洛沙坦对高氧致CLD新生大鼠肺纤维化影响的实验研究
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摘要
前言
早产儿慢性肺疾病(CLD)是早产儿在肺发育未成熟的基础上，因高氧、感染，气压伤等原因引起肺部的远期合并症。早产儿CLD作为早产儿肺透明膜病治疗的并发症在1967年首次被Northway描述，也称为支气管肺发育不良(BPD)。氧疗法是患有心肺疾病的早产儿最常应用的一种治疗手段。这一措施虽可改变患儿的低血氧状态、挽救患儿的生命，但长期吸入高浓度的氧气，可引起不同程度的肺损伤，严重者可发展为CLD。随着新生儿医学的不断发展，重症抢救技术水平的提高，使得CLD的发病率呈逐渐增高趋势(30％～40％)。由于目前尚无有效的预防和治疗方法。高氧后早产儿CLD已成为NICU最为棘手的问题之一。
目前CLD发生机制的研究已成为国内外研究的热点之一，虽然大量临床和动物实验从氧化应激性肺损伤、炎症反应、细胞凋亡和某些生长因子不足介导的肺发育障碍等诸多角度阐明了CLD的发生和发展过程，但其确切的发生机制仍不详。不论病因如何，CLD的特征性病理改变是肺发育受阻和肺间质纤维化。早产儿CLD目前尚无有效的治疗方法，早在上世纪90年代糖皮质激素是预防和治疗的治疗早产儿CLD的常规用药，用来减轻肺水肿、局部炎症反应并减少患者对氧的依赖程度，但越来越多临床研究表明：糖皮质激素影响肺分支发育、脑发育和体格发育，已很少应用。早期预防性应用表面活性物质替代疗法可改善肺功能，但不能降低CLD的发病率。因此研究其发病机制，寻求如何逆转高氧诱导的肺泡发育阻滞、减轻肺间质广泛纤维化、减少新生儿CLD发生成为新生儿医生面临的一个紧迫待解决的课题。
血管紧张素Ⅱ(AngⅡ)是作用于血管的辛肽化合物，由于其通过收缩血管和钠潴留来控制血压和容量自体稳定而一直受到关注。研究发现，在许多组织中存在局部的RAS能独立于循环系统产生AngⅡ的前体，并发现AngⅡ在组织损伤、纤维化过程中可能扮演重要的角色。体外实验证实AngⅡ通过AT1R促进培养的肺泡上皮细胞(AEC)调亡；刺激肺FB自分泌TGF-β1进而促进FB增殖和分化、胶原沉积；还有人认为AngⅡ可能通过与AT1R结合通过调控MMPs/TIMPs的合成及活化、参与组织重塑的过程；另外肺循环或肺实质内激活的局部RAS系统还能通过增加血管渗透性、增加血管紧张度等多个机制参与肺损伤的发生。应用ACEI和AT1R拮抗剂能够减轻博莱霉素诱导肺纤维化的进展。推测肺局部的RAS在肺纤维化发病过程中具有重要的作用。
关于RAS系统在高氧致新生大鼠CLD发病中的作用研究尚少。Li研究发现在高氧刺激后新生大鼠肺组织中AngⅡ和ACE水平上升，并且应用开博通可减轻肺纤维化。但其确切机制不清。本研究计划将新生鼠暴露于85％高氧环境发病中，制造CLD的动物模型，同时给特异性AT1R拮抗剂losartan干预，通过组织病理、形态学分析并且进一步从蛋白、基因水平等方法进行检测，从肺局部RAS的受体水平来研究在高氧致新生大鼠CLD发病机制，探讨losartan抗肺纤维化可能的作用机制，为寻求早产儿CLD的治疗提供新思路。
实验材料与方法
一、动物模型
本实验是在中国医科大学盛京医院实验动物中心完成。足月新生Wistar大鼠(连同母鼠)被随机分为4组：AIR组、空气对照组；O_2组、单纯高氧暴露组；AO_2组、高氧+注射用水组；LO_2组、高氧+losartan组。将2-4组在生后24 h内置于玻璃氧舱中，持续输入氧气，维持在FiO_2 0.85，每日两次监测氧浓度(美国OM-25ME型测氧仪监测)，用钙石灰吸收CO_2，使其浓度小于0.5％(美国Dapex气体分析仪)，温度20℃～25℃，湿度50％～70％，每天开箱0.5h，称重，添加水及饲料，更换垫料，与空气组互换母鼠以避免母鼠因氧中毒而致护理能力降低；空气对照组置于空气中(FiO_2 0.21)，饲养条件与高氧组相同。3、4组于生后6d每天分别经胃管灌服注射用水(3ml/kg)、losartan(5mg/kg/d，注射用水3ml/kg配成混悬液)，直至实验结束。
二、标本采集和处理
每组分别于实验开始后的第1、3、7、14、21天分别从中随机抽取8只，腹腔注射5％水合氯醛6ml/kg麻醉后处死。立即打开胸腔，无菌条件下分离肺组织，取右肺各叶置于无Rnase的Eppendorf管中于-80℃冰箱中保存。取左肺置于0.1％DEPC的4％多聚甲醛(0.1MPBS，pH7.0～7.6)固定，常规脱水、石蜡包埋。
三、实验方法
(一)、一般状况观察
动态观察各组新生大鼠的一般状态和死亡情况、监测体重，并比较时点生存率和生存曲线。
(二)、病理形态学研究
1、光镜观察：切片进行常规HE染色，光镜下动态观察肺组织的病理改变。
2、肺泡间隔厚度测定：
3、Masson三联染色动态观察肺组织内的胶原纤维沉积。
(三)、采用免疫组化、RT-PCR检测肺组织AT1R的表达
(四)、各组胶原蛋白含量和基因表达的动态变化
1、免疫组化方法检测肺组织α-平滑肌肌动蛋白(α-SMA)在肺组织中表达的强度和部位。
2、酸解法检测肺组织羟脯氨酸的含量，间接反映肺组织胶原的含量。
3、酶联免疫吸附法(ELISA)动态检测肺组织Ⅰ型胶原蛋白(ColI)的含量变化。
4、RT-PCR技术检测肺组织ColI mRNA的表达。
(五)、采用免疫组化、RT-PCR技术检测肺组织MMP-13和TIMP-1表达的变化
四、统计学分析
应用SSPS 11.5统计软件进行统计学处理，数据以均值±标准差((?)±s)表示，多组间比较采用方差分析(F检验)。两样本均数间比较采用Independent t test。相关性分析；生存率的估计使用寿命表法，时点生存率的比较用u检验。结果以P<0.05有意义。
结果
一、高氧及Losartan干预后新生大鼠一般状态和生存率的变化
O_2组新生鼠一般在5～7d后出现少动、呼吸急促、皮肤苍白以及离氧后不同程度的发绀，随高氧暴露时间延长而逐渐加重，在10d后出现对氧的依赖。LO_2组新生大鼠在离氧后的耐受能力好于高氧组。而AIR组则无以上异常表现。
随着高氧暴露时间的延长，O_2组新生大鼠生存率下降(P<0.05)；其中LO_2组新生大鼠存活率较O_2组增加；AO_2组同于O_2组；而AIR组，1天后无死亡。
O_2组在高氧暴露7d开始，新生大鼠体重与AIR组相比下降(P<0.05)，随着高氧暴露时间的延长，体重差异逐渐明显(P<0.01)。LO_2组体重与单纯O_2组相似。
二、各组新生大鼠肺组织病理形态学变化的比较
(一)肺组织的病理改变：
AIR组1d的肺组织出现小而浅的原始肺泡，形态不规则，肺泡间隔较厚；3d时终末气腔大小降低，数量渐增多，肺泡间隔渐薄；7d-21d肺泡化进一步完善，到21d肺泡间隔变薄，肺泡次级隔明显增多，肺泡数量增多，形态较规则；O_2组在高氧暴露1d后与AIR组无明显差别。3d可见小血管扩张、充血，肺泡腔内或间隔少量出血，以中性粒细胞为主的渗出、间质内细胞增多。7d时炎症反应进一步加重，肺泡间隔水肿变宽，出现终末气腔扩张、肺泡融合、肺泡分隔减少等。14d-21d间隔增宽，小灶或多灶状实变，肺组织正常结构破坏，且终末气腔扩张更为明显、小肺泡数量更少，肺泡次级隔明显减少。AO_2组与O_2组改变相似。而LO_2组在7d与高氧组区别不明显，14d、21d肺组织肺泡间隔变薄、纤维化程度明显减轻，但肺泡腔没有明显缩小，且肺泡次级隔仍较少。
(二)Masson染色：
蓝色的胶原纤维主要在支气管及血管壁的外周，肺泡壁有少许。O_2组随高氧时间的延长，胶原纤维在在支气管及血管壁的外周沉积增加；在高氧后14d、21d肺泡间隔蓝色胶原纤维所占比例较AIR组明显增加。LO_2组蓝色胶原纤维所占比例较O_2组减少且排列比较疏松，但仍明显高于AIR组。
三、高氧对CLD新生大鼠肺组织AT1R表达的影响
AT1R主要在肺泡上皮细胞、间质细胞、间质巨噬细胞、血管内皮细胞和血管平滑肌细胞、支气管和毛细血管外膜有免疫染色；AIR组3d表达增强，之后表达显著下降，21d仅在肺泡上皮细胞及血管外膜弱表达；而O2组在21d在损伤部位的间质巨噬细胞、FB仍有较强的免疫染色。O2组在1d、3d、7d AT1R蛋白和mRNA表达与AIR组无明显差别(P>0.05)，14d和21d时较同期AIR组表达显著增强(P<0.01)，
四、各组胶原蛋白含量和基因表达的动态变化
(一)α-SMA在肺组织中的表达
免疫组化结果示：AIR组肺组织，α-SMA表达部位主要在肺血管、气道壁及呼吸性支气管开口处平滑肌的胞浆中，同时1d-7d在初级肺泡隔间质中表达，到14d、21d时在次级隔顶端有免疫染色。O_2组7d后，α-SMA在管壁平滑肌中表达明显增强，同时在肺泡间隔有明显表达；到14d、21d在肺泡表面、间质出现弥漫性强的免疫染色。LO_2组α-SMA表达的范围和强度较O_2组明显减弱。
(二)酸解法检测肺组织HYP的含量
在1d-7d各组HYP的含量没有明显的区别。14d和21d O_2组肺组织HYP的含量明显高于同期AIR组(P<0.01)，AO_2组改变同于高氧组；LO_2组HYP的含量在14d较O_2组略有下降，但差异不明显，到21d则较O_2组下降显著P(0.01)，但仍明显高于AIR组(P<0.01)。
(三)检测各组肺组织ColI蛋白及mRNA表达的变化
AIR组ColI的含量均随日龄增加逐渐下降，而高氧各组逐渐增加(P<0.01)。14d O_2组较AIR组显著上升(P0.01)，21d时差异更为显著(P<0.001)；Losartan干预后14d较高氧组略有下降，但差异不明显，21d时则显著下降(P<0.01)。
AIR组各时间点ColImRNA表达无明显差异(P>0.05)。O_2组和AO2组ColImRNA表达随日龄的增加显著上升，7d差异显著(P<0.01)，21d时达高峰。LO2组在21d时较O_2组显著下降(P<0.01)。
肺组织α-SMA表达与ColImRNA之间呈较强直线相关关系(r=0.64，P<0.05)；而与ColI蛋白无直线相关关系(r=0.28，P>0.05)。
五、各组新生大鼠MMP-13和TIMP-1在肺组织表达的变化
(一)MMP-13和TIMP-1在肺组织中的蛋白表达比较
AIR组肺组织MMP-13和TIMP-1的表达部位主要在上皮细胞、毛细血管内皮和间质细胞胞浆中。
在AIR组各时间点MMP-13表达没有明显的变化(P>0.05)；O2和AO2组1d-14d与AIR组无明显差异(P>0.05)；到21d MMP-13表达显著下降(P<0.01)。LO2组在21d较O2和AO2组表达均明显增强(P<0.01)，与AIR组相近(P>0.05)。
TIMP-1在高氧7d后增强，14d和21d更为显著(P<0.01)；Losartan在21d抑制高氧肺组织TIMP-1表达(P<0.01)，但仍高于AIR组(P<0.05)。
(二)MMP-13和TIMP-1在肺组织中的mRNA表达比较
各组肺组织均有MMP-13，TIMP-1 mRNA表达；且mRNA表达与蛋白表达变化趋势具有一致性。
结论
1、新生大鼠持续高浓度氧气暴露，可导致新生大鼠肺组织出现肺泡发育障碍和肺纤维组织增生等病理形态学改变，符合早产儿CLD的发生发展特点和解剖学改变。
2、新生大鼠肺组织AT1R主要定位在肺泡上皮细胞、间质细胞、间质巨噬细胞、支气管和毛细血管外膜；高氧组在内皮细胞、支气管上皮细胞出现阳性染色。AT1R在高氧后期表达明显增强，与肺纤维化发生时间一致性的结果提示AngⅡ作为AT1R配体可能参与高氧CLD的发生。
3、在高氧暴露的中晚期，在肺泡腔的表面及间质内出现弥漫性、条索状α-SMA表达，而在空气对照组α-SMA在次级隔的顶端呈点状表达，而在肺泡腔和间质内却罕见表达。推测持续高浓度氧气暴露诱发肺组织MFB表达部位及表达的强度的改变可能是CLD发生的主要原因之一。
4、Losartan可明显降低高氧致CLD新生大鼠肺组织α-SMA、Col蛋白和mRNA的表达，降低肺组织HYP的含量；α-SMA与ColI mRNA表达具有显著相关性结果，提示Losartan可能通过抑制肺组织MFB的转化，进而抑制ColI mRNA的表达来减轻高氧CLD新生大鼠肺纤维化。结果同时暗示高氧致新生大鼠肺组织纤维化的发生可能通过AngⅡ与AT1R结合，调节ColI合成来介导完成的。
5、MMP-13和TIMP-1在正常肺组织有表达，提示MMP-13和TIMP-1可能在参与肺的形态构建，胶原代谢等过程。
6、在高氧晚期肺组织mmp-13基因和蛋白表达显著下降；而TIMP-1表达明显增强。猜测在高氧暴露晚期MMP-13/TIMP-1mRNA和蛋白表达失衡导致胶原降解减弱，这可能是高氧后期以ColI为代表的肺组织ECM异常沉积的关键所在。
7、Losartan可显著诱导高氧肺组织MMP-13mRNA和蛋白表达，下调TIMP-1mRNA和蛋白的表达，使得MMP-13/TIMP-1比值得到部分纠正，减少高氧肺组织中ColI的异常沉积。提示：肺局部AngⅡ可能通过与AT1R结合，通过调节MMP-13/TIMP-1的合成与活性来参与高氧CLD的发生。
Introduction
