维甲酸与丝裂原活化蛋白激酶调控高氧肺损伤机制的研究
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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。
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、p38
Western 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 mRNA
Both 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 expression
Similar 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 expression
p-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 mRNA
To 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 lung
Lungs 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 vitro
Quantitative 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.
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。
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、p38
Western 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 mRNA
Both 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 expression
Similar 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 expression
p-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 mRNA
To 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 lung
Lungs 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 vitro
Quantitative 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.
引文
1. Saugstad OD. Bronchopulmonary dysplasia and oxidative stress: are we closer to an understanding of the pathogenesis of BPD? [J]. Acta Paediatr, 1997, 86: 1277-1282.
2. Manji JS, O’Kelly CJ, Leung WI, Olson DM. Timing of hyperoxic exposure during alveolarization influences damage mediated by leukotrienes. Am J Physiol Lung Cell Mol Physiol, 2001, 281: L799–L806
3. Boros V, Burghardt JS, Morgan CJ, Olson DM. Leukotrienes are indicated as mediators of hyperoxia-inhibited alveolarization in newborn rats. Am J Physiol, 1997, 272: L433–L441
4. Parks WC, Shapiro SD. Matrix metalloproteinases in lung biology. Respir Res, 2001, 2: 10–19
5. Kheradmand F, Rishi K, Werb Z. Signaling through the EGF receptor controls lung morphogenesis in part by regulating MT1-MMP-mediated activation of gelatinase A/MMP2 [J]. J Cell Sci, 2002, 15: 839-848
6. Fukuda Y, Ishizaki M, Okada Y, Seiki M, Yamanaka N. Matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in fetal rabbit lung. Am J Physiol Lung Cell Mol Physiol, 2000, 279: L555–L561
7. Fassina G, Ferrari N, Brigati C, Benelli R, Santi L, Noonan DM, Albini A. Tissue inhibitors of metalloproteases: regulation and biological activities. Clin Exp Metastasis. 2000, 18:111–120
8. Hosford GE, Fang X, Olson DM. Hyperoxia Decreases Matrix Metalloproteinase-9 and Increases Tissue Inhibitor of Matrix Metalloproteinase-1 Protein in the Newborn Rat Lung: Association with Arrested Alveolarization. Pediatr Res, 2004, 56: 26-34
9. Melendez J, Maldonado V, Bingle CD, Selman M, Pardo A. Cloning and expression of guinea pig TIMP-2. Expression in normal and hyperoxic lung injury. Am J Physiol Lung Cell Mol Physiol, 2000, 278(4): L737-743
10. Pardo A, Barrios R, Maldonado V, Melendez J, Perez J, Ruiz V, Segura-Valdez L, Sznajder JI, Selman M. Gelatinases A and B are up-regulated in rat lungs by subacute hyperoxia: pathogenetic implications. Am J Pathol, 1998, 153: 833–844
11. Ekekezie II, Thibeault DW, Simon SD, Norberg M, Merrill JD, Ballard RA, Ballard PL, Truog WE. 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
12. Buckley S, Warburton D. Dynamics of metalloproteinase-2 and -9, TGF-beta, and uPA activities during normoxic vs. hyperoxic alveolarization. Am J PhysiolLung Cell Mol Physiol, 2002, 283: L747-754
13. Gushima Y, Ichikado K, Suga M, Okamoto T, Iyonaga K, Sato K, Miyakawa H, Ando M. Expression of matrix metalloproteinases in pigs with hyperoxia-induced acute lung injury. Eur Respir J, 2001, 18: 827-37
14. McGowan SE, Harvey CS, Jackson SK. Retinoids, retinoic acid receptors, and cytoplasmic retinoid binding proteins in perinatal rat lung fibroblasts. Am J Physiol, 1995, 269: L463–L472
15. Massaro GD, Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats. Am J Physiol, 1996, 270: L305–L310
16. Nabeyrat E, Corroyer S, Epaud R, et al. Retinoic acid induced proliferation of lung alveolar epithelial cells is linked to p21(CIP1) down regulation. Am J Physiol Lung Cell Mol Physiol, 2000, 278: L42–L50
17. Massaro GD, Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats. Nat Med, 1997, 3: 675–677
18. Ozer EA, Kumral A, Ozer E, et al. Effect of Retinoic Acid on Oxygen-Induced Lung Injury in the Newborn Rat. Pediatr Pulmonol, 2005, 39(2): 35–40
19. Frankenberger M, Hauck RW, Frankenberger B, Haussinger K, Maier KL, Heyder J, Ziegler-Heitbrock HW. All trans-retinoic acid selectively down-regulates matrix metalloproteinase-9 (MMP-9) and up-regulates tissue inhibitor of metalloproteinase-1 (TIMP-1) in human bronchoalveolar lavage cells. Mol Med, 2001, 7: 63-70
20. Mao JT, Tashkin DP, Belloni PN, Baileyhealy I, Baratelli F, Roth MD. All-trans retinoic acid modulates the balance of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in patients with emphysema. Chest, 2003 , 124: 1724-1732
21. Zhu YK, Liu X, Ertl RF, et al. Retinoic acid attenuates cytokine-driven fibroblast degradation of extracellular matrix in three-dimensional culture. Am J Respir Cell Mol Biol, 2001, 25: 620–627
22. 钱莉玲, 常立文, 容志惠, 张谦慎. 基质金属蛋白酶及其抑制剂在高氧暴露下的早产大鼠肺组织中的动态表达. 中华围产医学杂志, 2003, 6(5): 302-305
23. 钱莉玲, 常立文, 容志惠, 张谦慎. 维甲酸治疗新生大鼠高氧肺发育受抑对肺组织基质金属蛋白酶及其组织抑制剂表达的影响. 实用儿科临床杂志,2002, 17(6): 602-604
24. Warner BB, Stuart LA, Papes RA, Wispe JR. Functional and pathological effects of prolonged hyperoxia in neonatal mice. Am J Physiol, 1998, 275: L110-117
25. Williams MC. Development of the alveolar structure of the fetal rat in late gestation. Federation Proceedings, 1977, 36: 2653-2659
26. Vu TH, Werb Z. Matrix metalloproteinases: effectors of development and normalphysiology. Genes Dev, 2000, 14: 2123–2133
27. Fassina G, Ferrari N, Brigati C, Benelli R, Santi L, Noonan DM, Albini A. Tissue inhibitors of metalloproteases: regulation and biological activities. Clin Exp Metastasis, 2000, 18: 111–120
28. Buckley S, Driscoll B, Shi W, Anderson K, Warburton D. Migration and gelatinases in cultured fetal, adult, and hyperoxic alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2001, 281: L427-434
29. Minoo P, Penn R, deLemos DM, Coalson JJ, deLemos. RA Tissue inhibitor of metalloproteinase-1 mRNA is specifically induced in lung tissue after birth. Pediatr Res, 1993, 34: 729–734
30. Ekekezie II, Thibeault DW, Simon SD, Norberg M, Merrill JD, Ballard RA, Ballard PL, Truog WE. 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: 1709-1714
31. Benbow U, Schoenermark MP, Mitchell TI, Rutter JL, Shimokawa K, Nagase H, Brinckerhoff CE. A novel host/tumor cell interaction activates matrix metalloproteinase 1 and mediates invasion through type I collagen. J Biol Chem, 1999, 274: 25371-25378
32. Schoenermark MP, Mitchell TI, Rutter JL, Reczek PR, Brinckerhoff CE. Retinoid-mediated suppression of tumor invasion and matrix metalloproteinase synthesis. Ann N Y Acad Sci, 1999, 878: 466-486
33. Axel DI, Frigge A, Dittmann J, Runge H, Spyridopoulos I, Riessen R, Viebahn R, Karsch KR. All-trans retinoic acid regulates proliferation, migration, differentiation, and extracellular matrix turnover of human arterial smooth muscle cells. Cardiovasc Res, 2001, 49: 851-862
34. Dedieu S, Lefebvre P. Retinoids interfere with the AP1 signalling pathway in human breast cancer cells Cell Signal, 2006, 18: 889-898
35. Ho LJ, Lin LC, Hung LF, Wang SJ, Lee CH, Chang DM, Lai JH, Tai TY. Retinoic acid blocks pro-inflammatory cytokine-induced matrix metalloproteinase production by down-regulating JNK-AP-1 signaling in human chondrocytes. Biochem Pharmacol, 2005, 70:200-208
36. Varani J, Perone P, Merfert MG, Moon SE, Larkin D, Stevens MJ. All-trans retinoic acid improves structure and function of diabetic rat skin in organ culture. Diabetes, 2002,51: 3510-3516
1. Gary LJ, Razvan L. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science, 2002, 298: 1911-1912
2. Westermarck J, Li SP, Kallunki T, Han J, Kahari VM. p38 mitogen-activated protein kinase dependent activation of protein phosphatases 1 and 2A inhibits MEK1 and MEK2 activity and collagenase 1(MMP-1) gene expression. Mol Cell Biol, 2001; 21(7): 2373-2383
3. Reunanen N, Li SP, Ahonen M, Foschi M, Han J, Kahari VM. Activation of p38α MAPK enhances collagenase-1 (matrix metalloproteinase(MMP)-1) and stromelysin-1 (MMP-3) expression by mRNA stabilization. J Biol Chem, 2002; 277(35):32360-32368
4. Ranganathan AC, Nelson KK, Rodriguez AM, Kim KH, Tower GB, Rutter JL, Brinckerhoff CE, Huang TT, Epstein CJ, Jeffrey JJ, Melendez JA. Manganese superoxide dismutase signals matrix metalloproteinase expression via H2O2-dependent ERK1/2 activation. J Biol Chem, 2001; 276(17): 14264-14270
