【作者】 李文斌；

【导师】 常立文；

【作者基本信息】 华中科技大学 ， 儿科学， 2006， 博士

【摘要】 背景:急慢性肺损伤是造成新生儿尤其是早产儿死亡、伤残的主要原因之一,不成熟肺组织长时间暴露于高氧环境是导致急慢性肺损伤发生发展的重要因素。动物研究表明,在肺泡形成关键时期,长时间高氧暴露可阻碍肺泡间隔形成,使肺泡数目减少、呼吸膜内表面积减小和肺泡腔体积增大,与支气管肺发育不良(BPD)患儿肺部病理特征极其相似。同时,持续高氧暴露可促发肺部广泛炎症反应,导致肺泡毛细血管内皮细胞和上皮细胞坏死、凋亡,损害肺泡—毛细血管屏障功能;促使间质成纤维细胞向肺泡腔迁移、增生,并产生胶原蛋白沉积于肺泡腔,导致肺纤维化的发生。基质金属蛋白酶(MMPs)是一组依赖锌离子的中性蛋白酶,它们是降解细胞外基质(ECM)的主要介质,并与其特异性组织抑制物(TIMPs)一起参与了体内许多生物学过程,如胚胎发育、支气管分枝形成、血管发生、炎症过程以及损伤修复等。MMP-2、MMP-9又称明胶酶-A、明胶酶-B。在肺组织发育过程中,MMP-2和MMP-9的一个重要功能就是裂解ECM成分,产生具有生物学活性的片段,从而诱导肺泡上皮细胞的迁移以及肺分枝形态的发生,因此它们对于胚胎期以及出生后肺发育起至关重要的调控作用。我们以及其他研究者研究证实,高氧暴露引起早产大鼠急性肺损伤和肺发育阻滞同时,肺组织MMP-2、MMP-9表达水平和MMPs/TIMPs比值明显升高,ECM降解加速,肺组织发生广泛重塑,最终导致肺发育受阻。肺泡上皮主要由Ⅰ型(AECⅠ)和П型肺泡上皮细胞(AECⅡ)组成。AECⅡ是肺内主要干细胞,肺泡上皮损伤修复的唯一途径是AECⅡ增殖、迁移并向AECI转化,从而使肺泡壁重新上皮化以恢复气血屏障。在肺发育和损伤修复过程中,肺细胞增殖及凋亡行为受多种因素的精细调控。研究表明,高氧暴露可导致肺细胞DNA损伤,诱导肿瘤抑制蛋白p53及其下游靶基因表达,使细胞周期阻滞于G1/S期,以利于DNA修复;若DNA修复不成功,则诱导细胞发生凋亡。丝裂原活化蛋白激酶(MAPKs)是细胞增殖、分化、凋亡等信息传递途径的交汇点和共同通路,细胞外各种刺激信号可通过不同的细胞内信息传递系统,共同交汇于MAPKs。一旦被激活,MAPKs通过使其下游的转录因子磷酸化来调控靶基因表达。近年来研究表明,MAPK信号传递通路在MMPs/TIMPs的表达调控中扮演重要角色;并且还参与了肺发育和高氧肺损伤过程中细胞增殖和凋亡的调节。但MAPK信号传递通路是否参与高氧暴露下不成熟肺组织MMPs/TIMPs的表达调控?目前尚未见报道。两个大样本临床随机研究证实,维生素A是迄今为止唯一能降低BPD发生率和死亡率且无明显毒副作用的药物,但其作用机制尚未明了。维甲酸(RA)是维生素A在生物体内的重要活性形式,调节包括细胞生长、分化、发育和肿瘤发生等在内的多种重要生物学功能。研究表明,RA不仅能促进发育期大鼠肺泡形成,还能促进成熟肺组织损伤后的修复过程。给予外源性RA可改善肺泡结构、降低肺纤维化程度,对新生大鼠高氧肺损伤具有保护作用。进一步研究发现,RA对多种肺损伤的保护作用可能与调节MMPs/TIMPs的表达有关。那么,RA是否参与高氧暴露下肺组织MMPs/TIMPs表达调控?RA调节高氧暴露下肺组织MMPs/TIMPs的表达是否与调控MAPKs功能状态有关?迄今尚未见报道。另外,研究证实,RA还参与了细胞周期调控。那么,RA对高氧肺损伤时肺细胞凋亡、增殖的影响是否也与调节MAPKs的活性有关?目前尚不清楚。目的:通过建立早产大鼠高氧暴露动物和细胞模型,1、进一步阐明MMPs/TIMPs在高氧肺损伤中的作用;2、探讨MAPKs(ERK1/2、JNK1/2和p38)是否参与高氧暴露下MMPs/TIMPs的表达调控;3、探讨RA是否通过调控MAPKs功能状态调节MMPs/TIMPs的表达,从而发挥高氧肺损伤保护作用;4、从细胞增殖和凋亡角度,进一步论证RA高氧肺损伤保护作用机制。为RA的临床应用提供理论依据。方法:1、剖宫取出孕21 d(足月为22 d)SD早产鼠,随机分为4组:I、空气组;II、高氧组;III、空气+RA组;IV、高氧+RA组,I、III组置于空气中,II、IV组置于85 % O2中,III、IV组每日腹腔注射RA(500μg / kg)。于4 d、7 d、14 d收集肺组织标本,采用RT-PCR方法检测MMP-2、MMP-9、MT1-MMP、TIMP-1和TIMP-2 mRNA表达,采用明胶酶谱法检测MMP-2和MMP-9酶原及活酶表达,运用Western blot技术对TIMP-1、TIMP-2、p-ERK1/2、ERK1/2、p-JNK1/2、JNK1/2、p-p38、p38蛋白表达进行检测。2、原代培养早产鼠(孕19 d～20 d)AECII和肺成纤维细胞(LFs),待其生长至亚汇合状态时,将培养瓶中培养液换成含2 % FCS的MEM,并随机分为四组:I、空气组,II、高氧组,III、空气+RA组,IV、高氧+RA组。其中,III、IV组培养液中加入含有终浓度为1×10-6 mol/L的RA,I、II组培养液中加入含有相同终浓度的无水乙醇。I、III组置于空气中,II、IV组置于90 %高氧中。于培养2、6、12和24 h时提取细胞总RNA,采用RT-PCR方法检测MMP-2、MT1-MMP、和TIMP-2 mRNA表达;提取12 h和24 h细胞总蛋白及培养上清浓缩液,采用明胶酶谱法检测细胞总蛋白与培养上清混合液中MMP-2和MMP-9酶原及活酶表达,采用Western blot技术检测p-ERK1/2、ERK1/2、p-JNK1/2、JNK1/2、p-p38和p38蛋白表达;提取12 h和24 h细胞LFs核蛋白,采用Western blot方法检测p-c-Jun/c-Jun表达。3、分别以ERK1/2、JNK1/2和p38特异性阻断剂PD98059(10×10-6mol/L)、SP600125(10×10-6mol/L)和SB203580(10×10-6mol/L)作为干预方式,运用RT-PCR方法检测早产鼠LFs高氧暴露12 h MMP-2、MT1-MMP、和TIMP-2 mRNA表达,采用明胶酶谱法检测细胞总蛋白与培养上清混合液中MMP-2酶原及活酶表达和采用Western blot技术检测p-ERK1/2、ERK1/2、p-JNK1/2、JNK1/2、p-p38和p38蛋白表达。4、取上述各组4 d早产鼠肺组织标本,采用TUNEL法检测细胞凋亡,免疫组化法检测PCNA表达, Western blot技术检测p-ERK1/2、ERK1/2、p-JNK1/2、JNK1/2、p-p38和p38表达水平。