Chronic lung disease (CLD) of prematurity is a major long-term pulmonary consequence of preterm birth, in which pulmonary immaturity, oxygen toxicity, baro/volutrauma as a consequence of mechanical ventilation and/or infections are involved. Bronchopulmonary dysplasia (BPD) is one of a chronic lung disease first described in 1967 as a complication of therapy for premature infants with hyaline membrane disease, and treatment with high concentrations of oxygen was thought to be a major contributor to its development. Administration of supplemental oxygen as one of supportive therapies employ to elevate oxygen concentrations, which put them at risk for encountering pulmonary oxygen toxicity, cause significant lung damage, even develop to CLD.With neonatal medicine development, the incidence of premature CLD is at a rising tendency continously (reaching 30%～40% abroad). There is no ideal prevention and perfect therapeutic strategies due to unclear mechanism. Hyperoxic induced premature CLD is one of the most difficult problem in neonatal intensive care unit(NICU).
So far the mechanism research on CLD has been one of the hot topics all around the world, the accurate mechanism hasn't been identified although a great deal of clinical researches and animal experiments have been done on oxidative stress, inflammation, apoptosis, mechanism of collagen degradation and arrested lung development induced by the lackness of some growth factors to clarify the incidence and the development of CLD. However no matter what the mechanism is, histopathologic characteristics of the lung injury in CLD are arrested lung development and pulmonary interstitial fibrosis. At present, there are no effective treatment for CLD. Corticosteroids facilitate extubation and decrease neonatal respiratory support and oxygen exposure. However, more and more clinical research indicate glucocorticoids in premature infants has raised a host of concerns about effects on lung, brain and somatic growth, and substantially worse neuromotor and developmental outcomes in early childhood. Surfactant replacement therapy in early time after birth may improve pulmonary function, but cannot cut down incidence of CLD. So study the pathogenesy of CLD to explore how to reverse lung development arrest, to relieve lung fibrosis, decrease incidence of premature CLD become a urgent problem.
The vasoactive octapeptide, angiotensin II (AngII), has a well- described role in the control of systemic blood pressure and volume homeostasis. Local rennin angiotensin systems (RAS) have been described for a number of tissues in which AngII production is independent of circulating precursors. There are some Experimental evidence to suggest an important role for AngII in the fibrotic response to tissue injury. Ang II can stimulate lung fibroblast proliferation via activation of the AT1 receptor and involves the autocrine action of TGF-β1, and induces AEC apoptosis in response to Ang II is mediated by receptor subtype AT(1); some study consider that collagen remodeling may be facilitated by Ang II to effects on MMPs/TIMPs and collagen expression via the AT1 receptor to modulates profibrotic effects. Moreover local RAS take part in lung injury by increase vascular permeability, vascular tone and so on. Angiotensin-converting enzyme (ACE) inhibitors and AT1 receptor antagonist attenuate bleomycin-induced lung fibrosis in a number of animal models. So it is supposed that local RAS may have significant effect in course of lung fibrosis.
Li research showed that lung tissue of hyperoxia-exposed induced newborn rats lung injury AngII and ACE content increase signifencently, and Captopril may extenuate lung fibrosis induced by hyperoxia. But the mechanism is not clear completely. In this study we prepared an animal model of CLD with newborn rats induced by continuously inhaling high concentration of oxygen(FiO_285%)and at the 6th day after hyperoxia losartan (5mg/kg), a selective antagonist of AT1 receptors for Ang II, by intragastric administration until the end of experiment completed. Using special staining technic, morphology, immunoassay, immuno-histochemistry and Reverse transcription polymerase chain reaction (RT-PCR) to observing the dynamic changes of lung development and fibrosis, the MMPs/TIMPs, collagen expression are detected after losartan intervention. We try to provide new way for research pathogenesy of hyperoxia induced CLD.
Materials and Methods
1. Animal modle
The present study was performed in accordance with the guidelines provided by the Experimental Animal Laboratory of Shengjing Hospital China Medical University. Within 24 hours after birth, pups were randomly redistributed to the newly delivered mothers. Term neonatal Wistar rats were divided randomly into four groups. AIR group served as controls and were kept in air and not subjected to hyperoxia. Other groups were exposed to oxygen. The pups of hyperoxia exposures groups were maintained in glass chambers in which the oxygen was infused continuously to achieve 85% oxygen concentration, with oxygen monitor(OM-25ME, USA) twice daily. CO_2 was removed by soda lime absorption to keep CO_2 levels below 0.5%(Dapex Gas Monitor, USA). Temperature and Humidity was maintained at 20℃～25℃and 50%~70%, respectively. Every day, at clocked time of 0.5h opening chambers to exchange nursing mothers betweenin air and O_2-exposed groups every 24 hours to prevent maternal O_2 toxicity and eliminate maternal effects between groups, meanwhile change water, add food, clean dirty cages and record body weight. All animals were raised in the same room and all other conditions were the same. In the oxygen exposed groups, O_2 groups were simple oxygen exposed, AO_2groups and LO_2 were received daily aqua(3ml/kg) and losartan (5mg/kg) by intragastric administration from the sixth day after birth until the end of experiment completed, respectively.
2. tissue preparation
On postnatal 1d, 3d 7d, 14d, 21d. Each group of hyperoxia as well as air rats was anesthetized by intraperitoneal injection of 5% chloral hydrate (6ml/kg), and put to death. The thorax was opened and the lungs were resected, the right lung tissue were put in the Rnase-free Ependorf tubes, and immediately frozen in liquid N_2 for mRNA, Enzyme linked immunoadsorbent assay(ELISA), and so on. the left lung tissue were fixed in 4%formaldehydum polymerisatum which contains 0.1% DEPC(0.1MPBS, pH7.0～7.6), then were dehydrated, embedded in wax within 24 hours.
