the expression of iNOS in pulmonary artery of BLM+NS group was significantly increased (average optical density: 0.39±0.02, percentage of positive areas: 16.82±2.42) (P<0.01). Compared with NS+NS、NS+RSG group, the expression of iNOS in pulmonary artery of BLM+RSG group (average optical density: 0.33±0.02, percentage of positive areas: 11.03±2.06) were significiantly increased (P<0.05). Comparied with BLM+NS, the expression of iNOS protein in pulmonary artery of BLM+RSG group were singnificiantly reduced (P<0.05). In addition, sections showed that vascular endothelium was intact and continuous in NS+NS group; but vascular endothelial cells apeared losting in BLM+NS group; vascular endothelial cells were protected in BLM+RSG group. In incubation with DMEM present rosiglitazone group, the expression of iNOS was significantly reduced (average optical density: 0.32± 0.02,percentage of positive areas:11.09±1.58)compared with DMEM absent rosiglitazone (average optical density: 0.39±0.02,percentage of positive areas: 16.60±2.75) (p<0.05).
2 Rosiglitazone reduce the expression of iNOS in pulmonary artery in lungs induced by bleomycin. Small arteries in lung (diamerter<100um): In NS+NS group, the expression of iNOS in pulmonary artery was slight (average optical density: 0.30±0.05, percentage of positive areas: 5.27±0.44). The expression of iNOS in pulmonary arteries in NS+RSG group was similar with that in NS+NS group (P>0.05). Compared with NS+NS、NS+RSG group, the expression of iNOS in BLM+NS group increased significantly (average optical density: 0.36±0.01, percentage of positive areas:17.91±3.07) (P<0.01). Compared with NS+NS、NS+RSG group, the expression of iNOS in pulmonary artery (average optical density: 0.33±0.01, percentage of positive areas: 10.32±3.55) in BLM+RSG group were singnificantly increased (P<0.05). Compared with BLM+NS group, the expression of iNOS in pulmonary artery in BLM+RSG group was singnificiantly reduced (P<0.05). Larger arteries in Lung (diamerter>100um) : In NS+NS group, the expression of iNOS in pulmonary artery was slight (average optical density: 0.30±0.06, percentage of positive areas: 4.96±1.00). The expression of iNOS in pulmonary artery in NS+RSG group was similar to that in NS+NS group (P>0.05). Compared with NS+NS、NS+RSG group, the expression of iNOS in BLM+NS group increased significantly (average optical density: 0.37±0.02, percentage of positive areas: 16.71±5.00) (P<0.05 ) . Compared with BLM+NS group, the expression of iNOS in BLM+RSG group was markedly reduced (P<0.05). The expression of iNOS in BLM+RSG group was similar to that in NS+NS、NS+RSG group (P>0.05)
The results suggested that strong expression of iNOS and injury of endothelium in pulmonary arteries of rats on day 14 after intratracheal instillation of BLM, and rosiglitazone could significantly reduce the above changes.
PartⅡThe effects of RSG on reactivities of PARs in BLM-induced pulmonary hypertension rats and the roles of iNOS
Methods: 67 male Sprague-Dawley rats were randomly divided into six groups:①NS+NS with endothelium group: NS (0.5ml/kg) was administrated by single intratracheal instillation and NS (2ml/d, i.g) for 14 days;②NS+NS without endothelium group: NS (0.5ml/kg) was administrated by single intratracheal instillation and NS (2ml/d, i.g) for 14 days. The endothelium of pulmonary arteries was removed;③BLM+NS with endothelium group: BLM (5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and NS (2ml/d, i.g) for 14 days;④BLM+NS without endothelium group: BLM (5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and then NS (2ml/d, i.g) for 14 days. The endothelium of pulmonary artery was removed;⑤BLM+RSG with endothelium group: BLM (5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and then RSG (3mg·kg-1per rat,dissolved in 2ml NS, i.g.) for 14 days.⑥BLM+RSG without endothelium group: BLM (5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and then RSG (3mg·kg-1per rat,dissolved in 2ml NS, i.g.) for 14 days. The endothelium of pulmonary artery was removed. The rats were sacrificed on day 14 after single intratracheal instillation. Changs of vascular tension of pulmonary artery rings (PARs) were detected in vitro. The contraction responses to phenyleyphrine (PE 10-6mol/L) were then tested separatedly to observe the stability of the PARs reactivity.When the contraction responses had become stable, PARs of all groups were detected:①the contraction responses of PARs to PE (10-6mol/L) ;②the relaxation responses of PARs to acetylcholine (Ach 10-6mol/L);③the contraction responses of PARs to PE (10-6mol/L) after preincubation with aminoguanidine (AG 10-4mol/L) for 20 min;④the contraction responses of PARs to PE (10-6mol/L) after preincubation with N (omega) -nitro-L-arginine methyl ester (L-NAME 10-4mol/L) for 20 min.The PARs responses to PE were expressed as g/mg·d·w, and vascular relaxtion responses to Ach were expressed as percentage reduction of initial vascular tension induced by PE.
Sections were stained with immunohistochemistry for the expression of NT .