5、取上述各组12 h AECII和LFs,采用流式细胞术(Annexin V—PI双标记)检测细胞凋亡,Western blot检测AECII p-ERK1/2、p-JNK1/2、p-p38、PCNA、P53及Caspase-3表达。结果:1、高氧、RA对早产鼠肺组织MMP-2、MMP-9、MT1-MMP、TIMP-1和TIMP-2表达的影响mRNA水平:空气暴露4 d、7 d、14 d时,早产大鼠肺组织MMP-2 mRNA的表达呈明显下降趋势;MMP-9和MT1-MMP mRNA的表达变化不明显; TIMP-1 mRNA的表达呈升高趋势,而TIMP-2 mRNA的表达则呈下降趋势;RA对空气暴露下MMP-2、MMP-9、MT1-MMP、TIMP-1和TIMP-2 mRNA的表达均无明显影响;与空气组比较,高氧暴露后,MMP-2、MMP-9、MT1-MMP和TIMP-1 mRNA的表达均有不同程度升高;RA不同程度下调高氧暴露后MMP-2、MMP-9、MT1-MMP和TIMP-1 mRNA的表达;而高氧、RA对TIMP-2 mRNA的表达无明显影响。蛋白水平:空气暴露下,4 d、7 d、14 d时,MMP-2酶原和活酶的表达水平呈明显下降趋势;MMP-9酶原的表达在7 d、14 d时有所下降,而其活酶表达水平在14 d时才明显下降;RA对空气暴露下MMP-2和MMP-9酶原及其活酶的表达无明显影响;与空气组比较,高氧暴露明显提高了MMP-2活酶、MMP-9酶原及其活酶的表达,而对MMP-2酶原的表达无明显影响;RA不同程度下调高氧暴露后MMP-2活酶、MMP-9酶原及其活酶的表达;空气暴露下,4 d、7 d、14 d时,TIMP-1蛋白表达水平呈升高趋势;而TIMP-2的表达则无明显变化;RA对空气暴露下TIMP-1和TIMP-2表达无明显影响;高氧暴露显著提高TIMP-1的表达, RA则有进一步促进高氧暴露后TIMP-1蛋白表达升高趋势;而高氧、RA对TIMP-2蛋白表达无明显影响。2、高氧、RA对早产鼠肺组织p-ERK1/2、ERK1/2、p-JNK1/2、JNK1/2、p-p38、p38蛋白表达的影响高氧暴露显著提高p-ERK1/2、p-JNK1/2和p-p38表达水平;RA不同程度降低高氧暴露下p-JNK1/2和p-p38表达,但进一步上调p-ERK1/2表达;高氧、RA对总ERK1/2、JNK1/2和p38表达无影响。3、高氧、RA对早产鼠AECⅡ和LFs MMP-2、MT1-MMP和TIMP-2表达的影响高氧暴露使LFs和AECⅡMMP-2、MT1-MMP mRNA表达明显上调,RA则明显下调高氧暴露下MMP-2和MT1-MMP mRNA表达;高氧、RA对TIMP-2 mRNA表达无明显影响;高氧暴露明显增强LFs、AECⅡ细胞裂解蛋白和培养上清浓缩液混合物中MMP-2(LFs、AECⅡ)和MMP-9(AECⅡ)酶原及活酶表达,而RA则具有下调作用。4、高氧、RA对早产鼠AECⅡ和LFs p-ERK1/2、ERK1/2、p-JNK1/2、JNK1/2、p-p38、p38蛋白表达的影响高氧暴露显著提高LFs和AECⅡp-ERK1/2、p-JNK1/2和p-p38表达水平;RA不同程度降低高氧暴露下p-JNK1/2和p-p38表达,但进一步上调p-ERK1/2表达;高氧、RA对总ERK1/2、JNK1/2和p38表达无影响。5、高氧、RA对早产鼠LFs核蛋白p-c-Jun/c-Jun表达的影响高氧暴露显著提高LFs p-c-Jun表达水平,RA则明显下调高氧暴露下其表达。6、高氧、RA、PD98059、SP600125、SB203580对早产鼠LFs MMP-2、MT1-MMP以及TIMP-2表达的影响RA、SP600125、SB203580在下调p-JNK1/2和p-p38表达同时,LFs MMP-2和MT1-MMP mRNA表达也明显下调,而TIMP-2 mRNA表达则不受影响; PD98059对LFs MMP-2、MT1-MMP和TIMP-2 mRNA表达无明显影响。7、高氧、RA对早产鼠肺组织细胞增殖、凋亡的影响高氧暴露4 d,肺实质凋亡细胞显著增加,且以AECII、毛细血管内皮细胞和AECI为主;RA明显降低高氧暴露下肺细胞凋亡;高氧暴露4 d,PCNA阳性细胞指数明显下降;RA明显提高高氧暴露肺组织PCNA表达,同时高氧暴露使肺泡分隔第二嵴明显减少、气腔明显增大;RA使空气暴露早产鼠肺泡第二嵴明显增多、肺泡直径变小,而对高氧暴露动物气腔大小以及第二嵴形成没有明显影响。8、高氧、RA对原代培养早产鼠LFs和AECII增殖、凋亡的影响高氧暴露12 h, AECII以晚期凋亡和坏死为主,同时,与空气组比较,其早期凋亡细胞数也显著升高;RA明显下调高氧暴露下AECII坏死、凋亡;高氧暴露12 h,明显降低AECII PCNA表达,显著提高其P53和Caspase-3活性片段表达;RA则明显上调AECII PCNA表达,下调P53和Caspase-3活性片段表达;高氧、RA对LFs坏死、凋亡无明显影响。结论:1、MMP-2、MMP-9、MT1-MMP、TIMP-1和TIMP-2动态表达变化规律与其在肺泡化过程中的作用密不可分;2、高氧暴露明显改变MMPs/TIMPs的表达,在肺泡形成关键时期,MMPs/TIMPs之间平衡关系的破坏是造成肺发育受阻和肺纤维化的重要因素;3、高氧暴露激活MAPKs信号传递通路(主要是JNK1/2和p38)是导致MMPs/TIMPs表达失衡的重要原因;4、高氧暴露,导致AECⅡ大量凋亡、坏死,增殖受到抑制;同时,LFs所受影响较小,两种细胞对高氧暴露的差异性行为也是导致未成熟肺组织异常重构的重要原因;5、RA通过下调JNK1/2和p38磷酸化水平、上调ERK1/2磷酸化水平,进而下调MMP-2、MMP-9、MT1-MMP表达与活化,降低AECⅡ坏死、凋亡,从而发挥高氧肺损伤保护作用。更多还原