3. Experiment Methods
1.1 The appearance, survial rate and weight were monitored everyday in every experiment groups. Life table for survival analysis, point survival and survival curve were compared.
1.2 The changes of the lung pathology
1.2.1 For lung pathology assessment, lung sections from the left lobes were stained with HE (hematoxylin and eosin).
1.2.2 The thickness of inter alveolar septa: the hispathological changes of pulmonary fibrosis were evaluated by image analysis.
1.2.3 Masson staining: to evaluate degree of collagen fibers deposition in the lung.
3.3 Using Immonohistochemistry and RT-PCR to detect protein and mRNA of AT1R expression in the lung.
3.4 The measurement of collagen expression in the lung tissue
3.4.1 Immonohistochemistry: the detection intensity and position of the protein expression in lung tissue about alpha-SMA
3.4.2 Quantitation of total lung collagen: to detect hydroxyproline (HYP) of lung tissue by acidolysis assay, to express of total lung collage content.
3.4.3 ELISA: quantitation to detect the protein level of collagen I (ColI) in lung tissue.
3.4.4 RT-PCR: the detection at mRNA level of Coll.
3.5 The measurement of MMP-13 and TIMP-1 expression in the lung tissue.
3.5.1 Immonohistochemistry: the detection intensity and position of the protein expression in lung tissue about MMP-13 and TIMP-1.
3.5.2 RT-PCR: to detect mRNA level of MMP-13 and TIMP-1 in lung tissue of neonate rats .
4. Statistical analysis
SSPS version 11.5 was used to perform statistical analysis, with all data expressed as (mean±SD). Statistically significant differences in the mean values were analysis using the One-Way ANOVA, Independent-Samples T Test, bivariate. Life table was used to value survival rate, u-test for time comparison. Statistically significant differences set P<0.05.
Results
1. Survival rate and general status of rats in four groups
1.1 The hyperoxia rats almost began to present dyspnea, pale, cyanosis of different degrees after 5-7 days of oxygen exposure, some even depended high oxygen after 10 days of age. LO_2 groups was better than oxygen exposed and AO_2 groups withdraw from oxygen. The AIR groups didn't have the appearance above.
1.2 The death of hyperoxia rats increased with the time of oxygen exposure, and survival rate decreased significently (P<0.05); while there is no death after Id in AIR groups, and the survival curves were different in groups from 2 days of age(P<0.01). The survival rate of LO_2 groups increased comparing to oxygen exposure.
1.3 There was no difference in average birth weight between the four groups, but from 7 day of oxygen exposure, Body weights of the O_2 groups, AO_2 groups and LO_2 group animals showed a significant decrease compared with air controls at the sams time period(P<0.05). And the difference lasted to 21 days of age and more evident compared with air controls(P<0.01). There was no difference between the LO_2 groups and O_2 groups.
2. The pathological changes of the lung tissue
2.1 On day 1 of the experiment, it was observed in both room air and O_2 groups that the alveolar structure was irregular, terminal air space size was rather small, and the alveolar septum was thick. On 3 day, the alveolar structure of the each groups was more regular, the size of alveolus was equal, and alveolar septum was thinner, but in O_2 groups, there was a few inflammatory cells exuded out, blooding, interstitial cells increased. On 7 day, in room air rats, the alveolar size was equal, while the terminal air space size of the oxygen-exposed rat became large, there was inflammatory response and more interstitial cells. On 14 day and 21 day, air groups continue to progress alveolization; but in O_2 groups, the terminal air space size grew significantly large, secondary septum decreased, the quantity of alveolar reduced, with alveolus fusion, interstitial cells increased, and alveolar septa was thicker. There was no significant difference between the LO_2 groups and O_2 groups on 7 day. On 14 day d and 21 day, alveolar septum is thinner in LO2 groups, but alveolar space are not deflate obviously, and the sum of secondary septum is still less.
2.2 Masson staining: In air group, blue fibres mainly deposited in bronchil and vascular wall, little in alveolar septum. In O_2 groups, on 14 day and 21 day there were more collagen fibres along bronchil and vascular wall and alveolar septum, especially in alveolar septum. In LO_2 groups, the ratio of blue fibres is decreased and rarefied relatively compare with O_2 groups, but it is more than air group.
3. The dynamic changes of AT1R expression in the lung tissue
Immuonhistochemical studies shows AT1R expression in alveolar epithelial cells, fibroblast, interstitial macrophage and external of vessels and bronchus in control group; AT1R positive expression in endothelial cells and bronchial epithelial cells were also observed in fibrotic lesions in lungs. AT1R expression in air groups lung decreased but increased significantly in hyperoxia-exposed groups on 14 and 21 day.
4. The dynamic change of collagen expression in lung tissue
4.1 Immunohistochemical assessment of alpha-SMA expression
In air groups, alpha-SMA immunostaining was restricted to the smooth muscle of the pulmonary vasculature, airway walls, and openings to the respiratory bronchioles of terminal airway; meanwhile in 7-day-old rats, alpha-SMA positive cells are located in the slender elongated cells in the interstitium of primitive alveolar septa; in 21-day-old rats, alpha-SMA positive cells with a round shape are found at the tip of the secondary septa. Exposure to hyperoxia resulted in its expression a marked increasing in smooth muscle and alveolar septa myofibroblast foci and on the surface of alveolar significantly on 14 day and 21 day. Treatment of the hyperoxia-exposed animals with Losartan result in a significant decrease alpha-SMA expression compared with simple O_2 groups(P<0.01).
4.2 detect of HYP content in lung tissue by acidolysis.
There were no difference of HYP contents to four groups from 1 day to 7 day of the experiment. On 14 day and 21 day, HYP contents increased significantly in O_2 groups comparing to air groups(P<0.01); and changes of AO_2 groups similar to that of simple hyperoxia; in LO_2 groups it decreased slightly on 14 day; and on 21 day decreased significantly(P<0.01), however higher than that in air groups(P<0.01).
4.3 The dynamic change at the protein and mRNA level of ColI expression
The protein of Col I in the newborn lung tissue decreased with postnatal age rising in air groups; But in the O_2 and AO_2 groups it increased significantly than that in air control group(P<0.01), and its of larger difference on 21 day; in LO_2 groups it decreased slightly on 14 day; and on 21 day decreased significantly(P<0.01), but its higher than that in air group(P<0.01).
There was no different about mRNA expression of ColI at each spot in air groups; O_2 and AO_2 groups it increased with oxygen-exposed extending, and reach peak on 21 day. mRNA expression of ColI decreased significantly after Losartan-treated.
4.4 There was correlation between alpha-SMA and ColI mRNA in the lung (r=0.64, P<0.05), but none with the protein of ColI (r=0.28, P >0.05).
5.The dynamic changes of MMP-13 and TIMP-1 expression in the lung tissue
5.1 The dynamic change MMP-13 expression
Immunohistochemical staining shows, MMP-13 was expressed in the endochylema of epithelial cells, microvascular endothelial cells, interstitial cells and macrophages, in air lung tissues, there was no change of the MMP-13 expression at each time; In O_2 and AO_2 groups, it express at the same level from 1 day to 14 day, which was no difference with the air group. But on 21d, the expression of MMP-13 decreased significantly(P<0.01). In LO_2 group, it increased signifycantly comparing to O_2 group and reach the level of air groups. The curve change of MMP-13 mRNA is the same with that of protein.
5.2 The dynamic change TIMP-1 expression
TIMP-1 was expressed in epithelial cells, interstitial cells and pulmonary macrophages, the expression intensity of TIMP-1 protein and mRNA of all the rats were rising, from 7d after oxygen-exposed, its expression obvious increased, and significant on the 14 and 21d. losartan result in a significant decrease of the protein and mRNA expression of TIMP-1 compared with simple hyperoxia-exposed animals(P<0.01), but were higher than that of air group.
Concludsion
1. pathological findings charactered by the arrested alveolar development and deposited fibres in newborn rats after prolonged hyperoxia-exposed are consistent with those of CLD of prematurity in human.
2. Immuonhistochemical studies shows AT1R expression in alveolar epithelial cells, fibroblast, interstitial macrophage and external of vessels and bronchus in control group; AT1R positive expression in endothelial cells and bronchial epithelial cells were also observed in fibrotic lesions in lungs. AT1R expression increased significantly at the late stage of hyperoxia which is consist with the time of lung fibrosis genesis. It is hint that angiotensin II as a ligand of AT1R may take part in CLD genesis.
3. Exposure to hyperoxia resulted in a marked increase in smooth muscle, alveolar septa myofibroblast foci and on the surface of alveolar at the late phase of hyperoxia-exposed; however in air control groups, alpha-SMA positive cells with a round shape are found at the tip of the secondary septa, absent expression in alveolar space and mesenchymal. It is suppose that persistent hyperoxia exposed induced the position and intension change of myofibroblast may be one of the most reason of CLD genesis.
4. After Losartan administration, alpha-SMA, HYP content, and the protein and mRNA expression of ColI decreased significantly, meanwhile the degree of lung disorganization relieved. It is indicated that Losartan attenuated hyperoxia induced lung injury of neonate. It is also hint that hyperoxia induced lung fibrosis genesis may performed by the pathway Ang II binding to AT1 receptor regulating synthesis of ColI.
5. MMP-13 and TIMP-1 expression in the neonatal lung tissue, which hint MMP-13 and TIMP-1 invoved construction of lung morphous and collagen metabolism.
6. At the late phase of hyperoxia-exposed, the protein and mRNA expression disbalance of MMP-13/TIMP-1 in lung tissue may play an important role during the remodeling of CLD induced by hyperoxia.
7. losartan induced MMP-13 and down regulated TIMP-1 expression in hyperoxia-exposed lung tissue, attenuated extracellular matrix including ColI deposition at the late phase of hyperoxia-exposed, which is indicated that AngII may taked part in lung fibrosis induced by hyperoxia via AT1 receptor regulating synthesis and activity of MMP-13/TIMP-1.
