【作者】 郭军；

【导师】 陆慰萱； 郭子建； 施举红；

【作者基本信息】 中国协和医科大学 ， 内科学， 2010， 博士

【摘要】 背景和目的肺动脉高压(PH)是一组异源性疾病和不同发病机制引起的以肺血管阻力持续增加为特征的临床-病理生理综合征。动脉型肺动脉高压(PAH)是PH的常见的类型之一,以肺小动脉的增生、重构为特点。野百合碱(MCT)诱导PAH (MCT-PAH)大鼠是研究最常用模型之一。已证实肾素血管紧张素系统(RAS)在PAH发病中发挥一定作用。替米沙坦是新一代长效血管紧张素Ⅱ的1型受体(AT1R)拮抗剂,已经证实它有抗炎、抑制心脏和血管肥大、重构和细胞增殖等作用。本研究通过建立MCT-PAH大鼠模型,观察替米沙坦预防干预对PAH肺血管重构的保护效应,并进一步探讨可能的作用机制。方法1.实验动物分组：Sprague-Dawley大鼠32只,每组8只,随机分为：对照组(C组),皮下注射NS1次。PAH模型组(P组),皮下注射MCT (60mg/Kg体重)1次,NS灌胃3周。PAH+Tel高剂量组(PTh组),皮下注射MCT (60mg/Kg体重)1次,当日即开始Tel (10mg/kg/day)灌胃3周。PAH+Tel低剂量组(PTl组),皮下注射MCT (60mg/Kg体重)1次,当日同时开始Tel (5mg/kg/day)灌胃3周。2.干预3周后大鼠行右心导管检查测定右心室收缩压(RVSP)和肺动脉平均压(mPAP);分离心脏测定右心室肥大指数(RVHI)、肺小动脉中膜厚度百分比(WT%)、腺泡内肺小动脉肌化程度以及对肺小动脉周围炎性程度进行评分。3.应用ELISA试剂盒测定肺组织中IL-6、MCP-1和TNF-α蛋白水平的表达。4.应用免疫组化技术检测MMP-2和MMP-9在肺血管的表达；应用实时荧光定量PCR (RT-PCR)技术检测肺组织中MMP-2、MMP-9和TIMP-1 mRNA的表达；明胶酶谱法检测肺组织MMP-2和MMP-9的活性。5.应用免疫组化技术检测ACE2蛋白在肺血管的表达情况；应用RT-PCR技术检测肺组织中ACE2 mRNA的表达。结果1.替米沙坦对MCT-PAH大鼠mPAP、RVSP、RVHI、肺小动脉重构和肺小动脉周围炎症评分的影响：C组mPAP、RVSP和RVHI分别为17.13±3.30mmHg,40.33±5.77mmHg和0.25±0.02；P组此三项指标(37.00±4.98mmHg,67.04±4.87mmHg,0.40±0.02)均较C组明显增高,存在显著性差异(P＜0.01)；替米沙坦可抑制MCT诱导mPAP、RVSP、RVHI的升高,PTh和PTl组此三项指标分别为24.75±4.69mmHg,52.30±8.37mmHg,0.29±0.01和26.27±5.46mmHg,53.25±8.65mmHg,0.34±0.02,两组与P组比较均存在显著性差异(P＜0.01),两组与C组比较仍存在显著性差异(P<0.01); PTh与PTl组间mPAP、RVSP无显著性差异(P>0.05), RVHI在PTh组较PTl组更低,存在显著性差异(0.29±0.01 vs0.34±0.02,P＜0.01)。P组肺病理HE染色显示肌型肺动脉管壁明显增厚,平滑肌层增生肥厚明显,管腔狭窄,腺泡内肺微小动脉肌型化。P组肺小动脉中膜厚度百分比(WT%)较C组明显增加,存在显著性差异(45.52±5.48% vs 8.91±2.30%, P<0.01); PTh和PTl组的WT%分别为13.83±1.83%和20.844±15.21%,均较P组明显降低,存在显著性差异(P＜0.01)；两组均较C组仍增厚,存在显著性差异(P＜0.01)；PTh和PTl组间比较,WT%无显著性差异(P＞0.05)。P组腺泡内肺小动脉肌化程度较C组明显增加,存在显著性差异(67.55±2.95% vs 19.92±2.27%, P＜0.01); PTh和PTl组此指标分别为50.83±3.12%和53.22±1.97%,均较P组明显降低,存在显著性差异(P＜0.01)；两组与C组相比较,此项指标仍增加,存在显著性差异(P<0.01)；PTh和PTl组间相比较,此项指标无显著性差异(P＞0.05)。P组肺小动脉a-SMA免疫组化染色平均光密度(IOD/Area)值较C组有明显增加,存在显著性差异(0.49±0.03vs 0.22±0.04, P<0.01); PTh和PTl组IOD/Area值分别为0.34±0.02和0.36±0.02,均较P组有明显降低,存在显著性差异(P＜0.01)；两组与C组相比较,IOD/Area值仍增加,存在显著性差异(P<0.01); PTh组IOD/Area值和PTl组低,存在显著性差异(P＜0.05)。P组肺小动脉周围有明显的单核细胞浸润,血管周围炎症评分较C组明显增加,存在显著性差异(3.24±0.41 vs 0.40±0.16, P<0.01); PTh和PTl组评分分别为1.00±0.19和1.46±0.31,均较P组明显降低,存在显著性差异(P＜0.01)；两组的炎症评分仍高于C组,存在显著性差异(P＜0.01)。两组间相比较,PTh低于PTl组炎症评分,存在显著性差异(1.00±0.19 vs 1.46±0.31,P＜0.05)。2.替米沙坦对MCT-PAH大鼠肺组织IL-6、TNF-α和MCP-1表达的影响：P组IL-6、TNF-α和MCP-1与C组比较明显增高,存在显著性差异(459.40±94.77pg/ml vs 64.97±17.65pg/ml,436.46±62.20pg/ml vs 56.51±14.92pg/ml, 6265.53±2014.83pg/ml vs 65.18±23.37pg/ml, P<0.01);替米沙坦干预可抑制MCT大鼠肺组织IL-6、TNF-a和MCP-1的升高,PTh和PTl组分别为118.87±25.80pg/ml, 242.61±37.52pg/ml,1094.39±577.75pg/ml和155.87±50.64pg/ml,278.69±42.35pg/ml, 1288.57±593.14pg/ml,与P组比较均明显降低,存在显著性差异(P＜0.01),但三项均仍较C组增高,存在显著性差异(P<0.01); PTh和PTl组间比较IL-6、TNF-α和MCP-1均无显著性差异(P＞0.05)。3.替米沙坦对肺组织MMP-2.MMP-9和TIMP-1 mRNA的表达及MMP-2.MMP-9蛋白表达和活性的影响：P组MMP-2和MMP-9主要在肺血管内膜和外膜及周围的结缔组织表达,明显强于C组,PTh和PTl组均比P组明显降低但仍高于C组。P组肺组织MMP-2、MMP-9和TIMP-1 mRNA表达明显高于C组,均存在显著性差异(3.30±0.39vs0.96±0.08,7.07±0.54 vs 0.96±0.06,3.52±0.44 vs 1.02±0.14, P＜0.01); PTh和PTl组这三项分别为1.97±0.24,3.65±0.70,2.22±0.35和2.43±0.21,4.91±0.68,2.54±0.43,与P组比较均明显降低,存在显著性差异(P＜0.01),但仍均较C组增高,存在显著性差异(P＜0.01); MMP-2和MMP-9 mRNA在PTh组均较PTl组低,存在显著性差异(P＜0.05); TIMP-1 mRNA在这两组间无显著性差异(P＞0.05)。