5. Wenk J, Brenneisen P, Wlaschek M, Poswig A, Briviba K, Oberley TD, Scharffetter-Kochanek K. Stable overexpression of manganese superoxide dismutase in mitochondria identifies hydrogen peroxide as a major oxidant in the AP-1-mediated inductuin of matrix-degrading metalloprotease-1. J Biol Chem, 1999; 274(36): 25869-25876
6. Eberhardt W, Huwiler A, Beck KF, Walpen S, Pfeilschifter J. Amplification of IL-1β-induced matrix metalloproteinase-9 expresson by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-κB andactivating protein-1 and involves activation of the mitogen-activated protein kinase pathways. J Immunl, 2000;165(10):5788-5797
7. Buhimschi IA, Kramer WB, Buhimschi CS, Thompson LP, Weiner CP. Reduction-oxidation (redox) state regulation of matrix metalloproteinase activity in human fetal membranes. Am J Obstet Gynecol, 2000; 182(2): 458-464
8. Zhang HJ, Zhao W, Venkataraman S, Robbins ME, Buettner GR, Kregel KC, Oberley LW. Activation of matrix metalloproteinase-2 by overexpression of manganese superoxide dismutase in human breast cancer MCF-7 cells involes reactive oxygen species. J Biol Chem, 2002; 277(23): 20919-20926
9. Nabeyrat E, Besnard V, Corroyer S, Cazals V, Clement A. Retinoic acid-induced proliferation of lung alveolar epithelial cells: relation with the IGF system. Am J Physiol. 1998, 275 :L71-79
10. Zhu YK, Liu X, Ertl RF, Kohyama T, Wen FQ, Wang H, Spurzem JR. Retinoic acid attenuates cytokine-driven fibroblast degradation of extracellular matrix in three-dimensional culture. Am J Respir Cell Mol Biol, 2001, 25: 620–627
11. Massaro GD, Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats. Am J Physiol, 1996, 270: L305–L310
12. Massaro GD, Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats. Nat Med, 1997, 3: 675–677
13. Masuda M, Toh S, Koike K, Kuratomi Y, Suzui M, Deguchi A, Komiyama S, Weinstein IB. The roles of JNK1 and Stat3 in the response of head and neck cancer cell lines to combined treatment with all-trans-retinoic acid and 5-fluorouracil. Jpn J Cancer Res, 2002, 93:329-339
14. Benkoussa M, Brand C, Delmotte MH, Formstecher P, Lefebvre P. Retinoic acid receptors inhibit AP1 activation by regulating extracellular signal-regulated kinase and CBP recruitment to an AP1-responsive promoter. Mol Cell Biol , 2002, 22:4522-4534
15. Ortiz MA, Lopez-Hernandez FJ, Bayon Y, Pfahl M, Piedrafita FJ. Retinoid-related molecules induce cytochrome c release and apoptosis through activation of c-Jun NH(2)-terminal kinase/p38 mitogen-activated protein kinases. Cancer Res, 2001, 61: 8504-8512
16. Heussen C, Dowdle EB. Electrophoretic analysis of plasminogen activation in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem, 1980, 102:196-202
17. 钱莉玲, 常立文, 容志惠, 张谦慎. 基质金属蛋白酶及其抑制剂在高氧暴露下的早产大鼠肺组织中的动态表达. 中华围产医学杂志, 2003, 6(5): 302-305
18. 钱莉玲, 常立文, 容志惠, 张谦慎. 维甲酸治疗新生大鼠高氧肺发育受抑对肺组织基质金属蛋白酶及其组织抑制剂表达的影响. 实用儿科临床杂志,2002, 17(6): 602-604
19. Hong IK, Kim YM, Jeoung DI, Kim KC, Lee H. Tetraspanin CD9 induces MMP-2 expression by activating p38 MAPK, JNK and c-Jun pathways in humanmelanoma cells. Exp Mol Med, 2005, 37: 230-239
20. Denkert C, Siegert A, Leclere A, Turzynski A, Hauptmann S. An inhibitor of stress-activated MAP-kinases reduces invasion and MMP-2 expression of malignant melanoma cells. Clin Exp Metastasis, 2002, 19:79-85
21. Xie Z, Singh M, Singh K. Differential regulation of matrix metalloproteinase-2 and -9 expression and activity in adult rat cardiac fibroblasts in response to interleukin-1beta. J Biol Chem, 2004, 279: 39513-39519
22. Yao JS, Chen Y, Zhai W, Xu K, Young WL, Yang GY. Minocycline exerts multiple inhibitory effects on vascular endothelial growth factor-induced smooth muscle cell migration: the role of ERK1/2, PI3K, and matrix metalloproteinases. Circ Res, 2004 , 95: 364-371
23. Munshi HG, Wu YI, Mukhopadhyay S, Ottaviano AJ, Sassano A, Koblinski JE, Platanias LC, Stack MS. Differential regulation of membrane type 1-matrix metalloproteinase activity by ERK 1/2- and p38 MAPK-modulated tissue inhibitor of metalloproteinases 2 expression controls transforming growth factor-beta1-induced pericellular collagenolysis. J Biol Chem, 2004, 279: 39042-39050
24. Welty SE, Smith CV. Rationale for antioxidant therapy in premature infants to prevent bronchopulmonary dysplasia. Nutr Revi, 2001, 59: 10-17
25. Reunanen N, Li SP, Ahonen M, Foschi M, Han J, Kahari VM. Activation of p38α MAPK enhances collagenase-1 (matrix metalloproteinase (MMP)-1) and stromelysin-1 (MMP-3) expression by mRNA stabilization. J Biol Chem, 2002, 277:32360-32368
26. Ranganathan AC, Nelson KK, Rodriguez AM, Kim KH, Tower GB, Rutter JL, Brinckerhoff CE, Huang TT, Epstein CJ, Jeffrey JJ, Melendez JA. Manganese superoxide dismutase signals matrix metalloproteinase expression via H2O2-dependent ERK1/2 activation. J Biol Chem, 2001, 276(17): 14264-14270
27. Wenk J, Brenneisen P, Wlaschek M, Poswig A, Briviba K, Oberley TD, Scharffetter-Kochanek K. Stable overexpression of manganese superoxide dismutase in mitochondria identifies hydrogen peroxide as a major oxidant in the AP-1-mediated inductuin of matrix-degrading metalloprotease-1. J Biol Chem, 1999, 274: 25869-25876
28. Eberhardt W, Huwiler A, Beck KF, Walpen S, Pfeilschifter J. Amplification of IL-1β-induced matrix metalloproteinase-9 expresson by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-κB and activating protein-1 and involves activation of the mitogen-activated protein kinase pathways. J Immunl, 2000, 165:5788-5797
29. Buhimschi IA, Kramer WB, Buhimschi CS, Thompson LP, Weiner CP. Reduction-oxidation (redox) state regulation of matrix metalloproteinase activity in human fetal membranes. Am J Obstet Gynecol, 2000, 182: 458-464
30. Zhang HJ, Zhao W,et al. Activation of matrix metalloproteinase-2 byoverexpression of manganese superoxide dismutase in human breast cancer MCF-7 cells involes reactive oxygen species. J Biol Chem, 2002; 277(23): 20919-26
31. Lee HY, Walsh GL, Dawson MI, Hong WK, Kurie JM. All-trans-retinoic acid inhibits Jun N-terminal kinase-dependent signaling pathways. J Biol Chem, 1998, 273:7066-7071
32. Lee HY, Sueoka N, Hong WK, Mangelsdorf DJ, Claret FX, Kurie JM. All-trans-retinoic acid inhibits Jun N-terminal kinase by increasing dual-specificity phosphatase activity. Mol Cell Biol, 1999 , 19:1973-1980
33. Agadir A, Chen G, Bost F, Li Y, Mercola D, Zhang X. Differential effect of retinoic acid on growth regulation by phorbol ester in human cancer cell lines. J Biol Chem, 1999, 274: 29779-29785
34. Masuda M, Toh S, Koike K, Kuratomi Y, Suzui M, Deguchi A, Komiyama S, Weinstein IB. The roles of JNK1 and Stat3 in the response of head and neck cancer cell lines to combined treatment with all-trans-retinoic acid and 5-fluorouracil. Jpn J Cancer Res, 2002, 93:329-339
35. Benkoussa M, Brand C, Delmotte MH, Formstecher P, Lefebvre P. Retinoic acid receptors inhibit AP1 activation by regulating extracellular signal-regulated kinase and CBP recruitment to an AP1-responsive promoter. Mol Cell Biol , 2002, 22:4522-4534
36. Ortiz MA, Lopez-Hernandez FJ, Bayon Y, Pfahl M, Piedrafita FJ. Retinoid-related molecules induce cytochrome c release and apoptosis through activation of c-Jun NH (2)-terminal kinase/p38 mitogen-activated protein kinases. Cancer Res, 2001, 61: 8504-8512
37. Palm-Leis A, Singh US, Herbelin BS, et al. Mitogen-activated protein kinases and mitogen-activated protein kinase phosphatases mediate the inhibitory effects of all-trans retinoic acid on the hypertrophic growth of cardiomyocytes. J Biol Chem, 2004, 279:54905-54917
38. Yang G, Madan A, Dennery PA. Maturational differences in hyperoxic AP-1 activation in rat lung. Am J Physiol Lung Cell Mol Physiol, 2000, 278: L393-398
39. Bergman MR, Cheng S, Honbo N, Piacentini L, Karliner JS, Lovett DH. A functional activating protein 1 (AP-1) site regulates matrix metalloproteinase 2 (MMP-2) transcription by cardiac cells through interactions with JunB-Fra1 and JunB-FosB heterodimers. Biochem J, 2003, 369:485-496
1. Rush MG, Hazinski TA. Current therapy of bronchopulmonary dysplasia. Clin Perinatol, 1992,19: 563–590
2. Warner B, Stuart L, Papes R, Wispe J. Functional and pathological effects of prolonged hyperoxia in neonatal mice. Am J Physiol, 1998, 275: L110–L117
3. O’Brodovich HM, Mellins RB. Bronchopulmonary dysplasia.Unresolved neonatal acute lung injury. Am Rev Respir Dis, 1985, 132: 694–709
4. Mantell LL, Lee PJ. Signal transduction pathways in hyperoxia-induced lung cell death. Mol Genet Metab, 2000, 71:359-370
5. Wang X, Ryter SW, Dai C, Tang ZL, Watkins SC, Yin XM, Song R, Choi AM. Necrotic Cell Death in Response to Oxidant Stress Involves the Activation of the Apoptogenic Caspase-8/Bid Pathway. J Cell Biochem, 2003, 278: 29184-29191