早产儿慢性肺疾病(CLD)是早产儿在肺发育未成熟的基础上，因高氧、感染，气压伤等原因引起肺部的远期合并症。早产儿CLD作为早产儿肺透明膜病治疗的并发症在1967年首次被Northway描述，也称为支气管肺发育不良(BPD)。氧疗法是患有心肺疾病的早产儿最常应用的一种治疗手段。这一措施虽可改变患儿的低血氧状态、挽救患儿的生命，但长期吸入高浓度的氧气，可引起不同程度的肺损伤，严重者可发展为CLD。随着新生儿医学的不断发展，重症抢救技术水平的提高，使得CLD的发病率呈逐渐增高趋势(30％～40％)。由于目前尚无有效的预防和治疗方法。高氧后早产儿CLD已成为NICU最为棘手的问题之一。
目前CLD发生机制的研究已成为国内外研究的热点之一，虽然大量临床和动物实验从氧化应激性肺损伤、炎症反应、细胞凋亡和某些生长因子不足介导的肺发育障碍等诸多角度阐明了CLD的发生和发展过程，但其确切的发生机制仍不详。不论病因如何，CLD的特征性病理改变是肺发育受阻和肺间质纤维化。早产儿CLD目前尚无有效的治疗方法，早在上世纪90年代糖皮质激素是预防和治疗的治疗早产儿CLD的常规用药，用来减轻肺水肿、局部炎症反应并减少患者对氧的依赖程度，但越来越多临床研究表明：糖皮质激素影响肺分支发育、脑发育和体格发育，已很少应用。早期预防性应用表面活性物质替代疗法可改善肺功能，但不能降低CLD的发病率。因此研究其发病机制，寻求如何逆转高氧诱导的肺泡发育阻滞、减轻肺间质广泛纤维化、减少新生儿CLD发生成为新生儿医生面临的一个紧迫待解决的课题。
血管紧张素Ⅱ(AngⅡ)是作用于血管的辛肽化合物，由于其通过收缩血管和钠潴留来控制血压和容量自体稳定而一直受到关注。研究发现，在许多组织中存在局部的RAS能独立于循环系统产生AngⅡ的前体，并发现AngⅡ在组织损伤、纤维化过程中可能扮演重要的角色。体外实验证实AngⅡ通过AT1R促进培养的肺泡上皮细胞(AEC)调亡；刺激肺FB自分泌TGF-β1进而促进FB增殖和分化、胶原沉积；还有人认为AngⅡ可能通过与AT1R结合通过调控MMPs/TIMPs的合成及活化、参与组织重塑的过程；另外肺循环或肺实质内激活的局部RAS系统还能通过增加血管渗透性、增加血管紧张度等多个机制参与肺损伤的发生。应用ACEI和AT1R拮抗剂能够减轻博莱霉素诱导肺纤维化的进展。推测肺局部的RAS在肺纤维化发病过程中具有重要的作用。
关于RAS系统在高氧致新生大鼠CLD发病中的作用研究尚少。Li研究发现在高氧刺激后新生大鼠肺组织中AngⅡ和ACE水平上升，并且应用开博通可减轻肺纤维化。但其确切机制不清。本研究计划将新生鼠暴露于85％高氧环境发病中，制造CLD的动物模型，同时给特异性AT1R拮抗剂losartan干预，通过组织病理、形态学分析并且进一步从蛋白、基因水平等方法进行检测，从肺局部RAS的受体水平来研究在高氧致新生大鼠CLD发病机制，探讨losartan抗肺纤维化可能的作用机制，为寻求早产儿CLD的治疗提供新思路。
实验材料与方法
一、动物模型
本实验是在中国医科大学盛京医院实验动物中心完成。足月新生Wistar大鼠(连同母鼠)被随机分为4组：AIR组、空气对照组；O_2组、单纯高氧暴露组；AO_2组、高氧+注射用水组；LO_2组、高氧+losartan组。将2-4组在生后24 h内置于玻璃氧舱中，持续输入氧气，维持在FiO_2 0.85，每日两次监测氧浓度(美国OM-25ME型测氧仪监测)，用钙石灰吸收CO_2，使其浓度小于0.5％(美国Dapex气体分析仪)，温度20℃～25℃，湿度50％～70％，每天开箱0.5h，称重，添加水及饲料，更换垫料，与空气组互换母鼠以避免母鼠因氧中毒而致护理能力降低；空气对照组置于空气中(FiO_2 0.21)，饲养条件与高氧组相同。3、4组于生后6d每天分别经胃管灌服注射用水(3ml/kg)、losartan(5mg/kg/d，注射用水3ml/kg配成混悬液)，直至实验结束。
二、标本采集和处理
每组分别于实验开始后的第1、3、7、14、21天分别从中随机抽取8只，腹腔注射5％水合氯醛6ml/kg麻醉后处死。立即打开胸腔，无菌条件下分离肺组织，取右肺各叶置于无Rnase的Eppendorf管中于-80℃冰箱中保存。取左肺置于0.1％DEPC的4％多聚甲醛(0.1MPBS，pH7.0～7.6)固定，常规脱水、石蜡包埋。
三、实验方法
(一)、一般状况观察
动态观察各组新生大鼠的一般状态和死亡情况、监测体重，并比较时点生存率和生存曲线。
(二)、病理形态学研究
1、光镜观察：切片进行常规HE染色，光镜下动态观察肺组织的病理改变。
2、肺泡间隔厚度测定：
3、Masson三联染色动态观察肺组织内的胶原纤维沉积。
(三)、采用免疫组化、RT-PCR检测肺组织AT1R的表达
(四)、各组胶原蛋白含量和基因表达的动态变化
1、免疫组化方法检测肺组织α-平滑肌肌动蛋白(α-SMA)在肺组织中表达的强度和部位。
2、酸解法检测肺组织羟脯氨酸的含量，间接反映肺组织胶原的含量。
3、酶联免疫吸附法(ELISA)动态检测肺组织Ⅰ型胶原蛋白(ColI)的含量变化。
4、RT-PCR技术检测肺组织ColI mRNA的表达。
(五)、采用免疫组化、RT-PCR技术检测肺组织MMP-13和TIMP-1表达的变化
四、统计学分析
应用SSPS 11.5统计软件进行统计学处理，数据以均值±标准差((?)±s)表示，多组间比较采用方差分析(F检验)。两样本均数间比较采用Independent t test。相关性分析；生存率的估计使用寿命表法，时点生存率的比较用u检验。结果以P<0.05有意义。
结果
一、高氧及Losartan干预后新生大鼠一般状态和生存率的变化
O_2组新生鼠一般在5～7d后出现少动、呼吸急促、皮肤苍白以及离氧后不同程度的发绀，随高氧暴露时间延长而逐渐加重，在10d后出现对氧的依赖。LO_2组新生大鼠在离氧后的耐受能力好于高氧组。而AIR组则无以上异常表现。
随着高氧暴露时间的延长，O_2组新生大鼠生存率下降(P<0.05)；其中LO_2组新生大鼠存活率较O_2组增加；AO_2组同于O_2组；而AIR组，1天后无死亡。
O_2组在高氧暴露7d开始，新生大鼠体重与AIR组相比下降(P<0.05)，随着高氧暴露时间的延长，体重差异逐渐明显(P<0.01)。LO_2组体重与单纯O_2组相似。
二、各组新生大鼠肺组织病理形态学变化的比较
(一)肺组织的病理改变：
AIR组1d的肺组织出现小而浅的原始肺泡，形态不规则，肺泡间隔较厚；3d时终末气腔大小降低，数量渐增多，肺泡间隔渐薄；7d-21d肺泡化进一步完善，到21d肺泡间隔变薄，肺泡次级隔明显增多，肺泡数量增多，形态较规则；O_2组在高氧暴露1d后与AIR组无明显差别。3d可见小血管扩张、充血，肺泡腔内或间隔少量出血，以中性粒细胞为主的渗出、间质内细胞增多。7d时炎症反应进一步加重，肺泡间隔水肿变宽，出现终末气腔扩张、肺泡融合、肺泡分隔减少等。14d-21d间隔增宽，小灶或多灶状实变，肺组织正常结构破坏，且终末气腔扩张更为明显、小肺泡数量更少，肺泡次级隔明显减少。AO_2组与O_2组改变相似。而LO_2组在7d与高氧组区别不明显，14d、21d肺组织肺泡间隔变薄、纤维化程度明显减轻，但肺泡腔没有明显缩小，且肺泡次级隔仍较少。
(二)Masson染色：
蓝色的胶原纤维主要在支气管及血管壁的外周，肺泡壁有少许。O_2组随高氧时间的延长，胶原纤维在在支气管及血管壁的外周沉积增加；在高氧后14d、21d肺泡间隔蓝色胶原纤维所占比例较AIR组明显增加。LO_2组蓝色胶原纤维所占比例较O_2组减少且排列比较疏松，但仍明显高于AIR组。
三、高氧对CLD新生大鼠肺组织AT1R表达的影响
AT1R主要在肺泡上皮细胞、间质细胞、间质巨噬细胞、血管内皮细胞和血管平滑肌细胞、支气管和毛细血管外膜有免疫染色；AIR组3d表达增强，之后表达显著下降，21d仅在肺泡上皮细胞及血管外膜弱表达；而O2组在21d在损伤部位的间质巨噬细胞、FB仍有较强的免疫染色。O2组在1d、3d、7d AT1R蛋白和mRNA表达与AIR组无明显差别(P>0.05)，14d和21d时较同期AIR组表达显著增强(P<0.01)，
四、各组胶原蛋白含量和基因表达的动态变化
(一)α-SMA在肺组织中的表达
免疫组化结果示：AIR组肺组织，α-SMA表达部位主要在肺血管、气道壁及呼吸性支气管开口处平滑肌的胞浆中，同时1d-7d在初级肺泡隔间质中表达，到14d、21d时在次级隔顶端有免疫染色。O_2组7d后，α-SMA在管壁平滑肌中表达明显增强，同时在肺泡间隔有明显表达；到14d、21d在肺泡表面、间质出现弥漫性强的免疫染色。LO_2组α-SMA表达的范围和强度较O_2组明显减弱。
(二)酸解法检测肺组织HYP的含量
在1d-7d各组HYP的含量没有明显的区别。14d和21d O_2组肺组织HYP的含量明显高于同期AIR组(P<0.01)，AO_2组改变同于高氧组；LO_2组HYP的含量在14d较O_2组略有下降，但差异不明显，到21d则较O_2组下降显著P(0.01)，但仍明显高于AIR组(P<0.01)。
(三)检测各组肺组织ColI蛋白及mRNA表达的变化
AIR组ColI的含量均随日龄增加逐渐下降，而高氧各组逐渐增加(P<0.01)。14d O_2组较AIR组显著上升(P0.01)，21d时差异更为显著(P<0.001)；Losartan干预后14d较高氧组略有下降，但差异不明显，21d时则显著下降(P<0.01)。
AIR组各时间点ColImRNA表达无明显差异(P>0.05)。O_2组和AO2组ColImRNA表达随日龄的增加显著上升，7d差异显著(P<0.01)，21d时达高峰。LO2组在21d时较O_2组显著下降(P<0.01)。
肺组织α-SMA表达与ColImRNA之间呈较强直线相关关系(r=0.64，P<0.05)；而与ColI蛋白无直线相关关系(r=0.28，P>0.05)。
五、各组新生大鼠MMP-13和TIMP-1在肺组织表达的变化
(一)MMP-13和TIMP-1在肺组织中的蛋白表达比较
AIR组肺组织MMP-13和TIMP-1的表达部位主要在上皮细胞、毛细血管内皮和间质细胞胞浆中。
在AIR组各时间点MMP-13表达没有明显的变化(P>0.05)；O2和AO2组1d-14d与AIR组无明显差异(P>0.05)；到21d MMP-13表达显著下降(P<0.01)。LO2组在21d较O2和AO2组表达均明显增强(P<0.01)，与AIR组相近(P>0.05)。
TIMP-1在高氧7d后增强，14d和21d更为显著(P<0.01)；Losartan在21d抑制高氧肺组织TIMP-1表达(P<0.01)，但仍高于AIR组(P<0.05)。
(二)MMP-13和TIMP-1在肺组织中的mRNA表达比较
各组肺组织均有MMP-13，TIMP-1 mRNA表达；且mRNA表达与蛋白表达变化趋势具有一致性。
结论
1、新生大鼠持续高浓度氧气暴露，可导致新生大鼠肺组织出现肺泡发育障碍和肺纤维组织增生等病理形态学改变，符合早产儿CLD的发生发展特点和解剖学改变。
2、新生大鼠肺组织AT1R主要定位在肺泡上皮细胞、间质细胞、间质巨噬细胞、支气管和毛细血管外膜；高氧组在内皮细胞、支气管上皮细胞出现阳性染色。AT1R在高氧后期表达明显增强，与肺纤维化发生时间一致性的结果提示AngⅡ作为AT1R配体可能参与高氧CLD的发生。
3、在高氧暴露的中晚期，在肺泡腔的表面及间质内出现弥漫性、条索状α-SMA表达，而在空气对照组α-SMA在次级隔的顶端呈点状表达，而在肺泡腔和间质内却罕见表达。推测持续高浓度氧气暴露诱发肺组织MFB表达部位及表达的强度的改变可能是CLD发生的主要原因之一。
4、Losartan可明显降低高氧致CLD新生大鼠肺组织α-SMA、Col蛋白和mRNA的表达，降低肺组织HYP的含量；α-SMA与ColI mRNA表达具有显著相关性结果，提示Losartan可能通过抑制肺组织MFB的转化，进而抑制ColI mRNA的表达来减轻高氧CLD新生大鼠肺纤维化。结果同时暗示高氧致新生大鼠肺组织纤维化的发生可能通过AngⅡ与AT1R结合，调节ColI合成来介导完成的。
5、MMP-13和TIMP-1在正常肺组织有表达，提示MMP-13和TIMP-1可能在参与肺的形态构建，胶原代谢等过程。
6、在高氧晚期肺组织mmp-13基因和蛋白表达显著下降；而TIMP-1表达明显增强。猜测在高氧暴露晚期MMP-13/TIMP-1mRNA和蛋白表达失衡导致胶原降解减弱，这可能是高氧后期以ColI为代表的肺组织ECM异常沉积的关键所在。
7、Losartan可显著诱导高氧肺组织MMP-13mRNA和蛋白表达，下调TIMP-1mRNA和蛋白的表达，使得MMP-13/TIMP-1比值得到部分纠正，减少高氧肺组织中ColI的异常沉积。提示：肺局部AngⅡ可能通过与AT1R结合，通过调节MMP-13/TIMP-1的合成与活性来参与高氧CLD的发生。
Introduction
Chronic lung disease (CLD) of prematurity is a major long-term pulmonary consequence of preterm birth, in which pulmonary immaturity, oxygen toxicity, baro/volutrauma as a consequence of mechanical ventilation and/or infections are involved. Bronchopulmonary dysplasia (BPD) is one of a chronic lung disease first described in 1967 as a complication of therapy for premature infants with hyaline membrane disease, and treatment with high concentrations of oxygen was thought to be a major contributor to its development. Administration of supplemental oxygen as one of supportive therapies employ to elevate oxygen concentrations, which put them at risk for encountering pulmonary oxygen toxicity, cause significant lung damage, even develop to CLD.With neonatal medicine development, the incidence of premature CLD is at a rising tendency continously (reaching 30%～40% abroad). There is no ideal prevention and perfect therapeutic strategies due to unclear mechanism. Hyperoxic induced premature CLD is one of the most difficult problem in neonatal intensive care unit(NICU).
