Ⅶ因子活化蛋白酶在博莱霉素所致肺损伤大鼠的表达及其作用机制的研究
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摘要
前言
急性肺损伤/急性呼吸窘迫综合征(ALI/ARDS)是心源性以外的各种肺内外致病因素导致的急性进行性缺氧性呼吸衰竭,病死率高,存活下来的患者往往发生不同程度的肺纤维化。传统上将ARDS的病理变化分为三期：水肿出血期、机化和修复期以及纤维化期,一般认为机化和修复发生在病程第3-10天。研究显示急性肺损伤时炎症、抗炎和肺组织重塑及胶原的沉积是平行发生的,损伤的同时即伴有肺组织的修复,包括肺泡上皮细胞的更新、细胞外基质的修复。在这个过程中各种细胞因子如血小板源性生长因子(PDGF)、转化生长因子(TGF)等起着重要作用。过度的修复往往导致损伤的加重和纤维化的形成。。
Ⅶ因子活化蛋白酶(FactorⅦactivating protease, FSAP)是最近在血浆和组织中发现的一种丝氨酸蛋白酶,正常情况下以64kDa的单链形式存在,能通过自身活化的方式转化为具有蛋白水解活性的双链形式(tcFSAP)。后者是由一个46kDa的重链和一个具有活性结合区域的29kDa的轻链通过二硫键连接而成的,一旦形成,tcFSAP能很快自身降解。FSAP主要在肝脏内表达,但在其他器官如肺脏、肾脏、胎盘和胰腺中也有FSAP蛋白的表达。
FSAP在不同生理和病理生理状态下的准确作用目前还不十分清楚,最近发现其在凝血方面具有双重作用：首先FSAP是一种Ⅶ因子激活剂,能在不需要组织因子(tissue factor, TF)存在的情况下促进凝血系统激活；其次,FSAP还能通过激活组织型纤溶酶原激活物(uPA)来促进血凝块溶解。除了在凝血方面的作用外,与其他凝血途径中的丝氨酸蛋白酶一样,FSAP还具有与细胞迁移和增殖有关的细胞活性。FSAP能抑制血管平滑肌细胞和内皮细胞的迁移和增殖。研究发现局部应用FSAP能抑制股动脉损伤小鼠的血管新生内膜的增厚,这种保护作用是通过抑制新生血管内膜平滑肌细胞和炎性细胞的增殖来介导的,提示FSAP可能具有抗炎作用。除了上述作用外,还有研究发现在CCl4所致肝损伤早期FSAP表达增加,而在慢性肝纤维化小鼠的血浆中FSAP水平下降。体外实验中证实FSAP能抑制肝脏星状细胞的增殖和迁移,其作用机制与FSAP抑制血小板源性生长因子-BB(PDGF-BB)介导的p42/p44丝裂原活化蛋白激酶(p42/p44MAPK)磷酸化有关。提示FSAP可能是急慢性肝损伤导致肝纤维化过程中的重要调节因子,具有一定的抗纤维化活性。
在ARDS和肺纤维化中FSAP是否具有同样的调节作用目前尚不得而知。Wygrecka等发现在ARDS患者的血浆和支气管肺泡灌洗液(BAL)中均有FSAP蛋白水平的增加和活性增强,并且在ARDS患者的肺泡上皮细胞、肺血管内皮细胞和巨噬细胞内均有FSAP表达,提示FSAP与ARDS的发生发展可能有密切关系。有研究表明bFGF和PDGF-BB等生长因子在ARDS的发病过程中有诱导内皮细胞活化、刺激纤维母细胞增殖并可能影响ARDS后期的肺纤维化形成。而FSAP对上述生长因子的抑制作用提示FSAP在ALI/ARDS的发生及发展中可能扮演重要角色。本研究中我们试图应用博莱霉素导致的大鼠肺损伤和肺纤维化动物模型在细胞和分子水平来探讨FSAP在肺损伤和肺纤维化中的作用。
材料与方法
一、动物模型
健康清洁级雄性SD大鼠40只,体重200±20g,随机分为对照组和实验组,每组20只。实验组动物用2%戊巴比妥钠(0.2ml/kg)腹腔内注射麻醉,行气管切开置管,缓慢注入0.4%博来霉素0.25ml(5mg/kg,4mg/ml),注药后立即将动物直立旋转,使药物在肺内分布均匀,制成肺损伤和肺间质纤维化模型。对照组动物同样方法向气管内注入等量生理盐水。术后于干燥、温暖环境饲养,自由饮水和进食。
二、标本的采集与处理
每组分别于实验后3d、7d、14d、28d随机选取5只动物称重,5%水合氯醛腹腔注射(0.6ml/100mg)麻醉后,分离气管,打开胸腔,暴露心肺,经左主支气管缓慢注入4%多聚甲醛,然后取左肺置于4%多聚甲醛中,4℃过夜,石蜡包埋,制作4μm组织切片,用于肺形态学观察、免疫组织化学检测。收集另侧肺组织,置于无RNase的Eppendorf管中,液氮速冻,-800C冰箱保存,用于荧光定量PCR及Western-blot检测。
三、细胞培养
人肺纤维母细胞(HPF)购于上海拜力生物科技公司。生长于含10%小牛血清的DMEM培养基中,置37℃、5%CO2饱和湿度培养箱内培养。1-2天换液1次,取指数生长期细胞用于检测。
四、实验方法
1、组织水平
(1)肺组织形态学观察：HE染色。
(2)免疫组织化学技术：肺组织FSAP检测。
(3)荧光定量PCR：肺组织FSAP mRNA检测。
(4) Wetern-blot:肺组织FSAP蛋白检测。
2、细胞水平
(1) BrDU法检测FSAP对肺纤维母细胞增殖的影响。
(2) Transwell小室法检测FSAP对肺纤维母细胞迁移的影响。
(3) Western blotting法检测FSAP肺纤维母细胞p42/p44丝裂原活化蛋白激酶(MAPK)活化的影响
(4)荧光定量PCR法检测FSAP对人肺纤维母细胞胶原ⅢmRNA表达的影响。
(5) Western blotting法检测FSAP对肺纤维母细胞胶原Ⅲ蛋白表达的影响
五、统计学分析
应用SPSS13.0统计软件进行统计学处理,数据以均值士标准差(x±s)表示,单因素多水平比较采用One way ANOVA,两样本均数间比较采用independent t test。相关分析采用Spearman分析。结果以p<0.05有意义。