6. McGrath-Morrow SA, Stahl J. Apoptosis in Neonatal Murine Lung Exposed to Hyperoxia. Am J Physiol Lung Cell Mol Physiol, 2001, 25:150-155
7. Pardo A, Barrios R, Maldonado V, Melendez J, Perez J, Ruiz V, Segura-Valdez L, Sznajder JI, Selman M. Gelatinases A and B are up-regulated in rat lungs by subacute hyperoxia: pathogenetic implications. Am J Pathol, 1998, 153: 833-844
8. O’Reilly MA, Staversky RJ, Watkins RH, Reed CK, Mesy Jensen KL, Finkelstein JN, Keng PC. The Cyclin-Dependent Kinase Inhibitor p21 Protects the Lung from Oxidative Stress. Am J Physiol Lung Cell Mol Physiol, 2001, 24: 703-710
9. Buckley S, Barsky L, Driscoll B, Weinberg K, Anderson KD, Warburton D.Apoptosis and DNA damage in type 2 alveolar epithelial cells cultured from hyperoxic rats. Am J Physiol, 1998, 274:L714-720
10. O'Reilly MA, Staversky RJ, Stripp BR, Finkelstein JN. Exposure to hyperoxia induces p53 expression in mouse lung epithelium. Am J Respir Cell Mol Biol, 1998 , 18: 43-50
11. Staversky RJ, Watkins RH, Wright TW, Hernady E, LoMonaco MB, D'Angio CT, Williams JP, Maniscalco WM, O'Reilly MA. Normal remodeling of the oxygen-injured lung requires the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1). Am J Pathol, 2002, 161:1383-93
12. Shenberger JS, Dixon PS. Oxygen induces S-phase growth arrest and increases p53 and p21WAF1/CIP1 expression in human bronchial smooth-muscle cells. Am J Respir Cell Mol Biol, 1999, 21: 395-402
13. McGrath-Morrow SA, Cho C, Soutiere S, Mitzner W, Tuder R. The effect of neonatal hyperoxia on the lung of p21Waf1/Cip1/Sdi1-deficient mice. Am J Respir Cell Mol Biol, 2004, 30: 635-640
14. Chuang SM, Wang IC, Yang JL. Roles of JNK, p38 and ERK mitogen-activated protein kinases in the growth inhibition and apoptosis induced by cadmium. Carcinogenesis, 2000, 21:1423-1432
15. Kling DE, Lorenzo HK, Trbovich AM, Kinane TB, Donahoe PK, Schnitzer JJ. MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs.Am J Physiol Lung Cell Mol Physiol, 2002, 282:L370-L378
16. Morse D, Otterbein LE, Watkins S, Alber S, Zhou Z, Flavell RA, Davis RJ, Choi AM. Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice. Am J Physiol Lung Cell Mol Physiol, 2003, 285: L250-L257
17. Buder-Hoffmann S, Palmer C, Vacek P, Taatjes D, Mossman B. DifferentAccumulation of Activated Extracellular Signal–Regulated Kinases (ERK 1/2) and Role in Cell-Cycle Alterations by Epidermal Growth Factor, Hydrogen Peroxide, or Asbestos in Pulmonary Epithelial Cells. Am J Physiol Lung Cell Mol Physiol, 2001, 24: 405-413
18. Petrache I, Choi ME, Otterbein LE, Chin BY, Mantell LL, Horowitz S, Choi AM. Mitogen-activated protein kinase pathway mediates hyperoxia-induced apoptosis in cultured macrophage cells. Am J Physiol, 1999, 277: L589-L595
19. Nabeyrat E, Besnard V, Corroyer S, Cazals V, Clement A. Retinoic acid-induced proliferation of lung alveolar epithelial cells: relation with the IGF system. Am J Physiol, 1998, 275 : L71-L79
20. Liebeskind A, Srinivasan S, Kaetzel D, Bruce M. Retinoic acid stimulates immature lung fibroblast growth via a PDGF-mediated autocrine mechanism. Am J Physiol Lung Cell Mol Physiol, 2000, 279: L81-L90
21. Massaro GD, Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats. Am J Physiol Lung Cell Mol Physiol, 1996, 270: L305-L310
22. Robbins ST, Fletcher AB. Early vs. delayed vitaminA supplementation in very-low-birth-weight infants. J Parenter Enteral Nutr, 1993, 17: 220-225
23. Shenberger JS, Dixon PS. Oxygen induces S-phase growth arrest and increases p53 and p21(WAF1/CIP1) expression in human bronchial smooth-muscle cells. Am J Respir Cell Mol Biol, 1999, 21:395-402
24. Nabeyrat E, Corroyer S, Besnard V, Cazals-Laville V, Bourbon J, Clement A. Retinoic acid protects against hyperoxia-mediated cell-cycle arrest of lung alveolar epithelial cells by preserving late G1 cyclin activities. Am J Respir Cell Mol Biol, 2001, 25:507-514
25. Bui KC, Buckley S, Wu F, Uhal B, Joshi I, Liu J, Hussain M, Makhoul I, Warburton D. Induction of A- and D-type cyclins and cdc2 kinase activity during recovery from short-term hyperoxic lung injury. Am J Physiol, 1995, 268: L625-L635
26. Veness-Meehan KA, Pierce RA, Moats-Staats BM, Stiles AD. Retinoic acid attenuates O2-induced inhibition of lung septation. Am J Physiol Lung Cell Mol Physiol, 2002, 283(5): L971–980
27. Pierce RA, Shipley JM. Retinoid-enhanced alveolization: identifying relevant downstream Targets. Am J Respir Cell Mol Biol, 2000, 23(2): 137–141
28. Kresch MJ, Christian C, Wu F, Hussain N. Ontogeny of apoptosis during lung development. Pediatr Res, 1998, 43: 426–431
29. Bruce MC, Honaker CE, Cross RJ. Lung fibroblasts undergo apoptosis following alveolarization. Am J Respir Cell Mol Biol, 1999, 20: 228–236
30. De Paepe ME, Rubin LP, Jude C, Lesieur-Brooks AM, Mills DR, and Luks FI. Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development. Am J Physiol Lung Cell Mol Physiol, 2000, 279: L967–L976
31. Welty SE, Smith CV. Rationale for antioxidant therapy in premature infants to prevent bronchopulmonary dysplasia. Nutr Revi, 2001, 59: 10-17
32. Luo Y, Hurwitz J, Massague J. Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1. Nature, 1995, 375:159–161
33. Buccellato LJ, Tso M, Akinci OI, Chandel NS, Budinger GR. Reactive oxygen species are required for hyperoxia-induced Bax activation and cell death in alveolar epithelial cells. J Biol Chem, 2004, 279: 6753-6760
34. 常立文,祝华平,李文斌,刘汉楚,容志惠,张谦慎,陈红兵. 杏仁甙对高氧暴露的早产鼠肺泡 II 细胞的保护作用. 中华儿科杂志,2005,43(2):118-123
35. Massaro D, Massaro GD. Retinoids, alveolus formation, and alveolar deficiency: clinical implications. Am J Respir Cell Mol Biol, 2003, 28: 271–274
36. Nabeyrat E, Corroyer S, Epaud R, Besnard V, Cazals V, Clement A. Retinoic acid-induced proliferation of lung alveolar epithelial cells is linked to p21(CIP1) downregulation. Am J Physiol Lung Cell Mol Physiol, 2000, 278 : L42-L50
37. Cho SJ, George CL, Snyder JM, Acarregui MJ. Retinoic acid and erythropoietin maintain alveolar development in mice treated with an angiogenesis inhibitor. Am J Respir Cell Mol Biol. 2005, 33: 622-628
38. Liu M, Iavarones A, Freedman LP. Transcriptional activation of the human p21WAF1/CIP1 gene by retinoic acid receptor. J Biol Chem, 1996, 271: 31723-31728
39. Carvalho H, Evelson P, Sigaud S, et al. Mitogen-activated protein kinases modulate H2O2-induced apoptosis in primary rat alveolar epithelial cells. J Cell Biochem, 2004, 92: 502-513
40. Buckley S, Driscoll B, Barsky L, Weinberg K, Anderson K, Warburton D. ERK activation protects against DNA damage and apoptosis in hyperoxic rat AEC2. Am J Physiol, 1999, 277:L159-L166
41. Zhang X, Shan P, Sasidhar M, Chupp GL, Flavell RA, Choi AM, Lee PJ. Reactive oxygen species and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase mediate hyperoxia-induced cell death in lung epithelium, 2003 , 28: 305-315
42. Morse D, Otterbein LE, Watkins S, Alber S, Zhou Z, Flavell RA, Davis RJ, Choi AM. Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice, 2003, 285: L250-L257
43. Li Y, Arita Y, Koo HC, Davis JM, Kazzaz JA. Inhibition of c-Jun N-terminal kinase pathway improves cell viability in response to oxidant injury. Am J Respir Cell Mol Biol, 2003, 29: 779-783
44. Truong SV, Monick MM, Yarovinsky TO, Powers LS, Nyunoya T, Hunninghake GW. Extracellular signal-regulated kinase activation delays hyperoxia-induced epithelial cell death in conditions of Akt downregulation. Am J Respir Cell Mol Biol, 2004, 31: 611-618
45. Nyunoya T, Powers LS, Yarovinsky TO, Butler NS, Monick MM, Hunninghake GW. Hyperoxia induces macrophage cell cycle arrest by adhesion-dependent induction of p21Cip1 and activation of the retinoblastoma protein. J Biol Chem, 2003, 278:36099-360106
46. Cho SJ, George CL, Snyder JM, Acarregui MJ. Retinoic acid and erythropoietin maintain alveolar development in mice treated with an angiogenesis inhibitor. Am J Respir Cell Mol Biol. 2005, 33:622-628
47. Palm-Leis A, Singh US, Herbelin BS, et al. Mitogen-activated protein kinases and mitogen-activated protein kinase phosphatases mediate the inhibitory effects of all-trans retinoic acid on the hypertrophic growth of cardiomyocytes. J Biol Chem, 2004, 279:54905-54917
48. Crowe DL, Kim R, Chandraratna RA. Retinoic acid differentially regulates cancer cell proliferation via dose-dependent modulation of the mitogen-activated protein kinase pathway. Mol Cancer Res, 2003, 1:532-540
1. Schock BC, Sweet DG, et al. Oxidative stress and increased type-IV collagenase levels in bronchoalveolar lavage fluid from newborn babies. Pediatr Res, 2001; 50(1): 29-33
2. Gary LJ, Razvan L. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science, 2002; 298:1911-2