【Abstract】 Background:Acute and chronic lung injury are major causes of mortality and morbidity in both preterm and term neonates. Prolonged exposure to hyperoxia in the developing lung is believed to play critical roles in the development of acute and chronic lung injury. In animal models, we and others have demonstrated that exposure to hyperoxia during critical window of alveolarization impairs lung septation, decreases alveolar number and internal surface area, enlarges of alveolar ducts and results in emphysematous changes similar to those found in patients with bronchopulmonary dysplasia (BPD). Simultaneously, the pathological changes includes severe disruption of the alveolar-capillary barrier, parenchymal cell injury followed by an inflammatory response and later by fibroblast proliferation and collagen accumulation, with the consequent distortion of the lung architecture, and eventually to lung fibrosis.Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases with crucial roles in extracellular matrix remodeling, acting in concert with their tissue inhibitors (TIMPs). Some of their functions regulate processes associated with development, such as branching morphogenesis and angiogenesis as well as inflammatory processes and wound healing. MMP-2 and MMP-9, also called gelatinases-A and -B, respectively. A critical effect of MMP-2 and MMP-9 in prenatal and postnatal lung development is to induce alveolar epithylial cell migration and branching morphogenesis by proteolytically cleavage extracellular matrix components. We and others have demonstrated that hyperoxia exposure lead to acute lung injury and inhibited lung development, accompaniment with MMP-2 and MMP-9 level and MMPs/TIMPs ratio markedly increased, extracellular matrix degradation and extensive tissue remodeling.Alveolar epithelial cells include type I cells (AEC I) and type II cells (AECII). The type II epithelial cell (the stem cells of the alveolar epithelium) is essential for normal repair after lung injury because it repopulates dead AEC I through proliferation, migration and differentiation, subsequently recovery the alveolar-capillary barrier. Numerous factors delicately regulate alveolar epithelial cell proliferation and apoptosis. Evidences suggest that the response to DNA damage may be important because DNA fragmentation and increased expression of the tumor suppressor protein p53 and its downstream target genes have been observed in murine lungs exposed to hyperoxia. p53 responds to DNA damage by arresting the cell cycle in G1 /S phase to allow DNA repair to take place and may result in apoptosis if the cell is unable to repair the DNA damage.The processes of lung growth, development, injury and repair are extremely complex, involving a multitude of effectors. Many of these effectors activate signaling pathways that converge into mitogen-activated protein kinases (MAPKs). There are three major families of MAPKs: the extracellular signal-regulated kinases-1 and -2 (ERK-1/2), c-Jun NH2-terminal kinases (JNK), and p38 kinases. Both ERK-1 and -2 are thought to be involved primarily in proliferation and