So far the mechanism research on CLD has been one of the hot topics all around the world, the accurate mechanism hasn't been identified although a great deal of clinical researches and animal experiments have been done on oxidative stress, inflammation, apoptosis, mechanism of collagen degradation and arrested lung development induced by the lackness of some growth factors to clarify the incidence and the development of CLD. However no matter what the mechanism is, histopathologic characteristics of the lung injury in CLD are arrested lung development and pulmonary interstitial fibrosis. At present, there are no effective treatment for CLD. Corticosteroids facilitate extubation and decrease neonatal respiratory support and oxygen exposure. However, more and more clinical research indicate glucocorticoids in premature infants has raised a host of concerns about effects on lung, brain and somatic growth, and substantially worse neuromotor and developmental outcomes in early childhood. Surfactant replacement therapy in early time after birth may improve pulmonary function, but cannot cut down incidence of CLD. So study the pathogenesy of CLD to explore how to reverse lung development arrest, to relieve lung fibrosis, decrease incidence of premature CLD become a urgent problem.
The vasoactive octapeptide, angiotensin II (AngII), has a well- described role in the control of systemic blood pressure and volume homeostasis. Local rennin angiotensin systems (RAS) have been described for a number of tissues in which AngII production is independent of circulating precursors. There are some Experimental evidence to suggest an important role for AngII in the fibrotic response to tissue injury. Ang II can stimulate lung fibroblast proliferation via activation of the AT1 receptor and involves the autocrine action of TGF-β1, and induces AEC apoptosis in response to Ang II is mediated by receptor subtype AT(1); some study consider that collagen remodeling may be facilitated by Ang II to effects on MMPs/TIMPs and collagen expression via the AT1 receptor to modulates profibrotic effects. Moreover local RAS take part in lung injury by increase vascular permeability, vascular tone and so on. Angiotensin-converting enzyme (ACE) inhibitors and AT1 receptor antagonist attenuate bleomycin-induced lung fibrosis in a number of animal models. So it is supposed that local RAS may have significant effect in course of lung fibrosis.
Li research showed that lung tissue of hyperoxia-exposed induced newborn rats lung injury AngII and ACE content increase signifencently, and Captopril may extenuate lung fibrosis induced by hyperoxia. But the mechanism is not clear completely. In this study we prepared an animal model of CLD with newborn rats induced by continuously inhaling high concentration of oxygen(FiO_285%)and at the 6th day after hyperoxia losartan (5mg/kg), a selective antagonist of AT1 receptors for Ang II, by intragastric administration until the end of experiment completed. Using special staining technic, morphology, immunoassay, immuno-histochemistry and Reverse transcription polymerase chain reaction (RT-PCR) to observing the dynamic changes of lung development and fibrosis, the MMPs/TIMPs, collagen expression are detected after losartan intervention. We try to provide new way for research pathogenesy of hyperoxia induced CLD.
Materials and Methods
1. Animal modle
The present study was performed in accordance with the guidelines provided by the Experimental Animal Laboratory of Shengjing Hospital China Medical University. Within 24 hours after birth, pups were randomly redistributed to the newly delivered mothers. Term neonatal Wistar rats were divided randomly into four groups. AIR group served as controls and were kept in air and not subjected to hyperoxia. Other groups were exposed to oxygen. The pups of hyperoxia exposures groups were maintained in glass chambers in which the oxygen was infused continuously to achieve 85% oxygen concentration, with oxygen monitor(OM-25ME, USA) twice daily. CO_2 was removed by soda lime absorption to keep CO_2 levels below 0.5%(Dapex Gas Monitor, USA). Temperature and Humidity was maintained at 20℃～25℃and 50%~70%, respectively. Every day, at clocked time of 0.5h opening chambers to exchange nursing mothers betweenin air and O_2-exposed groups every 24 hours to prevent maternal O_2 toxicity and eliminate maternal effects between groups, meanwhile change water, add food, clean dirty cages and record body weight. All animals were raised in the same room and all other conditions were the same. In the oxygen exposed groups, O_2 groups were simple oxygen exposed, AO_2groups and LO_2 were received daily aqua(3ml/kg) and losartan (5mg/kg) by intragastric administration from the sixth day after birth until the end of experiment completed, respectively.
2. tissue preparation
On postnatal 1d, 3d 7d, 14d, 21d. Each group of hyperoxia as well as air rats was anesthetized by intraperitoneal injection of 5% chloral hydrate (6ml/kg), and put to death. The thorax was opened and the lungs were resected, the right lung tissue were put in the Rnase-free Ependorf tubes, and immediately frozen in liquid N_2 for mRNA, Enzyme linked immunoadsorbent assay(ELISA), and so on. the left lung tissue were fixed in 4%formaldehydum polymerisatum which contains 0.1% DEPC(0.1MPBS, pH7.0～7.6), then were dehydrated, embedded in wax within 24 hours.
3. Experiment Methods
1.1 The appearance, survial rate and weight were monitored everyday in every experiment groups. Life table for survival analysis, point survival and survival curve were compared.
1.2 The changes of the lung pathology
1.2.1 For lung pathology assessment, lung sections from the left lobes were stained with HE (hematoxylin and eosin).
1.2.2 The thickness of inter alveolar septa: the hispathological changes of pulmonary fibrosis were evaluated by image analysis.