3. Westermarck J, Li SP, et al. p38 mitogen-activated protein kinase dependent activation of protein phosphatases 1 and 2A inhibits MEK1 and MEK2 activity and collagenase 1(MMP-1) gene expression. Mol Cell Biol, 2001; 21(7): 2373-83
4. Dik WA, Krejger RR, et al. Localization and potential role of matrix metalloproteinase-1 and tissue inhibitors of metalloproteinase-1 and -2 in different phases of bronchopulmonary dysplasia. Pediatr Res, 2001; 50(6): 761-6
5. Cederqvist K, Sorsa T, et al. Matrix metalloproteinases-2, -8, -9 and TIMP-2 in tracheal aspirates from preterm infants with respiratory distress. Pediatrics, 2001; 108(3): 686-92
6. Welty SE, Smith CV. Rationale for antioxidant therapy in premature infants to prevent bronchopulmonary dysplasia. Nutr Revi, 2001; 59(1): 10-7
7. Reunanen N, Li SP, et al. Activation of p38α MAPK enhances collagenase-1 (matrix metalloproteinase(MMP)-1) and stromelysin-1 (MMP-3) expression by mRNA stabilization. J Biol Chem, 2002; 277(35):32360-8
8. Ranganathan AC, nelson KK, et al. Manganese superoxide dismutase signals matrix metalloproteinase expression via H2O2-dependent ERK1/2 activation. J Biol Chem, 2001; 276(17): 14264-70
9. Wenk J, Brenneisen P, et al. Stable overexpression of manganese superoxide dismutase in mitochondria identifies hydrogen peroxide as a major oxidant in the AP-1-mediated inductuin of matrix-degrading metalloprotease-1. J Biol Chem, 1999; 274(36): 25869-76
10. Eberhardt W, Huwiler A, et al. Amplification of IL-1 β -induced matrix metalloproteinase-9 expresson by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-κB and activating protein-1 and involves activation of the mitogen-activated protein kinase pathways. J Immunl, 2000;165(10):5788-97
11. Buhimschi IA, Kramer WB, et al. Reduction-oxidation (redox) state regulation of matrix metalloproteinase activity in human fetal membranes. Am J Obstet Gynecol, 2000; 182(2): 458-64
12. Eberhardt W, Huwiler A, et al. Amplification of IL-1 β -induced matrix metalloproteinase-9 expresson by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-κB and activating protein-1 and involvesactivation of the mitogen-activated protein kinase pathways. J Immunl, 2000 ; 165 (10):5788-97
13. Zhang HJ, Zhao W,et al. Activation of matrix metalloproteinase-2 by overexpression of manganese superoxide dismutase in human breast cancer MCF-7 cells involes reactive oxygen species. J Biol Chem, 2002; 277(23): 20919-26
14. Kresch MJ, Christian C, Wu F, Hussain N. Ontogeny of apoptosis during lung development. Pediatr Res, 1998, 43: 426–431
15. Bruce MC, Honaker CE, Cross RJ. Lung fibroblasts undergo apoptosis following alveolarization. Am J Respir Cell Mol Biol, 1999, 20: 228–236
16. De Paepe ME, Rubin LP, Jude C, Lesieur-Brooks AM, Mills DR, and Luks FI. Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development. Am J Physiol Lung Cell Mol Physiol, 2000, 279: L967–L976
17. Kling DE, Lorenzo HK, Trbovich AM, Kinane TB, Donahoe PK, Schnitzer JJ. MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs.Am J Physiol Lung Cell Mol Physiol, 2002, 282:L370-L378
18. Pardo A, Barrios R, Maldonado V, Melendez J, Perez J, Ruiz V, Segura-Valdez L, Sznajder JI, Selman M. Gelatinases A and B are up-regulated in rat lungs by subacute hyperoxia: pathogenetic implications. Am J Pathol, 1998, 153: 833-844
19. Wang X, Ryter SW, Dai C, Tang ZL, Watkins SC, Yin XM, Song R, Choi AM. Necrotic Cell Death in Response to Oxidant Stress Involves the Activation of the Apoptogenic Caspase-8/Bid Pathway. J Cell Biochem, 2003, 278: 29184-29191
20. Luo Y, Hurwitz J, Massague J. Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1. Nature, 1995, 375:159–161
21. Buccellato LJ, Tso M, Akinci OI, Chandel NS, Budinger GR. Reactive oxygen species are required for hyperoxia-induced Bax activation and cell death in alveolar epithelial cells. J Biol Chem, 2004, 279: 6753-6760
22. Kling DE, Lorenzo HK, Trbovich AM, Kinane TB, Donahoe PK, Schnitzer JJ. MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs.Am J Physiol Lung Cell Mol Physiol, 2002, 282:L370-L378
23. Morse D, Otterbein LE, Watkins S, Alber S, Zhou Z, Flavell RA, Davis RJ, Choi AM. Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice. Am J Physiol Lung Cell Mol Physiol, 2003, 285: L250-L257
24. Petrache I, Choi ME, Otterbein LE, Chin BY, Mantell LL, Horowitz S, Choi AM. Mitogen-activated protein kinase pathway mediates hyperoxia-induced apoptosis in cultured macrophage cells. Am J Physiol, 1999, 277: L589-L595
25. Zhang X, Shan P, Sasidhar M, Chupp GL, Flavell RA, Choi AM, Lee PJ. Reactive oxygen species and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase mediate hyperoxia-induced cell death in lung epithelium, 2003 , 28: 305-315
26. Buckley S, Driscoll B, Barsky L, Weinberg K, Anderson K, Warburton D. ERKactivation protects against DNA damage and apoptosis in hyperoxic rat AEC2. Am J Physiol, 1999, 277:L159-L166
27. Morse D, Otterbein LE, Watkins S, Alber S, Zhou Z, Flavell RA, Davis RJ, Choi AM. Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice, 2003, 285: L250-L257
28. Li Y, Arita Y, Koo HC, Davis JM, Kazzaz JA. Inhibition of c-Jun N-terminal kinase pathway improves cell viability in response to oxidant injury. Am J Respir Cell Mol Biol, 2003, 29: 779-783
29. Truong SV, Monick MM, Yarovinsky TO, Powers LS, Nyunoya T, Hunninghake GW. Extracellular signal-regulated kinase activation delays hyperoxia-induced epithelial cell death in conditions of Akt downregulation. Am J Respir Cell Mol Biol, 2004, 31: 611-618
30. Nyunoya T, Powers LS, Yarovinsky TO, Butler NS, Monick MM, Hunninghake GW. Hyperoxia induces macrophage cell cycle arrest by adhesion-dependent induction of p21Cip1 and activation of the retinoblastoma protein. J Biol Chem, 2003, 278:36099-360106
31. Cho SJ, George CL, Snyder JM, Acarregui MJ. Retinoic acid and erythropoietin maintain alveolar development in mice treated with an angiogenesis inhibitor. Am J Respir Cell Mol Biol. 2005, 33:622-628
32. Zhu YK, Liu X, Ertl RF, Kohyama T, Wen FQ, Wang H, Spurzem JR. Retinoic acid attenuates cytokine-driven fibroblast degradation of extracellular matrix in three-dimensional culture. Am J Respir Cell Mol Biol, 2001, 25: 620–627
33. Massaro GD, Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats. Am J Physiol, 1996, 270: L305–L310
34. Massaro GD, Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats. Nat Med, 1997, 3: 675–677
35. Ozer EA, Kumral A, Ozer E, et al. Effect of Retinoic Acid on Oxygen-Induced Lung Injury in the Newborn Rat. Pediatr Pulmonol, 2005, 39(2): 35–40
36. Schoenermark MP, Mitchell TI, Rutter JL, Reczek PR, Brinckerhoff CE. Retinoid-mediated suppression of tumor invasion and matrix metalloproteinase synthesis. Ann N Y Acad Sci, 1999, 878: 466-486
37. Axel DI, Frigge A, Dittmann J, Runge H, Spyridopoulos I, Riessen R, Viebahn R, Karsch KR. All-trans retinoic acid regulates proliferation, migration, differentiation, and extracellular matrix turnover of human arterial smooth muscle cells. Cardiovasc Res, 2001, 49: 851-862
38. Dedieu S, Lefebvre P. Retinoids interfere with the AP1 signalling pathway in human breast cancer cells Cell Signal, 2006, 18: 889-898
39. Ho LJ, Lin LC, Hung LF, Wang SJ, Lee CH, Chang DM, Lai JH, Tai TY. Retinoic acid blocks pro-inflammatory cytokine-induced matrix metalloproteinase production by down-regulating JNK-AP-1 signaling in human chondrocytes. Biochem Pharmacol, 2005, 70:200-208
40. Varani J, Perone P, Merfert MG, Moon SE, Larkin D, Stevens MJ. All-trans retinoic acid improves structure and function of diabetic rat skin in organ culture. Diabetes, 2002,51: 3510-3516
41. Mao JT, Tashkin DP, Belloni PN, Baileyhealy I, Baratelli F, Roth MD. All-trans retinoic acid modulates the balance of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in patients with emphysema. Chest, 2003 , 124: 1724-1732
42. 钱莉玲, 常立文, 容志惠, 张谦慎. 基质金属蛋白酶及其抑制剂在高氧暴露下的早产大鼠肺组织中的动态表达. 中华围产医学杂志, 2003, 6(5): 302-305
43. 钱莉玲, 常立文, 容志惠, 张谦慎. 维甲酸治疗新生大鼠高氧肺发育受抑对肺组织基质金属蛋白酶及其组织抑制剂表达的影响. 实用儿科临床杂志,2002, 17(6): 602-604
44. Nabeyrat E, Corroyer S, Epaud R, Besnard V, Cazals V, Clement A. Retinoic acid-induced proliferation of lung alveolar epithelial cells is linked to p21(CIP1) downregulation. Am J Physiol Lung Cell Mol Physiol, 2000, 278 : L42-L50
45. Nabeyrat E, Corroyer S, Besnard V, Cazals-Laville V, Bourbon J, Clement A. Retinoic acid protects against hyperoxia-mediated cell-cycle arrest of lung alveolar epithelial cells by preserving late G1 cyclin activities. Am J Respir Cell Mol Biol, 2001, 25:507-514
46. Liu M, Iavarones A, Freedman LP. Transcriptional activation of the human p21WAF1/CIP1 gene by retinoic acid receptor. J Biol Chem, 1996, 271: 31723-31728
47. Lee HY, Walsh GL, Dawson MI, Hong WK, Kurie JM. All-trans-retinoic acid inhibits Jun N-terminal kinase-dependent signaling pathways. J Biol Chem, 1998, 273:7066-7071