P组肺组织MMP-2、MMP-9的活性明显高于C组,存在显著性差异(54893.89±8480.12 vs 40669.68±4978.39, P<0.05; 23864.69±5121.83 vs 9511.13±3429.93, P<0.01). PTh和PTl组MMP-2活性分别为52401.41±6234.28和51651.18±5289.70,均较P组降低不明显,无显著性差异(P＞0.05),均较C组升高,存在显著性差异(P＜0.05)。PTh和PTl组MMP-9活性分别为14244.99±2601.82和14896.96±3625/29,仍均高于C组,存在显著性差异(P<0.05)；PTh和PTl组MMP-9活性较P组降低,存在显著性差异(分别P＜0.01和P＜0.05)；PTh和PTl组间MMP-2和MMP-9活性均无显著性差异(P＞0.05)。4.替米沙坦对肺组织ACE2 mRNA和蛋白表达的影响：ACE2主要在肺小动脉内膜阳性表达,P组表达量较C组明显下降,PTh和PTl组表达量较P组明显升高,甚至还高于C组。P组肺组织ACE2 mRNA表达明显低于C组,存在显著性差异(0.56±0.09 vs 1.04±0.14, P<0.01); ACE2 mRNA在PTh和PTl组分别为2.08±0.40和1.65±0.44,与P组比较均明显增高,存在显著性差异(P＜0.01),较C组亦均增高,存在显著性差异(P＜0.01)。PTh组mRNA表达较PTl组高,存在显著性差异(P＜0.05)。结论1.替米沙坦预防干预可延缓MCT-PAH大鼠mPAP和RVSP的升高,减轻右心室肥厚和肺血管重构。2.替米沙坦可抑制MCT-PAH大鼠肺组织IL-6、TNF-α和MCP-1升高及肺小动脉周围炎症,可能与其对肺血管的保护作用有关。3.替米沙坦可通过降低MMP-2、MMP-9 mRNA和蛋白表达和MMP-9活性而延缓肺血管重构。4.替米沙坦抑制PAH肺血管重构的机制还可能部分与上调ACE2表达有关。更多还原

【Abstract】 BackgroundPulmonary hypertension （PH） is a common pathophysiologic syndrome caused by a variety of diseases and characterized by a progressive increase of pulmonary vascular resistance and pulmonary artery pressure that finally causes right ventricular failure and premature death. Pulmonary arterial hypertension （PAH） is a disease of the small pulmonary arteries that is characterized by vascular remodeling. Characteristic features of this vascular remodeling include vessel wall thickening as a result of resident vessel wall cell proliferation, migration, and excessive deposition of extracellular matrix. In spite of recent advancements in the treatment of PAH, successful control has yet to be accomplished. The rennin angiotensin system （RAS） has been implicated in the pathogenesis of pulmonary vascular remodeling and PAH in a number of studies. Telmisartan is an angiotensin II type 1 receptor （AT1R） blocker, currently was used to treat patients with hypertension. Moreover, it has been associated with beneficial effects on anti-inflammation, vascular function, cardiac remodeling and renal function. However, little is known about the effects of telmisartan on PAH. To determine the effect of telmisartan on monocrotaline-induced PAH and its possible mechanism, the following study was carried out.Objective1. To investigate the interventional effects of telmisartan on monocrotaline-induced PAH in rats.2. To further explore the possible mechanisms of the protective effects of telmisartan on monocrotaline-induced PAH in rats.Methods1.32 male Sprague-Dawley rats were randomly divided into four groups:Group C （n=8） rats were injected subcutaneously with normal saline; Group P （n₈） rats were injected subcutaneously with monocrotaline （60mg/kg） and received with normal saline by daily oral gavage for 3 weeks; Group PTh （n=8） rats were injected subcutaneously with monocrotaline （60mg/kg） and received telmisartan （10mg/kg） by daily oral gavage for 3 weeks; Group PTl （n=8） rats were injected subcutaneously with monocrotaline （60mg/kg） and received telmisartan （5mg/kg） by daily oral gavage for 3 weeks. 