1.2.3 Masson staining: to evaluate degree of collagen fibers deposition in the lung.
3.3 Using Immonohistochemistry and RT-PCR to detect protein and mRNA of AT1R expression in the lung.
3.4 The measurement of collagen expression in the lung tissue
3.4.1 Immonohistochemistry: the detection intensity and position of the protein expression in lung tissue about alpha-SMA
3.4.2 Quantitation of total lung collagen: to detect hydroxyproline (HYP) of lung tissue by acidolysis assay, to express of total lung collage content.
3.4.3 ELISA: quantitation to detect the protein level of collagen I (ColI) in lung tissue.
3.4.4 RT-PCR: the detection at mRNA level of Coll.
3.5 The measurement of MMP-13 and TIMP-1 expression in the lung tissue.
3.5.1 Immonohistochemistry: the detection intensity and position of the protein expression in lung tissue about MMP-13 and TIMP-1.
3.5.2 RT-PCR: to detect mRNA level of MMP-13 and TIMP-1 in lung tissue of neonate rats .
4. Statistical analysis
SSPS version 11.5 was used to perform statistical analysis, with all data expressed as (mean±SD). Statistically significant differences in the mean values were analysis using the One-Way ANOVA, Independent-Samples T Test, bivariate. Life table was used to value survival rate, u-test for time comparison. Statistically significant differences set P<0.05.
Results
1. Survival rate and general status of rats in four groups
1.1 The hyperoxia rats almost began to present dyspnea, pale, cyanosis of different degrees after 5-7 days of oxygen exposure, some even depended high oxygen after 10 days of age. LO_2 groups was better than oxygen exposed and AO_2 groups withdraw from oxygen. The AIR groups didn't have the appearance above.
1.2 The death of hyperoxia rats increased with the time of oxygen exposure, and survival rate decreased significently (P<0.05); while there is no death after Id in AIR groups, and the survival curves were different in groups from 2 days of age(P<0.01). The survival rate of LO_2 groups increased comparing to oxygen exposure.
1.3 There was no difference in average birth weight between the four groups, but from 7 day of oxygen exposure, Body weights of the O_2 groups, AO_2 groups and LO_2 group animals showed a significant decrease compared with air controls at the sams time period(P<0.05). And the difference lasted to 21 days of age and more evident compared with air controls(P<0.01). There was no difference between the LO_2 groups and O_2 groups.
2. The pathological changes of the lung tissue
2.1 On day 1 of the experiment, it was observed in both room air and O_2 groups that the alveolar structure was irregular, terminal air space size was rather small, and the alveolar septum was thick. On 3 day, the alveolar structure of the each groups was more regular, the size of alveolus was equal, and alveolar septum was thinner, but in O_2 groups, there was a few inflammatory cells exuded out, blooding, interstitial cells increased. On 7 day, in room air rats, the alveolar size was equal, while the terminal air space size of the oxygen-exposed rat became large, there was inflammatory response and more interstitial cells. On 14 day and 21 day, air groups continue to progress alveolization; but in O_2 groups, the terminal air space size grew significantly large, secondary septum decreased, the quantity of alveolar reduced, with alveolus fusion, interstitial cells increased, and alveolar septa was thicker. There was no significant difference between the LO_2 groups and O_2 groups on 7 day. On 14 day d and 21 day, alveolar septum is thinner in LO2 groups, but alveolar space are not deflate obviously, and the sum of secondary septum is still less.
2.2 Masson staining: In air group, blue fibres mainly deposited in bronchil and vascular wall, little in alveolar septum. In O_2 groups, on 14 day and 21 day there were more collagen fibres along bronchil and vascular wall and alveolar septum, especially in alveolar septum. In LO_2 groups, the ratio of blue fibres is decreased and rarefied relatively compare with O_2 groups, but it is more than air group.
3. The dynamic changes of AT1R expression in the lung tissue
Immuonhistochemical studies shows AT1R expression in alveolar epithelial cells, fibroblast, interstitial macrophage and external of vessels and bronchus in control group; AT1R positive expression in endothelial cells and bronchial epithelial cells were also observed in fibrotic lesions in lungs. AT1R expression in air groups lung decreased but increased significantly in hyperoxia-exposed groups on 14 and 21 day.
4. The dynamic change of collagen expression in lung tissue
4.1 Immunohistochemical assessment of alpha-SMA expression
In air groups, alpha-SMA immunostaining was restricted to the smooth muscle of the pulmonary vasculature, airway walls, and openings to the respiratory bronchioles of terminal airway; meanwhile in 7-day-old rats, alpha-SMA positive cells are located in the slender elongated cells in the interstitium of primitive alveolar septa; in 21-day-old rats, alpha-SMA positive cells with a round shape are found at the tip of the secondary septa. Exposure to hyperoxia resulted in its expression a marked increasing in smooth muscle and alveolar septa myofibroblast foci and on the surface of alveolar significantly on 14 day and 21 day. Treatment of the hyperoxia-exposed animals with Losartan result in a significant decrease alpha-SMA expression compared with simple O_2 groups(P<0.01).
4.2 detect of HYP content in lung tissue by acidolysis.
There were no difference of HYP contents to four groups from 1 day to 7 day of the experiment. On 14 day and 21 day, HYP contents increased significantly in O_2 groups comparing to air groups(P<0.01); and changes of AO_2 groups similar to that of simple hyperoxia; in LO_2 groups it decreased slightly on 14 day; and on 21 day decreased significantly(P<0.01), however higher than that in air groups(P<0.01).
4.3 The dynamic change at the protein and mRNA level of ColI expression
The protein of Col I in the newborn lung tissue decreased with postnatal age rising in air groups; But in the O_2 and AO_2 groups it increased significantly than that in air control group(P<0.01), and its of larger difference on 21 day; in LO_2 groups it decreased slightly on 14 day; and on 21 day decreased significantly(P<0.01), but its higher than that in air group(P<0.01).
There was no different about mRNA expression of ColI at each spot in air groups; O_2 and AO_2 groups it increased with oxygen-exposed extending, and reach peak on 21 day. mRNA expression of ColI decreased significantly after Losartan-treated.
4.4 There was correlation between alpha-SMA and ColI mRNA in the lung (r=0.64, P<0.05), but none with the protein of ColI (r=0.28, P >0.05).
5.The dynamic changes of MMP-13 and TIMP-1 expression in the lung tissue
5.1 The dynamic change MMP-13 expression
Immunohistochemical staining shows, MMP-13 was expressed in the endochylema of epithelial cells, microvascular endothelial cells, interstitial cells and macrophages, in air lung tissues, there was no change of the MMP-13 expression at each time; In O_2 and AO_2 groups, it express at the same level from 1 day to 14 day, which was no difference with the air group. But on 21d, the expression of MMP-13 decreased significantly(P<0.01). In LO_2 group, it increased signifycantly comparing to O_2 group and reach the level of air groups. The curve change of MMP-13 mRNA is the same with that of protein.
5.2 The dynamic change TIMP-1 expression
TIMP-1 was expressed in epithelial cells, interstitial cells and pulmonary macrophages, the expression intensity of TIMP-1 protein and mRNA of all the rats were rising, from 7d after oxygen-exposed, its expression obvious increased, and significant on the 14 and 21d. losartan result in a significant decrease of the protein and mRNA expression of TIMP-1 compared with simple hyperoxia-exposed animals(P<0.01), but were higher than that of air group.
Concludsion
1. pathological findings charactered by the arrested alveolar development and deposited fibres in newborn rats after prolonged hyperoxia-exposed are consistent with those of CLD of prematurity in human.
2. Immuonhistochemical studies shows AT1R expression in alveolar epithelial cells, fibroblast, interstitial macrophage and external of vessels and bronchus in control group; AT1R positive expression in endothelial cells and bronchial epithelial cells were also observed in fibrotic lesions in lungs. AT1R expression increased significantly at the late stage of hyperoxia which is consist with the time of lung fibrosis genesis. It is hint that angiotensin II as a ligand of AT1R may take part in CLD genesis.
3. Exposure to hyperoxia resulted in a marked increase in smooth muscle, alveolar septa myofibroblast foci and on the surface of alveolar at the late phase of hyperoxia-exposed; however in air control groups, alpha-SMA positive cells with a round shape are found at the tip of the secondary septa, absent expression in alveolar space and mesenchymal. It is suppose that persistent hyperoxia exposed induced the position and intension change of myofibroblast may be one of the most reason of CLD genesis.
4. After Losartan administration, alpha-SMA, HYP content, and the protein and mRNA expression of ColI decreased significantly, meanwhile the degree of lung disorganization relieved. It is indicated that Losartan attenuated hyperoxia induced lung injury of neonate. It is also hint that hyperoxia induced lung fibrosis genesis may performed by the pathway Ang II binding to AT1 receptor regulating synthesis of ColI.
5. MMP-13 and TIMP-1 expression in the neonatal lung tissue, which hint MMP-13 and TIMP-1 invoved construction of lung morphous and collagen metabolism.
6. At the late phase of hyperoxia-exposed, the protein and mRNA expression disbalance of MMP-13/TIMP-1 in lung tissue may play an important role during the remodeling of CLD induced by hyperoxia.
7. losartan induced MMP-13 and down regulated TIMP-1 expression in hyperoxia-exposed lung tissue, attenuated extracellular matrix including ColI deposition at the late phase of hyperoxia-exposed, which is indicated that AngII may taked part in lung fibrosis induced by hyperoxia via AT1 receptor regulating synthesis and activity of MMP-13/TIMP-1.
引文
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42 Fukuda Y,Ishizaki M,Okada Y,et al.Matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in fetal rabbit lung.Am J Physiol Lung Cell Mol Physiol,2000,279(3) :L555-L561 43 Sweet DG,Pizzoti J,Wilbourn M,et al.Matrix metalloproteinase-9(MMP-9) in the airways of infants at risk of development chronic lung disease(CLD).Eur Respir J,1999,14(suppl 30) :248s.