48. Lee HY, Sueoka N, Hong WK, Mangelsdorf DJ, Claret FX, Kurie JM. All-trans-retinoic acid inhibits Jun N-terminal kinase by increasing dual-specificity phosphatase activity. Mol Cell Biol, 1999 , 19:1973-1980
49. Palm-Leis A, Singh US, Herbelin BS, et al. Mitogen-activated protein kinases and mitogen-activated protein kinase phosphatases mediate the inhibitory effects of all-trans retinoic acid on the hypertrophic growth of cardiomyocytes. J Biol Chem, 2004, 279:54905-54917
50. Crowe DL, Kim R, Chandraratna RA. Retinoic acid differentially regulates cancer cell proliferation via dose-dependent modulation of the mitogen-activated protein kinase pathway. Mol Cancer Res, 2003, 1:532-540
2. Manji JS, O’Kelly CJ, Leung WI, Olson DM. Timing of hyperoxic exposure during alveolarization influences damage mediated by leukotrienes. Am J Physiol Lung Cell Mol Physiol, 2001, 281: L799–L806
3. Boros V, Burghardt JS, Morgan CJ, Olson DM. Leukotrienes are indicated as mediators of hyperoxia-inhibited alveolarization in newborn rats. Am J Physiol, 1997, 272: L433–L441
4. Parks WC, Shapiro SD. Matrix metalloproteinases in lung biology. Respir Res, 2001, 2: 10–19
5. Kheradmand F, Rishi K, Werb Z. Signaling through the EGF receptor controls lung morphogenesis in part by regulating MT1-MMP-mediated activation of gelatinase A/MMP2 [J]. J Cell Sci, 2002, 15: 839-848
6. Fukuda Y, Ishizaki M, Okada Y, Seiki M, Yamanaka N. Matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in fetal rabbit lung. Am J Physiol Lung Cell Mol Physiol, 2000, 279: L555–L561
7. Fassina G, Ferrari N, Brigati C, Benelli R, Santi L, Noonan DM, Albini A. Tissue inhibitors of metalloproteases: regulation and biological activities. Clin Exp Metastasis. 2000, 18:111–120
8. Hosford GE, Fang X, Olson DM. Hyperoxia Decreases Matrix Metalloproteinase-9 and Increases Tissue Inhibitor of Matrix Metalloproteinase-1 Protein in the Newborn Rat Lung: Association with Arrested Alveolarization. Pediatr Res, 2004, 56: 26-34
9. Melendez J, Maldonado V, Bingle CD, Selman M, Pardo A. Cloning and expression of guinea pig TIMP-2. Expression in normal and hyperoxic lung injury. Am J Physiol Lung Cell Mol Physiol, 2000, 278(4): L737-743
10. Pardo A, Barrios R, Maldonado V, Melendez J, Perez J, Ruiz V, Segura-Valdez L, Sznajder JI, Selman M. Gelatinases A and B are up-regulated in rat lungs by subacute hyperoxia: pathogenetic implications. Am J Pathol, 1998, 153: 833–844
11. Ekekezie II, Thibeault DW, Simon SD, Norberg M, Merrill JD, Ballard RA, Ballard PL, Truog WE. 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
12. Buckley S, Warburton D. Dynamics of metalloproteinase-2 and -9, TGF-beta, and uPA activities during normoxic vs. hyperoxic alveolarization. Am J PhysiolLung Cell Mol Physiol, 2002, 283: L747-754
13. Gushima Y, Ichikado K, Suga M, Okamoto T, Iyonaga K, Sato K, Miyakawa H, Ando M. Expression of matrix metalloproteinases in pigs with hyperoxia-induced acute lung injury. Eur Respir J, 2001, 18: 827-37
14. McGowan SE, Harvey CS, Jackson SK. Retinoids, retinoic acid receptors, and cytoplasmic retinoid binding proteins in perinatal rat lung fibroblasts. Am J Physiol, 1995, 269: L463–L472
15. Massaro GD, Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats. Am J Physiol, 1996, 270: L305–L310
16. Nabeyrat E, Corroyer S, Epaud R, et al. Retinoic acid induced proliferation of lung alveolar epithelial cells is linked to p21(CIP1) down regulation. Am J Physiol Lung Cell Mol Physiol, 2000, 278: L42–L50
17. Massaro GD, Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats. Nat Med, 1997, 3: 675–677
18. Ozer EA, Kumral A, Ozer E, et al. Effect of Retinoic Acid on Oxygen-Induced Lung Injury in the Newborn Rat. Pediatr Pulmonol, 2005, 39(2): 35–40
19. Frankenberger M, Hauck RW, Frankenberger B, Haussinger K, Maier KL, Heyder J, Ziegler-Heitbrock HW. All trans-retinoic acid selectively down-regulates matrix metalloproteinase-9 (MMP-9) and up-regulates tissue inhibitor of metalloproteinase-1 (TIMP-1) in human bronchoalveolar lavage cells. Mol Med, 2001, 7: 63-70
20. Mao JT, Tashkin DP, Belloni PN, Baileyhealy I, Baratelli F, Roth MD. All-trans retinoic acid modulates the balance of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in patients with emphysema. Chest, 2003 , 124: 1724-1732
21. Zhu YK, Liu X, Ertl RF, et al. Retinoic acid attenuates cytokine-driven fibroblast degradation of extracellular matrix in three-dimensional culture. Am J Respir Cell Mol Biol, 2001, 25: 620–627
22. 钱莉玲, 常立文, 容志惠, 张谦慎. 基质金属蛋白酶及其抑制剂在高氧暴露下的早产大鼠肺组织中的动态表达. 中华围产医学杂志, 2003, 6(5): 302-305
23. 钱莉玲, 常立文, 容志惠, 张谦慎. 维甲酸治疗新生大鼠高氧肺发育受抑对肺组织基质金属蛋白酶及其组织抑制剂表达的影响. 实用儿科临床杂志,2002, 17(6): 602-604
24. Warner BB, Stuart LA, Papes RA, Wispe JR. Functional and pathological effects of prolonged hyperoxia in neonatal mice. Am J Physiol, 1998, 275: L110-117
25. Williams MC. Development of the alveolar structure of the fetal rat in late gestation. Federation Proceedings, 1977, 36: 2653-2659
26. Vu TH, Werb Z. Matrix metalloproteinases: effectors of development and normalphysiology. Genes Dev, 2000, 14: 2123–2133
27. Fassina G, Ferrari N, Brigati C, Benelli R, Santi L, Noonan DM, Albini A. Tissue inhibitors of metalloproteases: regulation and biological activities. Clin Exp Metastasis, 2000, 18: 111–120
28. Buckley S, Driscoll B, Shi W, Anderson K, Warburton D. Migration and gelatinases in cultured fetal, adult, and hyperoxic alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2001, 281: L427-434
29. Minoo P, Penn R, deLemos DM, Coalson JJ, deLemos. RA Tissue inhibitor of metalloproteinase-1 mRNA is specifically induced in lung tissue after birth. Pediatr Res, 1993, 34: 729–734
30. Ekekezie II, Thibeault DW, Simon SD, Norberg M, Merrill JD, Ballard RA, Ballard PL, Truog WE. 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: 1709-1714
31. Benbow U, Schoenermark MP, Mitchell TI, Rutter JL, Shimokawa K, Nagase H, Brinckerhoff CE. A novel host/tumor cell interaction activates matrix metalloproteinase 1 and mediates invasion through type I collagen. J Biol Chem, 1999, 274: 25371-25378
32. Schoenermark MP, Mitchell TI, Rutter JL, Reczek PR, Brinckerhoff CE. Retinoid-mediated suppression of tumor invasion and matrix metalloproteinase synthesis. Ann N Y Acad Sci, 1999, 878: 466-486
33. Axel DI, Frigge A, Dittmann J, Runge H, Spyridopoulos I, Riessen R, Viebahn R, Karsch KR. All-trans retinoic acid regulates proliferation, migration, differentiation, and extracellular matrix turnover of human arterial smooth muscle cells. Cardiovasc Res, 2001, 49: 851-862
34. Dedieu S, Lefebvre P. Retinoids interfere with the AP1 signalling pathway in human breast cancer cells Cell Signal, 2006, 18: 889-898
35. Ho LJ, Lin LC, Hung LF, Wang SJ, Lee CH, Chang DM, Lai JH, Tai TY. Retinoic acid blocks pro-inflammatory cytokine-induced matrix metalloproteinase production by down-regulating JNK-AP-1 signaling in human chondrocytes. Biochem Pharmacol, 2005, 70:200-208
36. Varani J, Perone P, Merfert MG, Moon SE, Larkin D, Stevens MJ. All-trans retinoic acid improves structure and function of diabetic rat skin in organ culture. Diabetes, 2002,51: 3510-3516
1. Gary LJ, Razvan L. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science, 2002, 298: 1911-1912
2. Westermarck J, Li SP, Kallunki T, Han J, Kahari VM. p38 mitogen-activated protein kinase dependent activation of protein phosphatases 1 and 2A inhibits MEK1 and MEK2 activity and collagenase 1(MMP-1) gene expression. Mol Cell Biol, 2001; 21(7): 2373-2383
3. Reunanen N, Li SP, Ahonen M, Foschi M, Han J, Kahari VM. Activation of p38α MAPK enhances collagenase-1 (matrix metalloproteinase(MMP)-1) and stromelysin-1 (MMP-3) expression by mRNA stabilization. J Biol Chem, 2002; 277(35):32360-32368
4. Ranganathan AC, Nelson KK, Rodriguez AM, Kim KH, Tower GB, Rutter JL, Brinckerhoff CE, Huang TT, Epstein CJ, Jeffrey JJ, Melendez JA. Manganese superoxide dismutase signals matrix metalloproteinase expression via H2O2-dependent ERK1/2 activation. J Biol Chem, 2001; 276(17): 14264-14270
5. Wenk J, Brenneisen P, Wlaschek M, Poswig A, Briviba K, Oberley TD, Scharffetter-Kochanek K. Stable overexpression of manganese superoxide dismutase in mitochondria identifies hydrogen peroxide as a major oxidant in the AP-1-mediated inductuin of matrix-degrading metalloprotease-1. J Biol Chem, 1999; 274(36): 25869-25876
6. Eberhardt W, Huwiler A, Beck KF, Walpen S, Pfeilschifter J. Amplification of IL-1β-induced matrix metalloproteinase-9 expresson by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-κB andactivating protein-1 and involves activation of the mitogen-activated protein kinase pathways. J Immunl, 2000;165(10):5788-5797