2. Right ventricular systolic pressure （RVSP） and mean pulmonary arterial pressure （mPAP） were measured by right heart catheter after 3 weeks. All rats were killed by exanguination and their hearts and lungs were harvested. The right ventriclular hypertrophy index （RVHI）, percentage of small pulmonary arteries media thickness （WT%）, muscularization degree of pulmonary vessels and score of pulmonary vascular inflammation were evaluated.3. ELISA kits were used to evaluate the proinflammatory cytokines （IL-6, TNF-a and MCP-1） in pulmonary homogenate at protein level.4. The protein expression of MMP-2 and MMP-9 at pulmonary vascular was examined by immunohistochemistry. The mRNA of MMP-2, MMP-9 and TIMP-1 in the lung were measured by real-time PCR. The enzymic activity of MMP-2 and MMP-9 were detected in gelatin zymography.5. The protein expression of ACE2 on pulmonary vascular was examined by immunohistochemistry. The mRNA of ACE2 of the lung was measured by real-time PCR.Results1. Effects of telmisartan on hemodynamic parameter, right ventricular hypertrophy, pulmonary vascular remodeling and perivascular inflammation:The mPAP, RVSP and RVHI of group C were 17.13±3.30mmHg, 40.33±5.77mmHg and 0.25±0.02. These indexes of group P were increased significantly compared with group C （37.00±4.98mmHg,67.04±4.87mmHg and 0.40±0.02, P<0.01）, and attenuated significantly with telmisartan administration （24.75±4.69mmHg, 52.30±8.37mmHg and 0.29±0.01 of group PTh; 26.27±5.46mmHg,53.25±8.65mmHg and 0.34±0.02 of group PTl; P<0.01）. No significant differences in mPAP and RVSP were observed in two telmisartan dosage groups （P>0.05）, but RVHI of group PTh was lower than group PT1 with statistical significance （0.29±0.01 vs 0.34±0.02, P<0.01）.Prominent medial wall hypertrophy, smooth muscle proliferation in muscular pulmonary arteries and muscularized arterioles were evident from rats treated with MCT （group P）. Compared with group C, percentage of small pulmonary arteries media thickness, muscularization of acinus pulmonary arterioles and a-smooth muscle actin expression （IOD/Area） of pulmonary arterioles of group P increased significantly （45.52±5.48% vs 8.91±2.30%,67.55±2.95% vs 19.92±2.27%,0.49±0.03 vs 0.22±0.04; P<0.01）. These increases were also prevented notably with telmisartan intervention （13.83±1.83%,50.83±3.12% and 0.34±0.02 of group PTh; 20.84±15.21%,53.22±1.97% and 0.36±0.02 of group PTl; P<0.01）.No significant differences in WT% and muscularization were observed in two telmisartan dosage groups （P>0.05）, but IOD/Area of group PTh was lower than group PTl with statistical significance （P<0.05）.Macrophages significantly increased in alveoli and primarily mononuclear cells infiltrated around the arterioles in group P. The perivascular inflammation score of group P was remarkably higher than that of group C with statistical significance （3.24±0.41 vs 0.40±0.16, P<0.01）, and was reduced significantly with telmisartan administration （1.00±0.19 of group PTh; 1.46±0.31 of group PTl; P<0.01）. The score of group PTh was lower than group PTl with statistical significance （P<0.05）.2. Effects of telmisartan on pulmonary homogenate IL-6, TNF-a and MCP-1 of rats injected with MCT:The IL-6, TNF-a and MCP-1 level of group P were significantly higher than that of group C with statistical significance （459.40±94.77pg/ml vs 64.97±17.65pg/ml, 436.46±62.20pg/ml vs 56.51±14.92pg/ml,6265.53±2014.83pg/ml vs 65.18±23.37pg/ml; P<0.01）. Also, MCT-induced increases in proinflammatory cytokines were significantly