Results: 1 Rosiglitazone improves PARs contraction responses to PE after BLM intracheal instillation Compared with the PARs in NS+NS group, the contraction reponses to PE of PARs in BLM+NS , either with endothelium or without endothelium, were significantly reduced (P<0.05). In BLM+RSG group, rosiglitazone administration reversed PARs contraction reponses to PE. The results suggested that PARs contraction responses to PE was damaged by BLM administratiom, and the damaged contraction reponses to PE was reversed by rosiglitazone.
2 Rosiglitazone protects endothelial cells of pulmonary artery and improves the endothelial cells-dependedent- relaxtion. Compared with PARs in NS+NS group with endothelium, the relaxation reponses to Ach of PARs in BLM+NS with endothelium group were significantly decreased (0.93±0.08 vs 0.43±0.14) (P<0.01) . In BLM+RSG group, the relaxtion responses to Ach of PARs were markedly improved compared with those in BLM+NS group (0.69±0.18 vs 0.43±0.14,P<0.05), which indicated that rosiglitazone impoves the function of pulmonary artery endothelial cells and reduce the damage to pulmonary artery endothelium induced by BLM, this may be one of mechanisms involved in rosiglitazone reversing the pulmonary artery hypertension induced by BLM.
3 Aminoguanidine (AG) has no effect on the PARs contraction reponses to PE in all groups. In all groups, the PARs contraction responses to PE were not changed after preincubation with AG(10-4mol/L), which indicated that in pulmonary artery hypertension induced by BLM NO derived by iNOS may not participate in the regulation of pulmoary artery vascular tension.
4 L-NAME preincubation enhance the PARs contraction responses to PE in groups with endothelium. After incubation with L-NAME, the contraction responses of PARs to PE in NS+NS group with endothelium significantly increased (1.20±0.50 g/mg·d·w vs 1.73±0.57 g/mg·d·w) (P<0.01) , while the contraction reponses of PARs in BLM+NS group with endothlium did not changed after preincubation with L-NAME. In BLM+RSG with endothelium group the contraction responses to PE markedly increased (1.31±0.38 vs 1.81±0.40) (P<0.05) . The contraction responses to PE in all groups without endothelium had no siginificant differences.The result indicated that rosiglitazone protect pulmonary artery vascular endothelial cells.
5 RSG reduces the expression of NT protein in pulmonary artery induced by BLM. In NS+NS group, the expression of NT in pulmonary artery was slight (average optical density: 0.31±0.01, percentage of positive areas:6.87±0.56). The expression of NT protein of pulmonary artery in BLM+NS group was significantly increased (average optical density: 0.39±0.02, percentage of positive areas: 21.42±1.84) compared with that in NS+NS group, (P<0.01). The expression of NT protein of pulmonary artery in BLM+RSG group (average optical density: 0.34±0.01, percentage of positive areas: 9.80±0.94) was lower than that in BLM+NS (P<0.05), and still higher than that in NS+NS group (P<0.05).
6 Rosiglitazone reduce the expression of NT in pulmonary artery in lungs induced by bleomycin. Small arteries in lung (diamerter<100um): In NS+NS group, the expression of NT in pulmonary artery was slight (average optical density: 0.31±0.01, percentage of positive areas: 4.89±1.00). Compared with NS+NS group, the expression of NT in BLM+NS group increased significantly (average optical density: 0.40±0.02, percentage of positive areas:14.60±2.55) (P<0.01). The expression of NT in pulmonary artery in BLM+RSG group (average optical density: 0.31±0.01, percentage of positive areas: 5.84±1.02) was lower than that in BLM+NS group (P<0.01) and was similar to that in NS+NS group(P>0.05). Larger arteries in Lung (diamerter>100um) : In NS+NS group, the expression of NT in pulmonary artery was slight (average optical density: 0.31±0.01, percentage of positive areas: 5.52±0.83).Compared with NS+NS group, the expression of NT in BLM+NS group increased significantly (average optical density: 0.38±0.02, percentage of positive areas: 16.47±1.82) (P<0.01). The expression of NT in BLM+RSG group was lower than that in BLM+NS group (P<0.01), and was similar to that in NS+NS group (P>0.05).
The results suggested that: (1) the contraction responses of PARs to PE and the endothlium-dependent relaxtion resposes of PARs to Ach were striking impaired by BLM, which led to PARs reactivties disorder; (2) the expression of NT in pulmonary arteries was markedly increased by BLM; (3) Rosiglitazone could reduce the expression of NT in pulmonary artery, partly protect the funtion of pulmonary artery endtholium, and reverse the abnormal funtion of smooth muscle induced by BLM.
Conclusions: 1 The expression of iNOS and NT in all kinds of pulmonary arteries was significantly increased and endothelium of pulmonary artery was injured by bleomycin, which maybe implicated in the bleomycin induced pulmonary artery hypertension.
2 Bleomycin induced the markedly impaired contraction to PE and the relaxtion to Ach of PARs, which had led to the disorder of pulmonary artery vascular reactivity. This might be one of the mechanisms of pulmonary hypertension in this model.
3 Rosiglitazone could significantly reduce the expression of iNOS and NT in all kinds of pulmonary arteries and reverse the impaired contraction responses of PARs. In addition, rosiglitazone could protect the function of pulmonary artery endothelial cells and correct the disorder of PARs induced by BLM. All above may be one of the mechanisms that rosiglitazone reverse the pulmonary artery hypertension induced by bleomycin.