7. Buhimschi IA, Kramer WB, Buhimschi CS, Thompson LP, Weiner CP. Reduction-oxidation (redox) state regulation of matrix metalloproteinase activity in human fetal membranes. Am J Obstet Gynecol, 2000; 182(2): 458-464
8. Zhang HJ, Zhao W, Venkataraman S, Robbins ME, Buettner GR, Kregel KC, Oberley LW. Activation of matrix metalloproteinase-2 by overexpression of manganese superoxide dismutase in human breast cancer MCF-7 cells involes reactive oxygen species. J Biol Chem, 2002; 277(23): 20919-20926
9. Nabeyrat E, Besnard V, Corroyer S, Cazals V, Clement A. Retinoic acid-induced proliferation of lung alveolar epithelial cells: relation with the IGF system. Am J Physiol. 1998, 275 :L71-79
10. Zhu YK, Liu X, Ertl RF, Kohyama T, Wen FQ, Wang H, Spurzem JR. Retinoic acid attenuates cytokine-driven fibroblast degradation of extracellular matrix in three-dimensional culture. Am J Respir Cell Mol Biol, 2001, 25: 620–627
11. Massaro GD, Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats. Am J Physiol, 1996, 270: L305–L310
12. Massaro GD, Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats. Nat Med, 1997, 3: 675–677
13. Masuda M, Toh S, Koike K, Kuratomi Y, Suzui M, Deguchi A, Komiyama S, Weinstein IB. The roles of JNK1 and Stat3 in the response of head and neck cancer cell lines to combined treatment with all-trans-retinoic acid and 5-fluorouracil. Jpn J Cancer Res, 2002, 93:329-339
14. Benkoussa M, Brand C, Delmotte MH, Formstecher P, Lefebvre P. Retinoic acid receptors inhibit AP1 activation by regulating extracellular signal-regulated kinase and CBP recruitment to an AP1-responsive promoter. Mol Cell Biol , 2002, 22:4522-4534
15. Ortiz MA, Lopez-Hernandez FJ, Bayon Y, Pfahl M, Piedrafita FJ. Retinoid-related molecules induce cytochrome c release and apoptosis through activation of c-Jun NH(2)-terminal kinase/p38 mitogen-activated protein kinases. Cancer Res, 2001, 61: 8504-8512
16. Heussen C, Dowdle EB. Electrophoretic analysis of plasminogen activation in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem, 1980, 102:196-202
17. 钱莉玲, 常立文, 容志惠, 张谦慎. 基质金属蛋白酶及其抑制剂在高氧暴露下的早产大鼠肺组织中的动态表达. 中华围产医学杂志, 2003, 6(5): 302-305
18. 钱莉玲, 常立文, 容志惠, 张谦慎. 维甲酸治疗新生大鼠高氧肺发育受抑对肺组织基质金属蛋白酶及其组织抑制剂表达的影响. 实用儿科临床杂志,2002, 17(6): 602-604
19. Hong IK, Kim YM, Jeoung DI, Kim KC, Lee H. Tetraspanin CD9 induces MMP-2 expression by activating p38 MAPK, JNK and c-Jun pathways in humanmelanoma cells. Exp Mol Med, 2005, 37: 230-239
20. Denkert C, Siegert A, Leclere A, Turzynski A, Hauptmann S. An inhibitor of stress-activated MAP-kinases reduces invasion and MMP-2 expression of malignant melanoma cells. Clin Exp Metastasis, 2002, 19:79-85
21. Xie Z, Singh M, Singh K. Differential regulation of matrix metalloproteinase-2 and -9 expression and activity in adult rat cardiac fibroblasts in response to interleukin-1beta. J Biol Chem, 2004, 279: 39513-39519
22. Yao JS, Chen Y, Zhai W, Xu K, Young WL, Yang GY. Minocycline exerts multiple inhibitory effects on vascular endothelial growth factor-induced smooth muscle cell migration: the role of ERK1/2, PI3K, and matrix metalloproteinases. Circ Res, 2004 , 95: 364-371
23. Munshi HG, Wu YI, Mukhopadhyay S, Ottaviano AJ, Sassano A, Koblinski JE, Platanias LC, Stack MS. Differential regulation of membrane type 1-matrix metalloproteinase activity by ERK 1/2- and p38 MAPK-modulated tissue inhibitor of metalloproteinases 2 expression controls transforming growth factor-beta1-induced pericellular collagenolysis. J Biol Chem, 2004, 279: 39042-39050
24. Welty SE, Smith CV. Rationale for antioxidant therapy in premature infants to prevent bronchopulmonary dysplasia. Nutr Revi, 2001, 59: 10-17
25. Reunanen N, Li SP, Ahonen M, Foschi M, Han J, Kahari VM. Activation of p38α MAPK enhances collagenase-1 (matrix metalloproteinase (MMP)-1) and stromelysin-1 (MMP-3) expression by mRNA stabilization. J Biol Chem, 2002, 277:32360-32368
26. Ranganathan AC, Nelson KK, Rodriguez AM, Kim KH, Tower GB, Rutter JL, Brinckerhoff CE, Huang TT, Epstein CJ, Jeffrey JJ, Melendez JA. Manganese superoxide dismutase signals matrix metalloproteinase expression via H2O2-dependent ERK1/2 activation. J Biol Chem, 2001, 276(17): 14264-14270
27. Wenk J, Brenneisen P, Wlaschek M, Poswig A, Briviba K, Oberley TD, Scharffetter-Kochanek K. Stable overexpression of manganese superoxide dismutase in mitochondria identifies hydrogen peroxide as a major oxidant in the AP-1-mediated inductuin of matrix-degrading metalloprotease-1. J Biol Chem, 1999, 274: 25869-25876
28. Eberhardt W, Huwiler A, Beck KF, Walpen S, Pfeilschifter J. Amplification of IL-1β-induced matrix metalloproteinase-9 expresson by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-κB and activating protein-1 and involves activation of the mitogen-activated protein kinase pathways. J Immunl, 2000, 165:5788-5797
29. Buhimschi IA, Kramer WB, Buhimschi CS, Thompson LP, Weiner CP. Reduction-oxidation (redox) state regulation of matrix metalloproteinase activity in human fetal membranes. Am J Obstet Gynecol, 2000, 182: 458-464
30. Zhang HJ, Zhao W,et al. Activation of matrix metalloproteinase-2 byoverexpression of manganese superoxide dismutase in human breast cancer MCF-7 cells involes reactive oxygen species. J Biol Chem, 2002; 277(23): 20919-26
31. Lee HY, Walsh GL, Dawson MI, Hong WK, Kurie JM. All-trans-retinoic acid inhibits Jun N-terminal kinase-dependent signaling pathways. J Biol Chem, 1998, 273:7066-7071
32. Lee HY, Sueoka N, Hong WK, Mangelsdorf DJ, Claret FX, Kurie JM. All-trans-retinoic acid inhibits Jun N-terminal kinase by increasing dual-specificity phosphatase activity. Mol Cell Biol, 1999 , 19:1973-1980
33. Agadir A, Chen G, Bost F, Li Y, Mercola D, Zhang X. Differential effect of retinoic acid on growth regulation by phorbol ester in human cancer cell lines. J Biol Chem, 1999, 274: 29779-29785
34. Masuda M, Toh S, Koike K, Kuratomi Y, Suzui M, Deguchi A, Komiyama S, Weinstein IB. The roles of JNK1 and Stat3 in the response of head and neck cancer cell lines to combined treatment with all-trans-retinoic acid and 5-fluorouracil. Jpn J Cancer Res, 2002, 93:329-339
35. Benkoussa M, Brand C, Delmotte MH, Formstecher P, Lefebvre P. Retinoic acid receptors inhibit AP1 activation by regulating extracellular signal-regulated kinase and CBP recruitment to an AP1-responsive promoter. Mol Cell Biol , 2002, 22:4522-4534
36. Ortiz MA, Lopez-Hernandez FJ, Bayon Y, Pfahl M, Piedrafita FJ. Retinoid-related molecules induce cytochrome c release and apoptosis through activation of c-Jun NH (2)-terminal kinase/p38 mitogen-activated protein kinases. Cancer Res, 2001, 61: 8504-8512
37. Palm-Leis A, Singh US, Herbelin BS, et al. Mitogen-activated protein kinases and mitogen-activated protein kinase phosphatases mediate the inhibitory effects of all-trans retinoic acid on the hypertrophic growth of cardiomyocytes. J Biol Chem, 2004, 279:54905-54917
38. Yang G, Madan A, Dennery PA. Maturational differences in hyperoxic AP-1 activation in rat lung. Am J Physiol Lung Cell Mol Physiol, 2000, 278: L393-398
39. Bergman MR, Cheng S, Honbo N, Piacentini L, Karliner JS, Lovett DH. A functional activating protein 1 (AP-1) site regulates matrix metalloproteinase 2 (MMP-2) transcription by cardiac cells through interactions with JunB-Fra1 and JunB-FosB heterodimers. Biochem J, 2003, 369:485-496
1. Rush MG, Hazinski TA. Current therapy of bronchopulmonary dysplasia. Clin Perinatol, 1992,19: 563–590
2. Warner B, Stuart L, Papes R, Wispe J. Functional and pathological effects of prolonged hyperoxia in neonatal mice. Am J Physiol, 1998, 275: L110–L117
3. O’Brodovich HM, Mellins RB. Bronchopulmonary dysplasia.Unresolved neonatal acute lung injury. Am Rev Respir Dis, 1985, 132: 694–709
4. Mantell LL, Lee PJ. Signal transduction pathways in hyperoxia-induced lung cell death. Mol Genet Metab, 2000, 71:359-370
5. Wang X, Ryter SW, Dai C, Tang ZL, Watkins SC, Yin XM, Song R, Choi AM. Necrotic Cell Death in Response to Oxidant Stress Involves the Activation of the Apoptogenic Caspase-8/Bid Pathway. J Cell Biochem, 2003, 278: 29184-29191
6. McGrath-Morrow SA, Stahl J. Apoptosis in Neonatal Murine Lung Exposed to Hyperoxia. Am J Physiol Lung Cell Mol Physiol, 2001, 25:150-155
7. Pardo A, Barrios R, Maldonado V, Melendez J, Perez J, Ruiz V, Segura-Valdez L, Sznajder JI, Selman M. Gelatinases A and B are up-regulated in rat lungs by subacute hyperoxia: pathogenetic implications. Am J Pathol, 1998, 153: 833-844
8. O’Reilly MA, Staversky RJ, Watkins RH, Reed CK, Mesy Jensen KL, Finkelstein JN, Keng PC. The Cyclin-Dependent Kinase Inhibitor p21 Protects the Lung from Oxidative Stress. Am J Physiol Lung Cell Mol Physiol, 2001, 24: 703-710
9. Buckley S, Barsky L, Driscoll B, Weinberg K, Anderson KD, Warburton D.Apoptosis and DNA damage in type 2 alveolar epithelial cells cultured from hyperoxic rats. Am J Physiol, 1998, 274:L714-720