attenuated by telmisartan intervention （118.87±25.80pg/ml,242.61±37.52pg/ml and 1094.39±577.75pg/ml of group PTh; 155.87±50.64pg/ml,278.69±42.35pg/ml and 1288.57±593.14pg/ml of group PTl; P＜0.01）. No significant differences in these proinflammatory cytokines were observed in two telmisartan dosage groups （P>0.05）.3. Effects of telmisartan on expression of MMP-2, MMP-9 and TIMP-1 mRNA in the lungs and MMP-2, MMP-9 protein and activity:Immunohistochemistry for MMP-2 and MMP-9 in pulmonary arterioles of group P revealed that positive staining was localized mainly in the tunica intima and adventitia, which showed a notable increase as compared to group C. Moreover, this expression was decreased after administration of telmisartan. The MMP-2, MMP-9 and TIMP-1 mRNA of group P were significantly higher than that of group C with statistical significance （3.30±0.39 vs 0.96±0.08,7.07±0.54 vs 0.96±0.06,3.52±0.44 vs 1.02±0.14;P<0.01）. These increases were also reduced notably with telmisartan intervention （1.97±0.24, 3.65±0.70 and 2.22±0.35 of group PTh; 2.43±0.21,4.91±0.68 and 2.54±0.43 of group PTl; P<0.01）. No significant difference in TIMP-1 mRNA was observed in two telmisartan dosage groups （P＞0.05）, but MMP-2 and MMP-9 mRNA of group PTh was lower than that of group PTl with statistical significance （P＜0.05）.MMP-2 and MMP-9 enzymatic activity in group P was significantly higher than that of group C （54893.89±8480.12 vs 40669.68±4978.39, P<0.05; 23864.69±5121.83 vs 9511.13±3429.93, P＜0.01）. MMP-2 activity in group PTh and PTl （52401.41±6234.28 and 51651.18±5289.70, respectively） was observed no significant differences compared with that of group P （P<0.05）. In contrast, MMP-9 activity was significantly lower in two telmisartan dosage groups compared with that of group P （14244.99±2601.82 of group PTh, P<0.01; 14896.96±3625.29 of group PTl, P<0.05）. No significant differences in MMP-2 and MMP-9 activity were observed in two telmisartan dosage groups （P>0.05）.4. Effects of telmisartan on expression of ACE2 mRNA or protein in the lung:In pulmonary arterioles ACE2 is preferentially localized in the endothelial layer. Immunohistochemistry results showed a notable decrease of ACE2 positive staining in group P as compared to that of control rats, and its expression is increased after administration of telmisartan. The ACE2 mRNA of group P were significantly lower than that of group C with statistical significance （0.56±0.09 vs 1.04±0.14, P＜0.01）, and this decrease was reversed notably with telmisartan intervention （2.08±0.40 of group PTh, 1.65±0.44 of group PTl; P＜0.01）, furthermore group PTh was higher than group PTl with statistical significance （P＜0.05）.Conclusion1. Telmisartan can attenuate MCT-induced pulmonary vascular remodeling and PAH.2. The mechanisms of protective effects partly by inhibiting the pervascular inflammation and proinflammatory cytokines in lung.3. Modulating the expression and activity of MMPs leads to amelioration of extracellular matrix remodeling was probably related with the protective effects of telmisartan.4. Telmisartan may be therapeutically useful in MCT-induced pulmonary vascular remodeling and PAH and ACE2 may be involved as part of its mechanisms.更多还原