引文
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2 Bonartsev AP, Slavutskaia A, Postnikov AB, et al. Effect of chronic administration of aminoguanidine on the reactivity of pulmonary vessels in rats with monocrotaline-induced pulmonary hypertension. Ross Fiziol Zh Im I M Sechenova, 2004, 90(7):908~915
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7 Zweier JL. Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci U S A, 1997, 94(13):6954~6958
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9 Cuzzocrea S, Pisano B, Dugo L, et al. Rosiglitazone, a ligand of the peroxisome proliferator-activated receptor-gamma, reduces the development of nonseptic shock induced by zymosan in mice. Crit Care Med, 2004, 32(2):457~466
10 Liu D, Zeng BX, Yao SL, et al. Rosiglitazone, an agonist of peroxisome proliferator-activated receptor gamma, reduces pulmonary inflammatory response in a rat model of endotoxemia. Inflamm Res, 2005, 54(11):464~470
11 Bonartsev AP, D'iakonov KB, Postnikov AB, et al. Effect of chronic administration of aminoguanidine on vascularreactivity of the greater circulation in rats with monocrotaline-induced pulmonary hypertension. Izv Akad Nauk Ser Biol, 2005, (3):316~322
12 Wang S, Jiang JL, Hu CP, et al. Relationship between protective effects of rosiglitazone on endothelium and endogenous nitric oxide synthase inhibitor in streptozotocin-induced diabetic rats and cultured endothelial cells. Diabetes Metab Res Rev, 2007, 23(2):157~164
13 Pistrosch F, Passauer J, Fischer S, et al. In type 2 diabetes, rosiglitazone therapy for insulin resistance ameliorates endothelial dysfunction independent of glucose control. Diabetes Care, 2004, 27(2):484~490
14 Zsolt Bagi, Akos Koller, Gabor Kaley. PPAR activation, by reducing oxidative stress, increases NO bioavailability in coronary arterioles of mice with Type 2 diabetes. Am J Physiol Heart Circ Physiol, 2004, 286(2):H742~748
15 Petrofsky JS, Lee S. The impact of rosiglitazone on cardiovascular responses and endurance during isometric exercise in patients with Type 2 diabetes. Med Sci Monit, 2006, 12(1):CR21~26
16 Bagi Z, Koller A, Kaley G. PPARgamma activation, by reducing oxidative stress, increases NO bioavailability in coronary arterioles of mice with Type 2 diabetes. Am J Physiol Heart Circ Physiol, 2004, 286(2):H742~748
17 Buga GM, Griscavage JM, Rogers NE, et al. Negative feedback regulation of endothelial cell function by nitric oxide. Circ Res, 1993, 73: 808~812
18 凌宏艳,胡必利,奉水东等. 罗格列酮对胰岛素抵抗大鼠血一氧化氮和内皮素的影响.中国动脉硬化杂志, 2005, 13(5):557~559
1 Simonneau G, Galien N, RubinLJ, et al. Clinical classification of pulmonary hypertension. J Am Coll Cardiol, 2004, 43(12):5S~12S1
2 Ignarro LJ, Harbison RG, Wood KS, et al. Activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid. J Pharmacol Exp Ther, 1986, 237:893~900
3 Dabrowska K, Hehre D, Young KC, et al. Effects of a nebulized NONOate, DPTA/NO, on group Bstreptococcus-induced pulmonary hypertension in newborn piglets. Pediatr Res, 2005, 57(3):378~383
4 陈运彬,杨杰,潘力.一氧化氮吸入法治疗新生儿肺动脉高压临床观察.临床儿科杂志, 2001, 19(2):78~80
5 Korhonen R, Lahti A, Kankaanranta H, et al. Nitric oxide production and signaling in inflammation. Curr Drug Targets Inflamm Allergy, 2005, 4(4): 471~479
6 Li J, Baud O, Vartanian T, et al. Peroxynitrite generated by inducible nitric oxide synthase and NADPH oxidase mediates microglial toxicity to oligodendrocytes. Proc Natl Acad Sci U S A, 2005, 102(28):9936~9941
7 Václav Hampl, Jana Bíbová, Alena Ba asová, et al. Pulmonary vascular iNOS induction participates in the onset of chronic hypoxic pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol, 2006, 290: L11~L20
8 David J. Vaughan, Thomas V. Brogan, Mark E. Kerr, et al. Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs. Am J Physiol Lung Cell Mol Physiol, 2003, 284:L834~L843
9 Muramatsu, M. R. C. Tyler, D. M. Rodman, et al. Thapsigargin stimulates increased NO activity in hypoxic hypertensive rat lungs and pulmonary arteries. J. Appl. Physiol, 1996, 80:1336~1344
10 Fagan KA, Tyler RC, Sato K, et al. Relative contributions of endothelial, inducible, and neuronal NOS to tone in the murine pulmonary circulation. Am J Physiol, 1999, 277(3 Pt 1):L472~478
11 Xia Y, Zweier JL. Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci U S A, 1997, 94(13):6954~6958
12 Govers R, Rabelink TJ. Cellular regulation of endothelial nitric oxide synthase. Am J Physiol Renal Physiol, 2001, 280:F193~F206
13 Shaul PW. Regulation of endothelial nitric oxide synthase: location, location, location. Annu Rev Physiol, 2002, 64: 749~774
14 Konduri GG, Bakhutashvili I, Eis A, et al. Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension. Am J Physiol Heart Circ Physiol, 2007, 292(4):H1812~820
15 Ozaki M, Kawashima S, Yamashita T, et al. Reduced hypo- xic pulmonary vascular remodeling by nitric oxide from the endothelium, 2001, 37(2):322~327