10. O'Reilly MA, Staversky RJ, Stripp BR, Finkelstein JN. Exposure to hyperoxia induces p53 expression in mouse lung epithelium. Am J Respir Cell Mol Biol, 1998 , 18: 43-50
11. Staversky RJ, Watkins RH, Wright TW, Hernady E, LoMonaco MB, D'Angio CT, Williams JP, Maniscalco WM, O'Reilly MA. Normal remodeling of the oxygen-injured lung requires the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1). Am J Pathol, 2002, 161:1383-93
12. Shenberger JS, Dixon PS. Oxygen induces S-phase growth arrest and increases p53 and p21WAF1/CIP1 expression in human bronchial smooth-muscle cells. Am J Respir Cell Mol Biol, 1999, 21: 395-402
13. McGrath-Morrow SA, Cho C, Soutiere S, Mitzner W, Tuder R. The effect of neonatal hyperoxia on the lung of p21Waf1/Cip1/Sdi1-deficient mice. Am J Respir Cell Mol Biol, 2004, 30: 635-640
14. Chuang SM, Wang IC, Yang JL. Roles of JNK, p38 and ERK mitogen-activated protein kinases in the growth inhibition and apoptosis induced by cadmium. Carcinogenesis, 2000, 21:1423-1432
15. Kling DE, Lorenzo HK, Trbovich AM, Kinane TB, Donahoe PK, Schnitzer JJ. MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs.Am J Physiol Lung Cell Mol Physiol, 2002, 282:L370-L378
16. Morse D, Otterbein LE, Watkins S, Alber S, Zhou Z, Flavell RA, Davis RJ, Choi AM. Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice. Am J Physiol Lung Cell Mol Physiol, 2003, 285: L250-L257
17. Buder-Hoffmann S, Palmer C, Vacek P, Taatjes D, Mossman B. DifferentAccumulation of Activated Extracellular Signal–Regulated Kinases (ERK 1/2) and Role in Cell-Cycle Alterations by Epidermal Growth Factor, Hydrogen Peroxide, or Asbestos in Pulmonary Epithelial Cells. Am J Physiol Lung Cell Mol Physiol, 2001, 24: 405-413
18. Petrache I, Choi ME, Otterbein LE, Chin BY, Mantell LL, Horowitz S, Choi AM. Mitogen-activated protein kinase pathway mediates hyperoxia-induced apoptosis in cultured macrophage cells. Am J Physiol, 1999, 277: L589-L595
19. Nabeyrat E, Besnard V, Corroyer S, Cazals V, Clement A. Retinoic acid-induced proliferation of lung alveolar epithelial cells: relation with the IGF system. Am J Physiol, 1998, 275 : L71-L79
20. Liebeskind A, Srinivasan S, Kaetzel D, Bruce M. Retinoic acid stimulates immature lung fibroblast growth via a PDGF-mediated autocrine mechanism. Am J Physiol Lung Cell Mol Physiol, 2000, 279: L81-L90
21. Massaro GD, Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats. Am J Physiol Lung Cell Mol Physiol, 1996, 270: L305-L310
22. Robbins ST, Fletcher AB. Early vs. delayed vitaminA supplementation in very-low-birth-weight infants. J Parenter Enteral Nutr, 1993, 17: 220-225
23. Shenberger JS, Dixon PS. Oxygen induces S-phase growth arrest and increases p53 and p21(WAF1/CIP1) expression in human bronchial smooth-muscle cells. Am J Respir Cell Mol Biol, 1999, 21:395-402
24. Nabeyrat E, Corroyer S, Besnard V, Cazals-Laville V, Bourbon J, Clement A. Retinoic acid protects against hyperoxia-mediated cell-cycle arrest of lung alveolar epithelial cells by preserving late G1 cyclin activities. Am J Respir Cell Mol Biol, 2001, 25:507-514
25. Bui KC, Buckley S, Wu F, Uhal B, Joshi I, Liu J, Hussain M, Makhoul I, Warburton D. Induction of A- and D-type cyclins and cdc2 kinase activity during recovery from short-term hyperoxic lung injury. Am J Physiol, 1995, 268: L625-L635
26. Veness-Meehan KA, Pierce RA, Moats-Staats BM, Stiles AD. Retinoic acid attenuates O2-induced inhibition of lung septation. Am J Physiol Lung Cell Mol Physiol, 2002, 283(5): L971–980
27. Pierce RA, Shipley JM. Retinoid-enhanced alveolization: identifying relevant downstream Targets. Am J Respir Cell Mol Biol, 2000, 23(2): 137–141
28. Kresch MJ, Christian C, Wu F, Hussain N. Ontogeny of apoptosis during lung development. Pediatr Res, 1998, 43: 426–431
29. Bruce MC, Honaker CE, Cross RJ. Lung fibroblasts undergo apoptosis following alveolarization. Am J Respir Cell Mol Biol, 1999, 20: 228–236
30. De Paepe ME, Rubin LP, Jude C, Lesieur-Brooks AM, Mills DR, and Luks FI. Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development. Am J Physiol Lung Cell Mol Physiol, 2000, 279: L967–L976
31. Welty SE, Smith CV. Rationale for antioxidant therapy in premature infants to prevent bronchopulmonary dysplasia. Nutr Revi, 2001, 59: 10-17
32. Luo Y, Hurwitz J, Massague J. Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1. Nature, 1995, 375:159–161
33. Buccellato LJ, Tso M, Akinci OI, Chandel NS, Budinger GR. Reactive oxygen species are required for hyperoxia-induced Bax activation and cell death in alveolar epithelial cells. J Biol Chem, 2004, 279: 6753-6760
34. 常立文,祝华平,李文斌,刘汉楚,容志惠,张谦慎,陈红兵. 杏仁甙对高氧暴露的早产鼠肺泡 II 细胞的保护作用. 中华儿科杂志,2005,43(2):118-123
35. Massaro D, Massaro GD. Retinoids, alveolus formation, and alveolar deficiency: clinical implications. Am J Respir Cell Mol Biol, 2003, 28: 271–274
36. Nabeyrat E, Corroyer S, Epaud R, Besnard V, Cazals V, Clement A. Retinoic acid-induced proliferation of lung alveolar epithelial cells is linked to p21(CIP1) downregulation. Am J Physiol Lung Cell Mol Physiol, 2000, 278 : L42-L50
37. Cho SJ, George CL, Snyder JM, Acarregui MJ. Retinoic acid and erythropoietin maintain alveolar development in mice treated with an angiogenesis inhibitor. Am J Respir Cell Mol Biol. 2005, 33: 622-628
38. Liu M, Iavarones A, Freedman LP. Transcriptional activation of the human p21WAF1/CIP1 gene by retinoic acid receptor. J Biol Chem, 1996, 271: 31723-31728
39. Carvalho H, Evelson P, Sigaud S, et al. Mitogen-activated protein kinases modulate H2O2-induced apoptosis in primary rat alveolar epithelial cells. J Cell Biochem, 2004, 92: 502-513
40. Buckley S, Driscoll B, Barsky L, Weinberg K, Anderson K, Warburton D. ERK activation protects against DNA damage and apoptosis in hyperoxic rat AEC2. Am J Physiol, 1999, 277:L159-L166
41. Zhang X, Shan P, Sasidhar M, Chupp GL, Flavell RA, Choi AM, Lee PJ. Reactive oxygen species and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase mediate hyperoxia-induced cell death in lung epithelium, 2003 , 28: 305-315
42. Morse D, Otterbein LE, Watkins S, Alber S, Zhou Z, Flavell RA, Davis RJ, Choi AM. Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice, 2003, 285: L250-L257
43. Li Y, Arita Y, Koo HC, Davis JM, Kazzaz JA. Inhibition of c-Jun N-terminal kinase pathway improves cell viability in response to oxidant injury. Am J Respir Cell Mol Biol, 2003, 29: 779-783
44. Truong SV, Monick MM, Yarovinsky TO, Powers LS, Nyunoya T, Hunninghake GW. Extracellular signal-regulated kinase activation delays hyperoxia-induced epithelial cell death in conditions of Akt downregulation. Am J Respir Cell Mol Biol, 2004, 31: 611-618
45. Nyunoya T, Powers LS, Yarovinsky TO, Butler NS, Monick MM, Hunninghake GW. Hyperoxia induces macrophage cell cycle arrest by adhesion-dependent induction of p21Cip1 and activation of the retinoblastoma protein. J Biol Chem, 2003, 278:36099-360106
46. Cho SJ, George CL, Snyder JM, Acarregui MJ. Retinoic acid and erythropoietin maintain alveolar development in mice treated with an angiogenesis inhibitor. Am J Respir Cell Mol Biol. 2005, 33:622-628
47. Palm-Leis A, Singh US, Herbelin BS, et al. Mitogen-activated protein kinases and mitogen-activated protein kinase phosphatases mediate the inhibitory effects of all-trans retinoic acid on the hypertrophic growth of cardiomyocytes. J Biol Chem, 2004, 279:54905-54917
48. Crowe DL, Kim R, Chandraratna RA. Retinoic acid differentially regulates cancer cell proliferation via dose-dependent modulation of the mitogen-activated protein kinase pathway. Mol Cancer Res, 2003, 1:532-540
1. Schock BC, Sweet DG, et al. Oxidative stress and increased type-IV collagenase levels in bronchoalveolar lavage fluid from newborn babies. Pediatr Res, 2001; 50(1): 29-33
2. Gary LJ, Razvan L. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science, 2002; 298:1911-2
3. Westermarck J, Li SP, et al. p38 mitogen-activated protein kinase dependent activation of protein phosphatases 1 and 2A inhibits MEK1 and MEK2 activity and collagenase 1(MMP-1) gene expression. Mol Cell Biol, 2001; 21(7): 2373-83
4. Dik WA, Krejger RR, et al. Localization and potential role of matrix metalloproteinase-1 and tissue inhibitors of metalloproteinase-1 and -2 in different phases of bronchopulmonary dysplasia. Pediatr Res, 2001; 50(6): 761-6
5. Cederqvist K, Sorsa T, et al. Matrix metalloproteinases-2, -8, -9 and TIMP-2 in tracheal aspirates from preterm infants with respiratory distress. Pediatrics, 2001; 108(3): 686-92
6. Welty SE, Smith CV. Rationale for antioxidant therapy in premature infants to prevent bronchopulmonary dysplasia. Nutr Revi, 2001; 59(1): 10-7