differentiation, whereas JNK and p38 are believed to be involved in stress responses and apoptosis. Once activated, MAPKs regulate gene expression through phosphorylation of downstream transcriptional factors. Recent studies suggest that MAPKs are involved in the regulation of MMPs/TIMPs expression. Furthermore, MAPKs activity contributes to growth arrest and apoptosis. But the roles of MAPKs in hyperoxia-mediated premature lung MMPs/TIMPs expression have not been explored.Vitamin A is to date the only intervention tested by randomized clinical trials demonstrated to produce a decrease in relative risk of death or BPD at 36 weeks of post-menstrual age and has no visible side effect, but the exact mechanism has not been elucidated. Retinoic acid (RA) is vitamin A derivatives that regulate important biological functions, including cell growth and differentiation, development, and carcinogenesis. Recent data suggests that exogenous RA can improve alveolar structure, decreases fibrosis and regulates MMPs/TIMPs expression in the newborn rat with oxygen-induced lung injury. In addition, it has been reported that RA was involved in cell cycle regulation. But whether the protection of RA was related to regulating hyperoxia-induced activation of MAPKs was yet unknown.Objective:Establishment of hyperoxia lung injury animal and cells model:1. To further explore the role of MMPs/TIMPs in hyperoxia lung injury;2. To investigate whether MAPKs are involved in regulation of MMPs/TIMPs expression;3. To prove whether the protective effect of RA on hyperoxia lung injury was related with regulation of MMPs/TIMPs expression by MAPKs;4. To further investigate the role of MAPKs as modulators of oxidant-mediated proliferation, differentiation and apoptosis and the protective effect of RA on hyperoxia lung injury.Methods:1. Gastation 21 d Sprague-Dawley (SD) fetuses (term = 22 d) were delivered by hysterotomy. Within 12 h～24 h of birth, premature rat pups were randomly divided into 4 groups: Group I, Air-exposed control group; GroupII, hyperoxia-exposed group; Group III, Air-exposed plus RA group, Group IV, hyperoxia-exposed plus RA group. Group I and III were remained in room air, and group IIand IV were placed in 85% oxygen. The pups in Group III and IV were injected with RA (500μg/kg, every day) intraperitoneally. All lung tissues of premature rat pups were collected at 4 d, 7 d and 14 d after birth. The levels of MMP-2、MMP-9、MT1-MMP、TIMP-1 and TIMP-2 mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). MMP-2 and MMP-9 activity was measured by zymography. The protein abundance of TIMP-1、TIMP-2、p-ERK1/2、ERK1/2、p-JNK1/2、JNK1/2、p-p38 and p38 was determined by western blot.2. The primary rat embryonic LFs and AECII (gestation 19 d-20 d) were cultured in vitro. Cells grew to subconfluence and then randomly divided