44 Radomski A,Sawicki G,Olson DM,et al.The role of nitric oxide and metalloproteinases in the pathogenesis of hyperoxia-induced lung injury in newborn rats.Br J Pharmacol,1998,125(7) :1455-1462．
45 Sweet DG,McMahon KJ,Curley AE,et al.Type I collagenases in bronchoalveolar lavage fluid from preterm babies at risk of developing chronic lung disease.Arch Dis Child Fetal Neonatal Ed,2001,84(3) :F168-F171．
46 Fu JH,Xue XD.Gene expressions and roles of matrix metalloproteinases-8 and tissue inhibitor of metalloproteinases-1 in hyperoxia-induced pulmonary fibrosis in neonatal rats.Zhongguo Dang Dai Er Ke Za Zhi.2007;9(1) :1-5．
47 Cole AA,Chubinskaya S,Schumacher B,et al.Chondrocyte matrix metalloproteinase-8． Human articular chondrocytes express neutrophil collagenase.J Biol Chem 1996;271:11023-11026．
48 Prikk K,Piril? E,Sepper R,et al.In vivo collagenase-2 expression by human bronchial epithelial cells and monocytes/macrophages in bronchiectasis.J Pathol 2001;194:232-238．
49 Wahlgren J,Maisi P,Sorsa T,et al.Expression and induction of collagenases(MMP-8 and-13) in plasma cells associated with bone destructive lesions.J Pathol.2001;194:217-224．
50 Schaefer B,Rivas-Estilla AM,Meraz-Cruz N,et al.Reciprocal modulation of matrix metalloproteinase-13 and type I collagen genes in rat hepatic stellate cells.Am J Pathol.2003;162(6) :1771-1780．
51 Lee HS,Miau LH,Chen CH,et al.Differential role of p38 in IL-1alpha induction of MMP-9 and MMP-13 in an established liver myofibroblast cell line.J Biomed Sci.2003;10(6 Pt 2) :757-65．
52 Liang C,Wu ZG,Ding J,et al.Losartan inhibited expression of matrix metalloproteinases in rat atherosclerotic lesions and angiotensin Ⅱ-stimulated macrophages.Acta Pharmacol Sin.2004;25(11) :1426-1432．
53 Zhang P,Yang YJ,Chen X,et al.Comparison of doxycycline,losartan,and their combination on the expression of matrix metalloproteinase,tissue inhibitor of matrix metalloproteinase,and collagen remodeling in the noninfarcted myocardium after acute myocardial infarction in rats.Zhongguo Yi Xue Ke Xue Yuan Xue Bao.2005;27(1) :53-61．
54 Wang M,Zhang J,Spinetti G,et al.Angiotensin Ⅱ Activates Matrix Metalloproteinase Type Ⅱ and Mimics Age-Associated Carotid Arterial Remodeling in Young Rats.Am J Pathol.2005;167(5) :1429-1442．
55 Browatzki M,Larsen D,Pfeiffer CA,et al.Angiotensin Ⅱ stimulates matrix metalloproteinase secretion in human vascular smooth muscle cells via nuclear factor-kappaB and activator protein 1 in a redox-sensitive manner.J Vasc Res.2005;42(5) :415-423．
56 Arenas IA,Xu Y,Lopez-Jaramillo P,et al.ANG Ⅱ-induced MMP-2 release from endothelial cells is mediated by TNF-α.Am J Physiol Cell Physiol.2004;286:C779-C784．
57 Papakonstantinou E,Roth M,Kokkas B,et al.Losartan inhibits the angiotensin Ⅱ-induced modifications on fibrinolysis and matrix deposition by primary human vascular smooth muscle cells.J Cardiovasc Pharmacol.2001 Nov;38(5) :715-728．
58 Singh R,Alavi N,Singh AK,et al..Role of angiotensin Ⅱ in glucose-induced inhibition of mesangial matrix degradation.Diabetes.1999;48(10) :2066-2073．
59 Funck RC,Wilke A,Rupp H,et al.Regulation and role of myocardial collagen matrix remodeling in hypertensive heart disease.Adv Exp Med Biol.1997;432:35-44．
60 Bancalari E,Claure N,Sosenko IR.Bronchopulmonary dysplasia:changes in pathogenesis,epidemiology and definition.Semin Neonatol.2003;8(1) :63-68．
1 Marshall RP, McAnulty RJ, Laurent GJ. Angiotensin Ⅱ is mitogenic for human lung fibroblasts via activation of the type 1 receptor. Am J Respir Crit Care Med. 2000; 161:1999-2004.
2 Cohen EP, Molteni A, Hill P, et al.Captopril preserves function and ultrastructure in experimental radiation nephropathy. Lab Invest. 1996; 75: 349-360.
3 Linz W,Scholkens BA,and Ganten D.Converting enzyme inhibition specifically prevents the development and induces regression of cardiac hypertrophy in rats.Clin Exp Hypertens A.1989;11:1325-1350．
4 Baker KM,Chernin MI,Wixson SK,et al.Renin-angiotensin system involvement in pressure-overload cardiac hypertrophy in rats.Am J Physiol.1990． 259:H324-H332．
5 Song L,Wang D,Cui X,et al.Kinetic alterations of angiotensin-Ⅱ and nitric oxide in radiation pulmonary fibrosis.J Environ Pathol Toxicol Oncol.1998;17:141-150．
6 Specks U,Martin WJ Ⅱ,Rohrbach MS.Bronchoalveolar lavage fluid angiotensin-converting enzyme in interstitial lung diseases.Am Rev Respir Dis.1990;141(1) :117-23．
7 Uhal BD,Gidea C,Bargout R,et al.Captopril inhibits apoptosis in human lung epithelial cells:a potential antifíbrotic mechanism.Am J Physiol.1998;275:L1013-L1017．
8 Otsuka M,Takahashi H,Shiratori M,et al.Reduction of bleomycin induced lung fibrosis by candesartan cilexetil,an angiotensin Ⅱ type 1 receptor antagonist.Thorax.2004;59(1) :31-38．
9 Bullock GR,Steyaert I,Bilbe G,et al.Distribution of type-1 and type-2 Angiotensin Ⅱ receptors in the normal human lung and in lung from patients with chronic obstructive pulmonary disease.J Histochem Cell Biol.2001;115(2) :117-124．
10 Campbell DJ and Habener JF.Hybridization in situ studies of angiotensinogen gene expression in rat adrenal and lung.J Endocrinology.1989;124:218-222．
11 Cassis L,Shenoy U,Lipke D,et al.Lung angiotensin receptor binding characteristics during the development of monocrotaline-induced pulmonary hypertension.J Biochem Pharmacol.1997;54:27-31．
12 Marshall RP,Gohlke P,Chambers RC,et al.Angiotensin Ⅱ and the fibroproliferative response to acute lung injury.Am J Physiol Lung Cell Mol Physiol.2004;286:L156-L164．
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40 Browatzki M,Larsen D,Pfeiffer CA,et al.Angiotensin Ⅱ stimulates matrix metalloproteinase secretion in human vascular smooth muscle cells via nuclear factor-kappaB and activator protein 1 in a redox-sensitive manner.J Vasc Res.2005;42(5) :415-423．
41 Liang C,Wu ZG,Ding J,et al.Losartan inhibited expression of matrix metalloproteinases in rat atherosclerotic lesions and angiotensin Ⅱ-stimulated macrophages.Acta Pharmacol Sin.2004;25(11) :1426-1432．
42 Wang M,Zhang J,Spinetti G,et al.Angiotensin Ⅱ Activates Matrix Metalloproteinase Type Ⅱ and Mimics Age-Associated Carotid Arterial Remodeling in Young Rats.Am J Pathol.2005;167(5) :1429-1442．
43 Zhang P,Yang YJ,Chen X,et al.Comparison of doxycycline,losartan,and their combination on the expression of matrix metalloproteinase,tissue inhibitor of matrix metalloproteinase,and collagen remodeling in the noninfarcted myocardium after acute myocardial infarction in rats.Zhongguo Yi Xue Ke Xue Yuan Xue Bao.2005;27(1) :53-61．
44 Funck RC,Wilke A,Rupp H,et al.Regulation and role of myocardial collagen matrix remodeling in hypertensive heart disease.Adv Exp Med Biol.1997;432:35-44．
45 Singh R,Alavi N,Singh AK,et al.Role of angiotensin Ⅱ in glucose-induced inhibition of mesangial matrix degradation.Diabetes.1999;48(10) :2066-2073．
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11 曹蕾，Schmidt B, speer CP应用分子生物学技术探讨慢性肺疾病和呼吸窘迫综合征的炎症反应机制．中华儿科杂志，2001，39(11)：672-674．
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13 Marshall RP, Mc Anulty RJ, Laurent GJ. Angiotensin Ⅱ is mitogenic for human lung fibroblasts via activation of the type 1 receptor. Am J Respir Crit Care Med. 2000; 161: 1999-2004.
14 Marshall RP, Gohlke P, Chambers RC, et al. Angiotensin Ⅱ and the fibroproliferative response to acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2004; 286: L156-L164.
15 Otsuka M, Takahashi H, Shiratori M, et al. Reduction of bleomycin induced lung fibrosis by candesartan cilexetil, an angiotensin Ⅱ type 1 receptor antagonist.Thorax. 2004; 59(1): 31-38.
16 Mancini GB, Khalil N. Angiotensin Ⅱ type 1 receptor blocker inhibits pulmonary injury. Clin Invest Med .2005; 28(3): 118-126.
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18 Wang R, Zagariya A, Ibarra-Sunga O, et al.Angiotensin Ⅱ induces apoptosis in human and rat alveolar epithelial cells. Am. J. Physiol. 1999.276: L885-L889.
19 Wang R, Ramos C, Joshi Ⅰ, et al. Human lung myofibroblast-derived inducers of alveolar epithelial apoptosis identified as angiotensin peptides. Am J Physiol. 1999;277: L1158-L1164.
20 Papp M, Li X, Zhuang J, et al. Angiotensin Ⅱ receptor subtype AT(1) mediates alveolar epithelial cell apoptosis in response to ANG Ⅱ.Am J Physiol Lung Cell Mol Physiol. 2002;282(4):L713-718.
21 Bullock GR, Steyaert Ⅰ, Bilbe G, et al. Distribution of type-1 and type-2 Angiotensin Ⅱ receptors in the normal human lung and in lung from patients with chronic obstructive pulmonary disease. J Histochem Cell Biol.2001; 115(2): 117-124.
22 Northway WH Jr, Rosan RC, Porter DY. Pulmonary disease following respirator therapy of hyaline-membrane disease: bronchopulmonary dysplasia. N Engl J Med. 1967; 276: 357-368.
23 Uhal, BD, Joshi I, True A, et al. Fibroblasts isolated after fibrotic lung injury induce apoptosis of alveolar epithelial cells in vitro. Am J Physiol. 1995; 269:L819-L828.