7. Reunanen N, Li SP, et al. Activation of p38α MAPK enhances collagenase-1 (matrix metalloproteinase(MMP)-1) and stromelysin-1 (MMP-3) expression by mRNA stabilization. J Biol Chem, 2002; 277(35):32360-8
8. Ranganathan AC, nelson KK, et al. Manganese superoxide dismutase signals matrix metalloproteinase expression via H2O2-dependent ERK1/2 activation. J Biol Chem, 2001; 276(17): 14264-70
9. Wenk J, Brenneisen P, et al. Stable overexpression of manganese superoxide dismutase in mitochondria identifies hydrogen peroxide as a major oxidant in the AP-1-mediated inductuin of matrix-degrading metalloprotease-1. J Biol Chem, 1999; 274(36): 25869-76
10. Eberhardt W, Huwiler A, et al. Amplification of IL-1 β -induced matrix metalloproteinase-9 expresson by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-κB and activating protein-1 and involves activation of the mitogen-activated protein kinase pathways. J Immunl, 2000;165(10):5788-97
11. Buhimschi IA, Kramer WB, et al. Reduction-oxidation (redox) state regulation of matrix metalloproteinase activity in human fetal membranes. Am J Obstet Gynecol, 2000; 182(2): 458-64
12. Eberhardt W, Huwiler A, et al. Amplification of IL-1 β -induced matrix metalloproteinase-9 expresson by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-κB and activating protein-1 and involvesactivation of the mitogen-activated protein kinase pathways. J Immunl, 2000 ; 165 (10):5788-97
13. Zhang HJ, Zhao W,et al. Activation of matrix metalloproteinase-2 by overexpression of manganese superoxide dismutase in human breast cancer MCF-7 cells involes reactive oxygen species. J Biol Chem, 2002; 277(23): 20919-26
14. Kresch MJ, Christian C, Wu F, Hussain N. Ontogeny of apoptosis during lung development. Pediatr Res, 1998, 43: 426–431
15. Bruce MC, Honaker CE, Cross RJ. Lung fibroblasts undergo apoptosis following alveolarization. Am J Respir Cell Mol Biol, 1999, 20: 228–236
16. De Paepe ME, Rubin LP, Jude C, Lesieur-Brooks AM, Mills DR, and Luks FI. Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development. Am J Physiol Lung Cell Mol Physiol, 2000, 279: L967–L976
17. Kling DE, Lorenzo HK, Trbovich AM, Kinane TB, Donahoe PK, Schnitzer JJ. MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs.Am J Physiol Lung Cell Mol Physiol, 2002, 282:L370-L378
18. Pardo A, Barrios R, Maldonado V, Melendez J, Perez J, Ruiz V, Segura-Valdez L, Sznajder JI, Selman M. Gelatinases A and B are up-regulated in rat lungs by subacute hyperoxia: pathogenetic implications. Am J Pathol, 1998, 153: 833-844
19. Wang X, Ryter SW, Dai C, Tang ZL, Watkins SC, Yin XM, Song R, Choi AM. Necrotic Cell Death in Response to Oxidant Stress Involves the Activation of the Apoptogenic Caspase-8/Bid Pathway. J Cell Biochem, 2003, 278: 29184-29191
20. Luo Y, Hurwitz J, Massague J. Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1. Nature, 1995, 375:159–161
21. Buccellato LJ, Tso M, Akinci OI, Chandel NS, Budinger GR. Reactive oxygen species are required for hyperoxia-induced Bax activation and cell death in alveolar epithelial cells. J Biol Chem, 2004, 279: 6753-6760
22. Kling DE, Lorenzo HK, Trbovich AM, Kinane TB, Donahoe PK, Schnitzer JJ. MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs.Am J Physiol Lung Cell Mol Physiol, 2002, 282:L370-L378
23. Morse D, Otterbein LE, Watkins S, Alber S, Zhou Z, Flavell RA, Davis RJ, Choi AM. Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice. Am J Physiol Lung Cell Mol Physiol, 2003, 285: L250-L257
24. Petrache I, Choi ME, Otterbein LE, Chin BY, Mantell LL, Horowitz S, Choi AM. Mitogen-activated protein kinase pathway mediates hyperoxia-induced apoptosis in cultured macrophage cells. Am J Physiol, 1999, 277: L589-L595
25. Zhang X, Shan P, Sasidhar M, Chupp GL, Flavell RA, Choi AM, Lee PJ. Reactive oxygen species and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase mediate hyperoxia-induced cell death in lung epithelium, 2003 , 28: 305-315
26. Buckley S, Driscoll B, Barsky L, Weinberg K, Anderson K, Warburton D. ERKactivation protects against DNA damage and apoptosis in hyperoxic rat AEC2. Am J Physiol, 1999, 277:L159-L166
27. Morse D, Otterbein LE, Watkins S, Alber S, Zhou Z, Flavell RA, Davis RJ, Choi AM. Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice, 2003, 285: L250-L257
28. Li Y, Arita Y, Koo HC, Davis JM, Kazzaz JA. Inhibition of c-Jun N-terminal kinase pathway improves cell viability in response to oxidant injury. Am J Respir Cell Mol Biol, 2003, 29: 779-783
29. Truong SV, Monick MM, Yarovinsky TO, Powers LS, Nyunoya T, Hunninghake GW. Extracellular signal-regulated kinase activation delays hyperoxia-induced epithelial cell death in conditions of Akt downregulation. Am J Respir Cell Mol Biol, 2004, 31: 611-618
30. Nyunoya T, Powers LS, Yarovinsky TO, Butler NS, Monick MM, Hunninghake GW. Hyperoxia induces macrophage cell cycle arrest by adhesion-dependent induction of p21Cip1 and activation of the retinoblastoma protein. J Biol Chem, 2003, 278:36099-360106
31. Cho SJ, George CL, Snyder JM, Acarregui MJ. Retinoic acid and erythropoietin maintain alveolar development in mice treated with an angiogenesis inhibitor. Am J Respir Cell Mol Biol. 2005, 33:622-628
32. Zhu YK, Liu X, Ertl RF, Kohyama T, Wen FQ, Wang H, Spurzem JR. Retinoic acid attenuates cytokine-driven fibroblast degradation of extracellular matrix in three-dimensional culture. Am J Respir Cell Mol Biol, 2001, 25: 620–627
33. Massaro GD, Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats. Am J Physiol, 1996, 270: L305–L310
34. Massaro GD, Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats. Nat Med, 1997, 3: 675–677
35. Ozer EA, Kumral A, Ozer E, et al. Effect of Retinoic Acid on Oxygen-Induced Lung Injury in the Newborn Rat. Pediatr Pulmonol, 2005, 39(2): 35–40
36. Schoenermark MP, Mitchell TI, Rutter JL, Reczek PR, Brinckerhoff CE. Retinoid-mediated suppression of tumor invasion and matrix metalloproteinase synthesis. Ann N Y Acad Sci, 1999, 878: 466-486
37. Axel DI, Frigge A, Dittmann J, Runge H, Spyridopoulos I, Riessen R, Viebahn R, Karsch KR. All-trans retinoic acid regulates proliferation, migration, differentiation, and extracellular matrix turnover of human arterial smooth muscle cells. Cardiovasc Res, 2001, 49: 851-862
38. Dedieu S, Lefebvre P. Retinoids interfere with the AP1 signalling pathway in human breast cancer cells Cell Signal, 2006, 18: 889-898
39. Ho LJ, Lin LC, Hung LF, Wang SJ, Lee CH, Chang DM, Lai JH, Tai TY. Retinoic acid blocks pro-inflammatory cytokine-induced matrix metalloproteinase production by down-regulating JNK-AP-1 signaling in human chondrocytes. Biochem Pharmacol, 2005, 70:200-208
40. Varani J, Perone P, Merfert MG, Moon SE, Larkin D, Stevens MJ. All-trans retinoic acid improves structure and function of diabetic rat skin in organ culture. Diabetes, 2002,51: 3510-3516
41. Mao JT, Tashkin DP, Belloni PN, Baileyhealy I, Baratelli F, Roth MD. All-trans retinoic acid modulates the balance of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in patients with emphysema. Chest, 2003 , 124: 1724-1732
42. 钱莉玲, 常立文, 容志惠, 张谦慎. 基质金属蛋白酶及其抑制剂在高氧暴露下的早产大鼠肺组织中的动态表达. 中华围产医学杂志, 2003, 6(5): 302-305
43. 钱莉玲, 常立文, 容志惠, 张谦慎. 维甲酸治疗新生大鼠高氧肺发育受抑对肺组织基质金属蛋白酶及其组织抑制剂表达的影响. 实用儿科临床杂志,2002, 17(6): 602-604
44. Nabeyrat E, Corroyer S, Epaud R, Besnard V, Cazals V, Clement A. Retinoic acid-induced proliferation of lung alveolar epithelial cells is linked to p21(CIP1) downregulation. Am J Physiol Lung Cell Mol Physiol, 2000, 278 : L42-L50
45. Nabeyrat E, Corroyer S, Besnard V, Cazals-Laville V, Bourbon J, Clement A. Retinoic acid protects against hyperoxia-mediated cell-cycle arrest of lung alveolar epithelial cells by preserving late G1 cyclin activities. Am J Respir Cell Mol Biol, 2001, 25:507-514
46. Liu M, Iavarones A, Freedman LP. Transcriptional activation of the human p21WAF1/CIP1 gene by retinoic acid receptor. J Biol Chem, 1996, 271: 31723-31728
47. Lee HY, Walsh GL, Dawson MI, Hong WK, Kurie JM. All-trans-retinoic acid inhibits Jun N-terminal kinase-dependent signaling pathways. J Biol Chem, 1998, 273:7066-7071
48. Lee HY, Sueoka N, Hong WK, Mangelsdorf DJ, Claret FX, Kurie JM. All-trans-retinoic acid inhibits Jun N-terminal kinase by increasing dual-specificity phosphatase activity. Mol Cell Biol, 1999 , 19:1973-1980
49. Palm-Leis A, Singh US, Herbelin BS, et al. Mitogen-activated protein kinases and mitogen-activated protein kinase phosphatases mediate the inhibitory effects of all-trans retinoic acid on the hypertrophic growth of cardiomyocytes. J Biol Chem, 2004, 279:54905-54917
50. Crowe DL, Kim R, Chandraratna RA. Retinoic acid differentially regulates cancer cell proliferation via dose-dependent modulation of the mitogen-activated protein kinase pathway. Mol Cancer Res, 2003, 1:532-540