into 4 groups: Group I, Air-exposed control group; GroupII, hyperoxia-exposed group; Group III, Air-exposed plus RA group, Group IV, hyperoxia-exposed plus RA group. For the study of RA effects, sub-confluence growing cells were cultured for 24 h in medium with or without 1μM RA. Cells were then exposed to hyperoxia in the presence or absence of RA for the indicated durations. Cells cultured without RA were cultured in medium containing the same amount of ethanol. The levels of MMP-2、MT1-MMP and TIMP-2 mRNA were detected by RT-PCR; MMP-2 and MMP-9 activity was measured by zymography; The abundance of p-ERK1/2、ERK1/2、p-JNK1/2、JNK1/2、p-p38 and p38 was determined by western blot. LFs nuclear proteins were prepared and p-c-Jun/c-Jun was detected by western blot.3. The LFs were treated by PD98059(10×10-6mol/L), a specific inhibitor of MKK1 and MKK2 (ERK upstream kinases), SP600125(10×10-6mol/L), a specific inhibitor of JNK, and SB203580(10×10-6mol/L), a specific inhibitor of p38. The levels of MMP-2、MT1-MMP and TIMP-2 mRNA were detected by RT-PCR; MMP-2 and MMP-9 activity was measured by zymography; The abundance of p-ERK1/2、ERK1/2、p-JNK1/2、JNK1/2、p-p38 and p38 was determined by western blot.4. All lung tissues of premature rat pups were collected at 4 d after birth. Terminal Transferase d-UTP nick end labeling (TUNEL) staining detected cell apoptosis. The expression of PCNA was detected by Immunohistochemistry. Western blot analyses for phosphorylated and total nonphosphorylated ERKs, JNKs or p38.5. AEC II and LFs Apoptosis were analyzed by Annexin V/Propidium Iodide double Staining and flow cytometry. the expression of p-ERK1/2、p-JNK1/2、p-p38、PCNA、P53 and Caspase-3 in AEC II were determined by western blot. Results:1. The effect of hyperoxia and RA on the expression of lung tissue MMP-2、MMP-9、MT1-MMP、TIMP-1 and TIMP-2 mRNA levels:In room air, from 4 d to 14 d, Expression of message for MMP-2 was decreased, MMP-9 and MT1-MMP mRNA expression levels did not change, TIMP-1 mRNA expression levels was increased, and TIMP-2 mRNA expression levels was declined in pups. Treatment with RA did not significantly change expression of message for MMP-2、MMP-9、MT1-MMP、TIMP-1 and TIMP-2 in air-exposure.Exposure to oxygen resulted in levels of MMP-2、MMP-9、MT1-MMP and TIMP-1 mRNA consistently greater than levels expressed from lungs of normoxic pups. but rat pups treatment with RA from the hyperoxic environment expressed significantly lower levels of mRNA for MMP-2、MMP-9、MT1-MMP and TIMP-1 than the hyperoxic control pups on each experimental day. But hyperoxia and RA had not changed the expression of TIMP-2 mRNA.Protein levels:In room air, levels of pro-MMP-2, active MMP-2 and pro-MMP-9 were declined from 4 d to 14 d, active MMP-9 in 14 d was decreased. There were not significantly different between animals