16 Xia Y, Dawson VL, Dawson TM, et al. Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury. Proc Natl Acad Sci USA, 1996, 93: 6770~6774
17 Konduri GG, Bakhutashvili I, Eis A, et al. Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension, 2007, 292(4):H1812~820
18 Muzaffar S, Shukla N, Angelini GD, et al. Acute hypoxia simultaneously induces the expression of gp91phox and endothelial nitric oxide synthase in the porcine pulmonary artery. Thorax, 2005, 60(4):265~267
19 Bitar MS, Wahid S, Mustafa S, et al. Nitric oxide dynamics and endothelial dysfunction in type II model of genetic diabetes. Eur J Pharmacol, 2005, 511(1):53~64
20 Bauersachs J, Bouloumié A, Fraccarollo D, et al. Endo- thelial dysfunction in chronic myocardial infarction despite increased vascular endothelial nitric oxide synthase and soluble guanylate cyclase expression: role of enhanced vascular superoxide production. Circulation, 1999, 100(3):292~298
21 Patel JD, Krupka T, Anderson JM. iNOS-mediated gen- eration of reactive oxygen and nitrogen species by biomaterial-adherent neutrophils. J Biomed Mater Res A, 2007, 80(2):381~390
22 Miller AA, Megson IL, Gray GA, et al. Inducible nitric oxide synthase-derived superoxide contributes to hypereactivity in small mesenteric arteries from a rat model of chronic heart failure. Br J Pharmacol, 2000, 131(1):29~36
23 Hoshikawa Y, Ono S, Suzuki S, et al. Generation of oxidative stress contributes to the development of pulmonary hypertension induced by hypoxia. J Appl Physiol, 2001, 90:1299~1306
24 Lachmanová V, Hnili ková O, Pov ilová V, et al. N-acetylcysteine inhibits hypoxic pulmonary hypertension most effectively in the initial phase of chronic hypoxia. Life Sci, 2005, 77:175~182
25 Herget J, Wilhelm J, Novotná J, et al. A possible role of the oxidant tissue injury in the development of hypoxic pulmonary hypertension. Physiol Res, 2000, 49:493~501
26 Bowers R, Cool C, Murphy RC, et al. Oxidative stress in severe pulmonary hypertension. Am J Respir Crit Care Med,2004, 169:764~769
27 Bonartsev AP, Slavutskaia A, Postnikov AB, et al. Effect of chronic administration of aminoguanidine on the reactivity of pulmonary vessels in rats with monocrotaline-induced pulmonary hypertension. Ross Fiziol Zh Im I M Sechenova, 2004, 90(7):908~915
28 Raquel Hernanz, Ana M. Briones, María J. Alonso, et al. Hypertension alters role of iNOS, COX-2, and oxidative stress in bradykinin relaxation impairment after LPS in rat cerebral arteries. Am J Physiol Heart Circ Physiol, 2004, 287:H225~H234
29 Ichihara A, Hayashi M, Navar G, et al. Inducible nitric oxide synthase attenuates endothelium-dependent renal microvascular vasodilation. J Am Soc Nephrol, 2000, 11: 1807~1812
30 Gunnett CA, Lund DD, Chu Y, et al. NO-dependent vasorelaxation is impaired after gene transfer of inducibleNO-synthase. Arterioscler Thromb Vasc Biol, 2001, 21(8):1259~1260
31 Gunnett CA, Lund DD, Howard MA, et al. Gene transfer of inducible nitric oxide synthase impairs relaxation in human and rabbit cerebral arteries. Stroke, 2002, 33:2292~2296
32 Hong HJ, Loh SH, Yen MH,et al. Suppression of the development of hypertension by the inhibitor of inducible nitric oxide synthase. Br J Pharmacol, 2000, 131(3):631~637
33 Chauhan SD, Seggara G, Vo PA, et al. Protection against lipopolysaccharide-induced endothelial dysfunction in resistance and conduit vasculature of iNOS knockout mice. FASEB J, 2003, 17:773~775
34 Jukka S. Luoma, Pontus Str?lin, Stefan L. Marklund, et al . Expression of Extracellular SOD and iNOS in Macrophages and Smooth Muscle Cells in Human and Rabbit. Atherosclerotic Lesions Arteriosclerosis, Thrombosis, and Vascular Biology, 1998, 18:157~167
35 Simonet S, Rupin A, Badier-Commander C, et al. Evidence for superoxide anion generation in aortas of cholesterol-fed rabbits treated with L-arginine. Eur J Pharmacol, 2004, 492(2-3):211~216
36 Ferro TJ, Gertzberg N, Selden L, et al. Endothelial barrier dysfunction and p42 oxidation induced by TNF-alpha are mediated by nitric oxide. Am J Physiol, 1997, 272(5 Pt 1):L979~988
37 Chakraborti T, Das S, Chakraborti S. Proteolytic activation of protein kinase Calpha by peroxynitrite in stimulating cytosolic phospholipase A2 in pulmonary endothelium: involvement of a pertussis toxin sensitive protein. Biochemistry, 2005, 44(13):5246~5257
38 Gu ZY, Ling YL, Xu XH, et al. Endogenous peroxynitrite mediates lipopolysaccharide-induced injury in cultured pulmonary artery endothelial cells. Sheng Li Xue Bao, 2003, 55(4):475~480
39 Gu Z, Ling Y, Cong B. Peroxynitrite mediated acute lung injury induced by lipopolysaccharides in rats. Zhong hua Yi Xue Za Zhi, 2000, 80(1):58~61
40 James L. Knepler Jr., Loui N. Taher, Mahesh P. Gupta, et al. Peroxynitrite causes endothelial cell monolayer barrier dysfunction. Am J Physiol Cell Physiol, 2001, 281: C1064~C1075