结果
一、肺组织形态改变
1、肺组织病理改变
大鼠气管内注射博莱霉素后3天光镜下观察即有急性肺损伤表现,肺泡间隔增宽,小血管充血,肺泡和间质内少量出血,中性粒细胞渗出。7天后肺胞壁水肿、增厚比较明显,主要以巨噬细胞(AM)和中性粒细胞(PMN)为主的炎性细胞浸润,伴有毛细血管增生；部分肺胞腔变小或消失,成纤维细胞数量增多；14天后,肺胞间隔明显增宽,肺胞炎达高峰,淋巴细胞(LYM)及成纤维细胞明显增多,胶原纤维纵横交错排列；28天后肺间质纤维成分明显增多,肺纤维化最重,肺胞结构紊乱,部分塌陷和消失,毛细血管腔明显增厚。
2、免疫组化检测FSAP表达
免疫组化显示正常肺组织仅在巨噬细胞内有少量的FSAP表达,博莱霉素造成肺损伤后3天肺泡上皮细胞和微血管内皮细胞中有FSAP表达明显,7天和14天上述部位FSAP表达与3天时未见明显变化,而28天时FSAP表达明显减弱。
二、细胞培养
1、FSAP抑制HPF的DNA合成和细胞增殖
PDGF-BB能明显刺激HPF细胞增殖而肝素对HPF细胞增殖有轻度抑制作用,FSAP本身无论是否加用肝素对HPF增殖无太多影响,但能明显抑制PDGF-BB所引起的HPF细胞增殖。加用FSAP酶抑制剂Aprotitin后FSAP的抑制细胞增殖作用能被逆转。
2、FSAP抑制HPF迁移
PDGF-BB能明显刺激HPF的迁移,FSAP单独应用无论有无肝素存在对HPF迁移并无明显抑制作用；但在肝素存在的情况下FSAP能明显抑制PDGF-BB所刺激的HPF迁移,该抑制作用能被酶抑制剂Aprotitin逆转。提示FSAP能通过与PDGF-BB的作用来抑制细胞迁移。
3、FSAP对HPFp42/44MAPK磷酸化的影响
PDGF-BB能刺激HPF的p42/44 MAPK磷酸化,FSAP本身不能抑制PDGF-BB对MAPK的磷酸化作用,但当肝素与FSAP共同作用时,PDGF-BB的活性受到明显的抑制,加用酶抑制剂Aprotitin后FSAP的抑制作用被逆转。
4、FSAP对HPF胶原Ⅲ合成的影响
PDGF-BB作用72h后,HPF胶原ⅢmRNA和蛋白水平均显著增加,FSAP和肝素单独应用对胶原Ⅲ合成有轻度抑制作用,但当PDGF-BB与FSAP和肝素共同作用时,PDGF-BB刺激胶原合成的作用受到明显的抑制,加用Aprotitin则能逆转FSAP的抑制作用。
结论
本实验首先应用博莱霉素气管内注射的方法制备大鼠的肺损伤和肺纤维化动物模型,研究了肺损伤各个时期的病理特点,并运用免疫组化法和分子生物学方法比较了大鼠肺损伤各个时期FSAP表达的变化；在体外实验部分观察了FSAP对肺纤维母细胞迁移、增殖以及胶原合成的影响,得出以下结论：
1、博莱霉素引起的大鼠肺组织损伤包括炎性反应和纤维化病理发展过程,早期损伤以急性炎性渗出和出血为主,后期以慢性炎症和纤维组织增生为主。
2、在博莱霉素所致大鼠肺损伤早期,肺组织内FSAP表达明显增加,但在以慢性炎症和纤维增生为主的纤维化过程中FSAP水平下降。
3、FSAP能抑制PDGF-BB介导的人肺纤维母细胞增殖和迁移,并能抑制人肺纤维母细胞胶原Ⅲ的合成。
4、FSAP对人肺纤维母细胞活化的抑制作用与其抑制PDGF-BB介导的人肺纤维母细胞p42/P44MAPK磷酸化有关。
5、FSAP是肺损伤和肺纤维化过程HPF活化的重要调节因子,FSAP的表达不足可能与肺损伤加重和肺纤维化形成有关。
Acute lung injury (ALI) and its more severe form acute respiratory distress syndrome (ARDS) are common, devastating clinical syndromes which characterized by non-cardiogenic pulmonary edema, respiratory distress and hypoxemia (Gao & Barnes, 2009). Although the mortality rate associated with ARDS has improved in the last decade,40-70% of patients still die from this syndrome and survivors encounter significant physical and psychological impairments (Rubenfeld et al.,2005). ALI/ARDS are caused by a number of processes that direct or indirect injure to lung (Pietropaoli & Georas,2009). Traditional model of ARDS suggested that lung injury follows an orderly, sequential pattern in which damage to the alveolar capillary membrane accompanied by edema formation was followed by active clearance of edema fluid and subsequent repair of the alveolar-capillary membrane with a varying degree of fibrosis (Marshall et al.,1998). It is now recognized that these processes occur