exposed to room air in the presence or absence of RA.The mean levels of active MMP-2, pro-MMP-9 and active MMP-9 after exposure to O2 were higher than air groups on each experimental day, and Pro-MMP-2 activity levels did not change. The levels of active MMP-2, pro-MMP-9 and active MMP-9 were decreased markedly after RA treatment in hyperoxia exposure rat pups.Protein expression of TIMP-1 was increased from 4 d to 14 d in room air exposure pups. RA had no effect on the protein levels of TIMP-1 in the pups exposed to room air. Hyperoxic exposure, however, caused a rapid increase in TIMP-1 mean protein levels on each experimental day, and RA treatment lead to a further elevate. Hyperoxia and RA did not change the protein expression of TIMP-2. 2. The effect of hyperoxia and RA on the expression of lung tissue p-ERK1/2、ERK1/2、p-JNK1/2、JNK1/2、p-p38、p38Western blot analyses showed that the amounts of JNK, p38 and ERK proteins in hyperoxia-exposure or RA-treated lung tissues were the same as in untreated lung tissues, whereas activation of these MAPKs was markedly altered by hyperoxia and RA. After hyperoxia exposure, p-ERK1/2, p-JNK1/2 and p-p38 were dramatically increased on each experimental day, p-JNK1/2 and p-p38 were markedly declined, but p-ERK1/2 was further elevated by RA treatment.3. The effect of hyperoxia and RA on the expression of AECII and LFs MMP-2、MT1-MMP and TIMP-2 mRNABoth MMP-2 and MT1-MMP mRNA expression levels were increased in cells exposed to hyperoxia for 2 h, 6 h, 12 h and 24 h, and decreased after RA treatment. The expression of TIMP-2 mRNA was not change by hyperoxia or RA treatment. Gelatinase zymography analyses showed that the levels of pro-MMP-2, active MMP-2, pro-MMP-9 and active MMP-9 were higher after exposure to O2 for 6 h and 12 h, and were lower by RA treatment.4. The effect of hyperoxia and RA on the protein levels of AECII and LFs p-ERK1/2、ERK1/2、p-JNK1/2、JNK1/2、p-p38、p38 expressionSimilar to premature lung tissues, hyperoxia-exposure or RA-treatment did not change the total of JNK, p38 and ERK proteins in AECII and LFs. After hyperoxia exposure, p-ERK1/2, p-JNK1/2 and p-p38 were significantly increased on each experimental hour, p-JNK1/2 and p-p38 were markedly decreased, but p-ERK1/2 was further increased by RA treatment.5. The effect of hyperoxia and RA on the LFs nuclear protein levels of p-c-Jun/c-Jun expressionp-c-Jun was elevated after exposure to O2 for 6 h and 12 h, and were decreased by RA treatment.6. The effect of hyperoxia, RA, PD98059, SP600125 and SB203580 on the expression of LFs MMP-2, MT1-MMP and TIMP-2 mRNATo inspect the roles of ERK, JNK and p38 signaling pathways in regulation the expression of MMP-2, MT1-MMP and TIMP-2 after hyperoxia exposure, LFs were exposed to hyperoxia or room air for 12 h in the presence of the kinase