41 Joseph S. Beckman. –OONO. Rebounding From Nitric Oxide. Editoria 2001 American Heart Association, Inc.
42 Wedgwood S, McMullan DM, Bekker JM, et al. Role for endothelin-1 induced superoxide and peroxynitrite production in rebound pulmonary hypertension associated with inhaled nitric oxide therapy. Circ Res, 2001 , 89(4):295~267
43 Peter Oishi, Albert Grobe, Eileen Benavidez, et al. Inhaled nitric oxide induced NOS inhibition and rebound pulmonary hypertension: a role for superoxide andperoxynitrite in the intact lamb. Am J Physiol Lung Cell Mol Physiol, 2006, 290: L359~L366
2 Bonartsev AP, D'iakonov KB, Postnikov AB, et al. Effect of chronic administration of aminoguanidine on vascular reactivity of the greater circulation in rats with monocrotaline-induced pulmonary hypertension. Izv Akad Nauk Ser Biol, 2005, (3):316~322
3 Gunnett CA, Lund DD, Chu Y, et al. NO-dependent vasorelaxation is impaired after gene transfer of inducible NO-synthase. Arterioscler Thromb Vasc Biol, 2001, 21(8):1259~1260
4 Tsai BM, Wang M, Pitcher JM, et al. Disparate IL-1betaand iNOS gene expression in the aorta and pulmonary artery after endotoxemia. Surg Infect (Larchmt), 2006, 7(1):21~27
5 Genovese T, Cuzzocrea S, Di Paola R, et al. Effect of rosiglitazone and 15-deoxy-Delta12,14-prostaglandin Ja on bleomycin-induced lung injury. Eur Respir J, 2005, 25(2):225~234
6 Chang PC, Chen TH, Chang CJ, et al. Advanced glycosylation end products induce inducible nitric oxide synthase (iNOS) expression via a p38 MAPK-dependent pathway. Kidney Int, 2004, 65(5):1664~1675
7 Folch-Puy E, Granell S, Iovanna JL, et al. Peroxisome proliferator-activat-ed receptor gamma agonist reduces the severity of post-ERCP pancreatitis in rats. World J Gastroenterol, 2006, 12(40):6458~6463
8 魏经国, 崔光彬, 王玮等. 博莱霉素所致大鼠肺纤维化及与肺血管内皮损伤的关系.中化劳动卫生职业病杂志, 2004, 22(5):354~357
9 Cuzzocrea S, Genovese T, Mazzon E, et al. Glycogen synthase kinase-3beta inhibition attenuates the development of bleomycin-induced lung injuryInt. J Immunopathol Pharmacol, 2007, 20(3):619~630
10 Browner NC, Sellak H, Lincoln TM. Downregulation of cGMP-dependent protein kinase expression by inflammatory cytokines in vascular smooth muscle cells. Am J Physiol Cell Physiol, 2004, 287(1):C88~96
11 孔璐, 王继峰, 牛建照等.气管内一次性注入博莱霉素诱发大鼠肺损伤的动态变化.中国现代医学杂志, 2004, 14(24):14~18
12 Zhang Y, Liu LM, Ming J, et al. Regulatory role of hypoxia inducible factor-1alpha in the changes of contraction of vascular smooth muscle cell induced by hypoxiaZhongguo Wei Zhong Bing Ji Jiu Yi Xue, 2007, 19(11):647~651
13 田宏,杜军保,范瑾等.一氧化氮诱导大鼠肺动脉平滑肌细 胞 凋 亡 机 制 研 究 . 中 国 病 理 生 理 杂 志 , 2003, 19(7):870~874
14 Idel S, Ellinghaus P, Wolfrum C, et al. Branched chain fatty acids induce nitric oxide-dependent apoptosis in vascular smooth muscle cells. J Biol Chem, 2002, 277(51):49319~49325
15 Yang YM, Wang SJ, Jin DD, et al. Inhibitory effects of emodin on proliferation of rat vascular smooth muscle cell induced by angiotensin II. Zhong guo Zhong Yao Za Zhi, 2008, 33(1):63~67
16 Hampl V, Bibova J, Banasova A, et al. Pulmonary vascular iNOS induction participates in the onset of chronic hypoxic pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol, 2006, 290(1):L11~20
17 Hong HJ, Loh SH, Yen MH. Suppression of the development of hypertension by the inhibitor of inducible nitric oxide synthase. Br J Pharmacol, 2000, 131(3):631~637
18 Zweier JL. Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci U S A, 1997, 94(13):6954~6958
19 Tang ZL, Wasserloos KJ, Liu X, et al. Nitric oxide decreases thesensitivity of pulmonary endothelial cells to LPS-inducedapoptosis in a zinc-dependent fashion. Mol Cell Biochem, 2002, 234(1):211~217
20 Valledor AF, Sánchez-Tilló E, Arpa L, et al. Selective Roles of MAPKs during the Macrophage Response to IFN-{gamma}. J Immunol, 2008, 180(7):4523~4529
21 Natarajan M, Gibbons CF, Mohan S, et al. Oxidative stress signalling: a potential mediator of tumour necrosis factor alpha-induced genomic instability in primary vascular endothelial cells. Br J Radiol, 2007, 80:S13-22