24 王昌明，黄慧，张珍祥等．槲皮素对博莱霉素致鼠肺纤维化的防治作用．中国药理学通报．2000：16(1)：94-96．
25 Akkula M, Le Cras TD, Gebb S, et al. Inhibition of angiogenesis decreases alveolarization in t he developing rat lung. Am J Physiol Lung Cell Mol Physiol, 2000, 279(3): 600-607.
26 Li X,Rayford H,Uhal BD.Essential roles for angiotensin receptor AT1a in bleomycin-induced apoptosis and lung fibrosis in mice.Am J Pathol.2003;163(6) :2523-2530．
27 Phan SH.The myofibroblast in pulmonary fibrosis.Chest 2002;122:286S-289S.
28 Hu B,Wu Z,Phan SH.Smad3 mediates transforming growth factor-beta-induced alpha-smooth muscle actin expression.Am J Respir Cell Mol Biol.2003;29:397-404．
29 Epperly MW,Guo H,Gretton JE,Greenberger JS.Bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis.Am J Respir Cell Mol Biol 2003;29:213-224．
30 Willis BC,duBois RM,Borok Z.Epithelial Origin of Myofibroblasts during Fibrosis in the Lung.Proceedings of the ATS,2006;3(4) :377-382．
31 Yamada M,Kurihara H,Kinoshita K,et al.Temporal Expression of Alpha-Smooth Muscle Actin and Drebrin in Septal Interstitial Cells during Alveolar Maturation.Journal of Histo-chemistry and Cytochemistry.2005;53(6) :735-744．
32 Lindahl P,Karlsson L,Hellstrom M,et al.Alveogenesis failure in PDGF-A-deficient mice is coupled to lack of distal spreading of alveolar smooth muscle cell progenitors during lung development.Development,1997,124:3943-3953．
33 Wenzel S,Taimor G,Piper HM,et al.Redox-sensitive intermediates mediate Angiotensin Ⅱ-induced p38 MAP kinase activation,AP-1 binding activity,and TGF-beta expression in adult ventricular cardiomyocytes.J FASEB.2001;15:2291-2293．
34 Hu B,Wu Z,Phan SH.Smad3 Mediates Transforming Growth Factor-β1 Induced α-Smooth Muscle Actin Expression.American Journal of Respiratory Cell and Molecular Biology.2003;29,397-404．
35 Mancini GB,Khalil N.Angiotensin Ⅱ type 1 receptor blocker inhibits pulmonary injury.Clin Invest Med.2005;28(3) :118-126．
36 Keogh KA,Standing J,Kane GC,et al.Angiotensin Ⅱantagonism fails to ameliorate bleomycin-induced pulmonary fibrosis in mice.Eur Respir J.2005;25(4) :708-714．
37 Chen CM,Chou HC,Hsu HH,et al.Transforming growth factor-betal upregulation is independent of angiotensin in paraquat-induced lung fibrosis.Toxicology.2005;216(2-3) :181-187．
38 Arthur MJP.Fibrosis and altered matrix degradation.Digestion,1998;59:376-380．
39 Ekekezie Ⅱ, Thibeault DW,Simon SD, et al.Low levels of tissue inhibitors of metalloproteinases with a high matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio are present in tracheal aspirate fluids of infants who develop chronic lung disease.Pediatrics.2004;113(6) :1709-1714．
41 Pardo A,Barrios R,Maldonado V,et al.Gelatinases A and B are up-regulated in rat lungs by subacute hyperoxia:pathogenetic implications.Am J Pathol,1998,153(3) :833-844．
42 Fukuda Y,Ishizaki M,Okada Y,et al.Matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in fetal rabbit lung.Am J Physiol Lung Cell Mol Physiol,2000,279(3) :L555-L561 43 Sweet DG,Pizzoti J,Wilbourn M,et al.Matrix metalloproteinase-9(MMP-9) in the airways of infants at risk of development chronic lung disease(CLD).Eur Respir J,1999,14(suppl 30) :248s.
44 Radomski A,Sawicki G,Olson DM,et al.The role of nitric oxide and metalloproteinases in the pathogenesis of hyperoxia-induced lung injury in newborn rats.Br J Pharmacol,1998,125(7) :1455-1462．
45 Sweet DG,McMahon KJ,Curley AE,et al.Type I collagenases in bronchoalveolar lavage fluid from preterm babies at risk of developing chronic lung disease.Arch Dis Child Fetal Neonatal Ed,2001,84(3) :F168-F171．
46 Fu JH,Xue XD.Gene expressions and roles of matrix metalloproteinases-8 and tissue inhibitor of metalloproteinases-1 in hyperoxia-induced pulmonary fibrosis in neonatal rats.Zhongguo Dang Dai Er Ke Za Zhi.2007;9(1) :1-5．
47 Cole AA,Chubinskaya S,Schumacher B,et al.Chondrocyte matrix metalloproteinase-8． Human articular chondrocytes express neutrophil collagenase.J Biol Chem 1996;271:11023-11026．
48 Prikk K,Piril? E,Sepper R,et al.In vivo collagenase-2 expression by human bronchial epithelial cells and monocytes/macrophages in bronchiectasis.J Pathol 2001;194:232-238．
49 Wahlgren J,Maisi P,Sorsa T,et al.Expression and induction of collagenases(MMP-8 and-13) in plasma cells associated with bone destructive lesions.J Pathol.2001;194:217-224．
50 Schaefer B,Rivas-Estilla AM,Meraz-Cruz N,et al.Reciprocal modulation of matrix metalloproteinase-13 and type I collagen genes in rat hepatic stellate cells.Am J Pathol.2003;162(6) :1771-1780．
51 Lee HS,Miau LH,Chen CH,et al.Differential role of p38 in IL-1alpha induction of MMP-9 and MMP-13 in an established liver myofibroblast cell line.J Biomed Sci.2003;10(6 Pt 2) :757-65．
52 Liang C,Wu ZG,Ding J,et al.Losartan inhibited expression of matrix metalloproteinases in rat atherosclerotic lesions and angiotensin Ⅱ-stimulated macrophages.Acta Pharmacol Sin.2004;25(11) :1426-1432．
53 Zhang P,Yang YJ,Chen X,et al.Comparison of doxycycline,losartan,and their combination on the expression of matrix metalloproteinase,tissue inhibitor of matrix metalloproteinase,and collagen remodeling in the noninfarcted myocardium after acute myocardial infarction in rats.Zhongguo Yi Xue Ke Xue Yuan Xue Bao.2005;27(1) :53-61．
54 Wang M,Zhang J,Spinetti G,et al.Angiotensin Ⅱ Activates Matrix Metalloproteinase Type Ⅱ and Mimics Age-Associated Carotid Arterial Remodeling in Young Rats.Am J Pathol.2005;167(5) :1429-1442．
55 Browatzki M,Larsen D,Pfeiffer CA,et al.Angiotensin Ⅱ stimulates matrix metalloproteinase secretion in human vascular smooth muscle cells via nuclear factor-kappaB and activator protein 1 in a redox-sensitive manner.J Vasc Res.2005;42(5) :415-423．
56 Arenas IA,Xu Y,Lopez-Jaramillo P,et al.ANG Ⅱ-induced MMP-2 release from endothelial cells is mediated by TNF-α.Am J Physiol Cell Physiol.2004;286:C779-C784．
57 Papakonstantinou E,Roth M,Kokkas B,et al.Losartan inhibits the angiotensin Ⅱ-induced modifications on fibrinolysis and matrix deposition by primary human vascular smooth muscle cells.J Cardiovasc Pharmacol.2001 Nov;38(5) :715-728．
58 Singh R,Alavi N,Singh AK,et al..Role of angiotensin Ⅱ in glucose-induced inhibition of mesangial matrix degradation.Diabetes.1999;48(10) :2066-2073．
59 Funck RC,Wilke A,Rupp H,et al.Regulation and role of myocardial collagen matrix remodeling in hypertensive heart disease.Adv Exp Med Biol.1997;432:35-44．
60 Bancalari E,Claure N,Sosenko IR.Bronchopulmonary dysplasia:changes in pathogenesis,epidemiology and definition.Semin Neonatol.2003;8(1) :63-68．
1 Marshall RP, McAnulty RJ, Laurent GJ. Angiotensin Ⅱ is mitogenic for human lung fibroblasts via activation of the type 1 receptor. Am J Respir Crit Care Med. 2000; 161:1999-2004.
2 Cohen EP, Molteni A, Hill P, et al.Captopril preserves function and ultrastructure in experimental radiation nephropathy. Lab Invest. 1996; 75: 349-360.
3 Linz W,Scholkens BA,and Ganten D.Converting enzyme inhibition specifically prevents the development and induces regression of cardiac hypertrophy in rats.Clin Exp Hypertens A.1989;11:1325-1350．
4 Baker KM,Chernin MI,Wixson SK,et al.Renin-angiotensin system involvement in pressure-overload cardiac hypertrophy in rats.Am J Physiol.1990． 259:H324-H332．
5 Song L,Wang D,Cui X,et al.Kinetic alterations of angiotensin-Ⅱ and nitric oxide in radiation pulmonary fibrosis.J Environ Pathol Toxicol Oncol.1998;17:141-150．
6 Specks U,Martin WJ Ⅱ,Rohrbach MS.Bronchoalveolar lavage fluid angiotensin-converting enzyme in interstitial lung diseases.Am Rev Respir Dis.1990;141(1) :117-23．
7 Uhal BD,Gidea C,Bargout R,et al.Captopril inhibits apoptosis in human lung epithelial cells:a potential antifíbrotic mechanism.Am J Physiol.1998;275:L1013-L1017．
8 Otsuka M,Takahashi H,Shiratori M,et al.Reduction of bleomycin induced lung fibrosis by candesartan cilexetil,an angiotensin Ⅱ type 1 receptor antagonist.Thorax.2004;59(1) :31-38．
9 Bullock GR,Steyaert I,Bilbe G,et al.Distribution of type-1 and type-2 Angiotensin Ⅱ receptors in the normal human lung and in lung from patients with chronic obstructive pulmonary disease.J Histochem Cell Biol.2001;115(2) :117-124．
10 Campbell DJ and Habener JF.Hybridization in situ studies of angiotensinogen gene expression in rat adrenal and lung.J Endocrinology.1989;124:218-222．
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