inhibitors PD98059, SP600125, SB203580, and RA respectively. The results showed that SP600125, SB203580, and RA inhibited p-JNK1/2 and p-p38, simultaneously decreased the expression of LFs MMP-2 and MT1-MMP mRNA, but PD98059 did not change their expression. In addition, PD98059, SP600125 and SB203580 had no effect on the the expression of TIMP-2 mRNA.7. The effect of hyperoxia and RA on the proliferation and apoptosis of prenatal lungLungs from pups exposed to hyperoxia for 4 d exhibited TUNEL-positive nuclei increased markedly throughout the parenchyma. TUNEL-positive nuclei in the parenchyma were mainly observed in alveolar type II cells, endothelial cells surrounding capillaries and type I cells. After RA treatment, TUNEL-positive nuclei decreased significantly in hyperoxia-exposed lung.In lung sections, after exposure to 85 % O2 for 4 d, the number of PCNA positive cells index was obviously decreased, and increased markedly by RA treatment. The air-space size was significantly enlarged and secondary crests were markedly decreased in hyperoxia-exposed animals. RA treatment improved lung air spaces and secondary crests in air-exposed pups, but had no effect on hyperoxia exposure pups. 8. The effect of hyperoxia and RA on the proliferation and apoptosis of AECII and LFs in vitroQuantitative data from flow cytometry analyses (PI/Annexin-V double staining) demonstrated that there was a significant increase in signs of both early apoptosis, as designated by quadrant II, and late apoptosis/necrosis (quadrant III) after AECII 12 h of hyperoxia. RA markedly decreased hyperoxia-induced AECII apoptosis and necrosis.Western blot analyses showed that the protein levels of PCNA was reduced, that of p53 and active fragment of Caspase-3 were increased after 12 h of hyperoxia in AECII; RA improved the expression of PCNA, and decreased the expression of p53 and active fragment of Caspase-3.The apoptosis and proliferation of LFs were not changed by hyperoxia exposure and/or RA treatment.Conclusions:1. MMP-2, MMP-9, MT1-MMP, TIMP-1 and TIMP-2 are all involved in alveolarization of premature rat lung development;2. the balance of MMPs/TIMPs was broken by hyperoxia during alveolarization of premature rat lung development, which lead to lung development inhibition and lung fibrosis;3. Hyperoxia exposure activated MAPKs (mainly JNK and p38), which played a role in broken the balance of MMPs/TIMPs;4. hyperoxia exposure lead to numerous AECII apoptosis and necrosis, but did not change LFs survival, both of which were involved in abnormal lung remodeling;5. RA had a protective effect on hyperoxia lung injury by which decrease active levels of JNK and p38, increase active levels of ERK, subsequently reduce the expression and activation of MMP-2, MMP-9 and MT1-MMP, and decline AECII apoptosis and necrosis.更多还原