22 Amore A, Cirina P, Conti G, et al. Amadori-configurated albumin induces nitric oxide-dependent apoptosis of endothelial cells: a possible mechanism of diabetic vasculopathy. Nephrol Dial Transplant, 2004, 19(1):53~60
23 Hong HJ, Loh SH, Yen MH. Suppression of the development of hypertension by the inhibitor of inducible nitric oxide synthase. Br J Pharmacol, 2000, 131(3):631~637
24 Dilley R J, Jennings GL, Law RE, et al. Inhibitory activity of clinical thiazolidinedione peroxisome proliferators zativating receptor-gammar ligands toward internal mammary artery, radial artery and saphenous vein smoothmuscle cell proliferation. J.Circulation, 2002, 107(20):2448~2550
25 Wedgwood S, McMullan DM, Bekker JM, et al. Role for endothelin-1-induced superoxide and peroxynitrite production in rebound pulmonary hypertension associated with inhaled nitric oxide therapy. Circ Res, 2001, 89(4):295~297
26 Wang S, Jiang JL, Hu CP, et al. Relationship between protective effects of rosiglitazone on endothelium and endogenous nitric oxide synthase inhibitor in streptozotocin-induced diabetic rats and cultured endothelial cells. Diabetes Metab Res Rev, 2007, 23(2):157~164
1 Raquel Hernanz, Ana M. Briones, María J. Alonso, et al. Hypertension alters role of iNOS, COX-2, and oxidative stress in bradykinin relaxation impairment after LPS in rat cerebral arteries. Am J Physiol Heart Circ Physiol, 2004, 287:H225~H234
2 Bonartsev AP, Slavutskaia A, Postnikov AB, et al. Effect of chronic administration of aminoguanidine on the reactivity of pulmonary vessels in rats with monocrotaline-induced pulmonary hypertension. Ross Fiziol Zh Im I M Sechenova, 2004, 90(7):908~915
3 Gunnett CA, Lund DD, Chu Y, et al. NO-dependent vasorelaxation is impaired after gene transfer of inducible NO-synthase. Heistad DDArterioscler Thromb Vasc Biol, 2001, 21(8):1259~1260
4 谷振勇,凌亦凌,许小虎等.过氧亚硝基阴离子对离体兔肺动脉反应性变化的影响.生理学报, 2003, 55(4):469~474
5 Václav Hampl, Jana Bíbová, Alena Ba asová, et al. Pulmonary vascular iNOS induction participates in the onset of chronic hypoxic pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol, 2006, 290: L11~L20
6 Herget J, Wilhelm J, Novotná J, et al. A possible role of the oxidant tissue injury in the development of hypoxic pulmonary hypertension. Physiol Res, 2000, 49: 493~501
7 Zweier JL. Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci U S A, 1997, 94(13):6954~6958
8 Genovese T, Cuzzocrea S, Di Paola R, et al. Effect of rosiglitazone and 15-deoxy-Delta12,14-prostaglandin Ja on bleomycin-induced lung injury. Eur Respir J, 2005, 25(2):225~234
9 Cuzzocrea S, Pisano B, Dugo L, et al. Rosiglitazone, a ligand of the peroxisome proliferator-activated receptor-gamma, reduces the development of nonseptic shock induced by zymosan in mice. Crit Care Med, 2004, 32(2):457~466
10 Liu D, Zeng BX, Yao SL, et al. Rosiglitazone, an agonist of peroxisome proliferator-activated receptor gamma, reduces pulmonary inflammatory response in a rat model of endotoxemia. Inflamm Res, 2005, 54(11):464~470
11 Bonartsev AP, D'iakonov KB, Postnikov AB, et al. Effect of chronic administration of aminoguanidine on vascularreactivity of the greater circulation in rats with monocrotaline-induced pulmonary hypertension. Izv Akad Nauk Ser Biol, 2005, (3):316~322
12 Wang S, Jiang JL, Hu CP, et al. Relationship between protective effects of rosiglitazone on endothelium and endogenous nitric oxide synthase inhibitor in streptozotocin-induced diabetic rats and cultured endothelial cells. Diabetes Metab Res Rev, 2007, 23(2):157~164
13 Pistrosch F, Passauer J, Fischer S, et al. In type 2 diabetes, rosiglitazone therapy for insulin resistance ameliorates endothelial dysfunction independent of glucose control. Diabetes Care, 2004, 27(2):484~490
14 Zsolt Bagi, Akos Koller, Gabor Kaley. PPAR activation, by reducing oxidative stress, increases NO bioavailability in coronary arterioles of mice with Type 2 diabetes. Am J Physiol Heart Circ Physiol, 2004, 286(2):H742~748
15 Petrofsky JS, Lee S. The impact of rosiglitazone on cardiovascular responses and endurance during isometric exercise in patients with Type 2 diabetes. Med Sci Monit, 2006, 12(1):CR21~26
16 Bagi Z, Koller A, Kaley G. PPARgamma activation, by reducing oxidative stress, increases NO bioavailability in coronary arterioles of mice with Type 2 diabetes. Am J Physiol Heart Circ Physiol, 2004, 286(2):H742~748
17 Buga GM, Griscavage JM, Rogers NE, et al. Negative feedback regulation of endothelial cell function by nitric oxide. Circ Res, 1993, 73: 808~812
18 凌宏艳,胡必利,奉水东等. 罗格列酮对胰岛素抵抗大鼠血一氧化氮和内皮素的影响.中国动脉硬化杂志, 2005, 13(5):557~559
1 Simonneau G, Galien N, RubinLJ, et al. Clinical classification of pulmonary hypertension. J Am Coll Cardiol, 2004, 43(12):5S~12S1
2 Ignarro LJ, Harbison RG, Wood KS, et al. Activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid. J Pharmacol Exp Ther, 1986, 237:893~900
3 Dabrowska K, Hehre D, Young KC, et al. Effects of a nebulized NONOate, DPTA/NO, on group Bstreptococcus-induced pulmonary hypertension in newborn piglets. Pediatr Res, 2005, 57(3):378~383
4 陈运彬,杨杰,潘力.一氧化氮吸入法治疗新生儿肺动脉高压临床观察.临床儿科杂志, 2001, 19(2):78~80
5 Korhonen R, Lahti A, Kankaanranta H, et al. Nitric oxide production and signaling in inflammation. Curr Drug Targets Inflamm Allergy, 2005, 4(4): 471~479
6 Li J, Baud O, Vartanian T, et al. Peroxynitrite generated by inducible nitric oxide synthase and NADPH oxidase mediates microglial toxicity to oligodendrocytes. Proc Natl Acad Sci U S A, 2005, 102(28):9936~9941
7 Václav Hampl, Jana Bíbová, Alena Ba asová, et al. Pulmonary vascular iNOS induction participates in the onset of chronic hypoxic pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol, 2006, 290: L11~L20
8 David J. Vaughan, Thomas V. Brogan, Mark E. Kerr, et al. Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs. Am J Physiol Lung Cell Mol Physiol, 2003, 284:L834~L843
9 Muramatsu, M. R. C. Tyler, D. M. Rodman, et al. Thapsigargin stimulates increased NO activity in hypoxic hypertensive rat lungs and pulmonary arteries. J. Appl. Physiol, 1996, 80:1336~1344
10 Fagan KA, Tyler RC, Sato K, et al. Relative contributions of endothelial, inducible, and neuronal NOS to tone in the murine pulmonary circulation. Am J Physiol, 1999, 277(3 Pt 1):L472~478
11 Xia Y, Zweier JL. Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci U S A, 1997, 94(13):6954~6958
12 Govers R, Rabelink TJ. Cellular regulation of endothelial nitric oxide synthase. Am J Physiol Renal Physiol, 2001, 280:F193~F206
13 Shaul PW. Regulation of endothelial nitric oxide synthase: location, location, location. Annu Rev Physiol, 2002, 64: 749~774
14 Konduri GG, Bakhutashvili I, Eis A, et al. Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension. Am J Physiol Heart Circ Physiol, 2007, 292(4):H1812~820
15 Ozaki M, Kawashima S, Yamashita T, et al. Reduced hypo- xic pulmonary vascular remodeling by nitric oxide from the endothelium, 2001, 37(2):322~327
16 Xia Y, Dawson VL, Dawson TM, et al. Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury. Proc Natl Acad Sci USA, 1996, 93: 6770~6774
17 Konduri GG, Bakhutashvili I, Eis A, et al. Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension, 2007, 292(4):H1812~820
18 Muzaffar S, Shukla N, Angelini GD, et al. Acute hypoxia simultaneously induces the expression of gp91phox and endothelial nitric oxide synthase in the porcine pulmonary artery. Thorax, 2005, 60(4):265~267
19 Bitar MS, Wahid S, Mustafa S, et al. Nitric oxide dynamics and endothelial dysfunction in type II model of genetic diabetes. Eur J Pharmacol, 2005, 511(1):53~64
20 Bauersachs J, Bouloumié A, Fraccarollo D, et al. Endo- thelial dysfunction in chronic myocardial infarction despite increased vascular endothelial nitric oxide synthase and soluble guanylate cyclase expression: role of enhanced vascular superoxide production. Circulation, 1999, 100(3):292~298
21 Patel JD, Krupka T, Anderson JM. iNOS-mediated gen- eration of reactive oxygen and nitrogen species by biomaterial-adherent neutrophils. J Biomed Mater Res A, 2007, 80(2):381~390
22 Miller AA, Megson IL, Gray GA, et al. Inducible nitric oxide synthase-derived superoxide contributes to hypereactivity in small mesenteric arteries from a rat model of chronic heart failure. Br J Pharmacol, 2000, 131(1):29~36
23 Hoshikawa Y, Ono S, Suzuki S, et al. Generation of oxidative stress contributes to the development of pulmonary hypertension induced by hypoxia. J Appl Physiol, 2001, 90:1299~1306
24 Lachmanová V, Hnili ková O, Pov ilová V, et al. N-acetylcysteine inhibits hypoxic pulmonary hypertension most effectively in the initial phase of chronic hypoxia. Life Sci, 2005, 77:175~182
25 Herget J, Wilhelm J, Novotná J, et al. A possible role of the oxidant tissue injury in the development of hypoxic pulmonary hypertension. Physiol Res, 2000, 49:493~501
26 Bowers R, Cool C, Murphy RC, et al. Oxidative stress in severe pulmonary hypertension. Am J Respir Crit Care Med,2004, 169:764~769
27 Bonartsev AP, Slavutskaia A, Postnikov AB, et al. Effect of chronic administration of aminoguanidine on the reactivity of pulmonary vessels in rats with monocrotaline-induced pulmonary hypertension. Ross Fiziol Zh Im I M Sechenova, 2004, 90(7):908~915
28 Raquel Hernanz, Ana M. Briones, María J. Alonso, et al. Hypertension alters role of iNOS, COX-2, and oxidative stress in bradykinin relaxation impairment after LPS in rat cerebral arteries. Am J Physiol Heart Circ Physiol, 2004, 287:H225~H234
29 Ichihara A, Hayashi M, Navar G, et al. Inducible nitric oxide synthase attenuates endothelium-dependent renal microvascular vasodilation. J Am Soc Nephrol, 2000, 11: 1807~1812
30 Gunnett CA, Lund DD, Chu Y, et al. NO-dependent vasorelaxation is impaired after gene transfer of inducibleNO-synthase. Arterioscler Thromb Vasc Biol, 2001, 21(8):1259~1260
31 Gunnett CA, Lund DD, Howard MA, et al. Gene transfer of inducible nitric oxide synthase impairs relaxation in human and rabbit cerebral arteries. Stroke, 2002, 33:2292~2296
32 Hong HJ, Loh SH, Yen MH,et al. Suppression of the development of hypertension by the inhibitor of inducible nitric oxide synthase. Br J Pharmacol, 2000, 131(3):631~637
33 Chauhan SD, Seggara G, Vo PA, et al. Protection against lipopolysaccharide-induced endothelial dysfunction in resistance and conduit vasculature of iNOS knockout mice. FASEB J, 2003, 17:773~775
34 Jukka S. Luoma, Pontus Str?lin, Stefan L. Marklund, et al . Expression of Extracellular SOD and iNOS in Macrophages and Smooth Muscle Cells in Human and Rabbit. Atherosclerotic Lesions Arteriosclerosis, Thrombosis, and Vascular Biology, 1998, 18:157~167
35 Simonet S, Rupin A, Badier-Commander C, et al. Evidence for superoxide anion generation in aortas of cholesterol-fed rabbits treated with L-arginine. Eur J Pharmacol, 2004, 492(2-3):211~216
36 Ferro TJ, Gertzberg N, Selden L, et al. Endothelial barrier dysfunction and p42 oxidation induced by TNF-alpha are mediated by nitric oxide. Am J Physiol, 1997, 272(5 Pt 1):L979~988
37 Chakraborti T, Das S, Chakraborti S. Proteolytic activation of protein kinase Calpha by peroxynitrite in stimulating cytosolic phospholipase A2 in pulmonary endothelium: involvement of a pertussis toxin sensitive protein. Biochemistry, 2005, 44(13):5246~5257
38 Gu ZY, Ling YL, Xu XH, et al. Endogenous peroxynitrite mediates lipopolysaccharide-induced injury in cultured pulmonary artery endothelial cells. Sheng Li Xue Bao, 2003, 55(4):475~480
39 Gu Z, Ling Y, Cong B. Peroxynitrite mediated acute lung injury induced by lipopolysaccharides in rats. Zhong hua Yi Xue Za Zhi, 2000, 80(1):58~61
40 James L. Knepler Jr., Loui N. Taher, Mahesh P. Gupta, et al. Peroxynitrite causes endothelial cell monolayer barrier dysfunction. Am J Physiol Cell Physiol, 2001, 281: C1064~C1075
41 Joseph S. Beckman. –OONO. Rebounding From Nitric Oxide. Editoria 2001 American Heart Association, Inc.
42 Wedgwood S, McMullan DM, Bekker JM, et al. Role for endothelin-1 induced superoxide and peroxynitrite production in rebound pulmonary hypertension associated with inhaled nitric oxide therapy. Circ Res, 2001 , 89(4):295~267
43 Peter Oishi, Albert Grobe, Eileen Benavidez, et al. Inhaled nitric oxide induced NOS inhibition and rebound pulmonary hypertension: a role for superoxide andperoxynitrite in the intact lamb. Am J Physiol Lung Cell Mol Physiol, 2006, 290: L359~L366