simultaneously in the lung of patient with ARDS (Chesnutt et al.,1997). Previous studies indicated that the changes of various cytokine such as platelet derived growth factor (PDGF) and transforming growth factor (TGF) are involved in these processes (Zagai et al.,2003, Budinger et al.,2005). Therefore, understanding the mechanisms of cytokine changes in these processes will provide possible opportunities for therapeutic intervention of ARDS.
FactorⅦactivating protease (FSAP) is a plasma-derived protease structurally homologous to members of the haemostasis family (Romisch et al.,1999, Romisch, 2002). It serves to active pro-urokinase as well as factorⅦand might play a role in the regulation of both coagulation and fibrinolysis (Parahuleva et al.,2008). Recently, Wygrecka et al reported that FSAP protein level and activity were markedly increased in the plasma and BAL fluid of patients with ARDS, suggesting a role for FSAP in the development of ARDS (Wygrecka et al.,2007). Nonetheless, the precise role of FSAP in ARDS needs to be further elucidated. Accumulating evidences implicated that basic fibroblast growth factor (bFGF) and platelet-derived growth factor-BB (PDGF-BB) participate in the pathogenesis of ARDS by induction of endothelial cells activation, stimulation of fibroblasts proliferation as well as promotion pulmonary fibrosis (Henke et al.,1993, Madtes et al.,1994). Moreover, FSAP can inhibit hepatic stellate cells and vascular smooth muscle cells proliferation and migration by cleavage of PDGF-BB, thereby functions as a suppressor of fibrosis and inflammation response (Roderfeld et al.,2009). These findings led us to explore whether similar mechanisms of FSAP also play a role in the progression of ARDS. Additionally, since the pulmonary fibrosis is a long-term dynamic and progressive disease, the investigation of changing levels of the FSAP along the time course may have significance in understanding the real pathophysiology of the condition. To this end, in the present study we investigated the expression of FSAP in bleomycin-induced acute lung injury and fibrosis in rat model. The influence of FSAP on cultured fibroblasts was also further examined.
Materials and Methods
1.Animals models
Pathogen free male Sprague-Dawley rats (200±20g) were purchased from the Experimental Animal Center of China Medical University. Forty rats were randomly divided into control group (n=20) and bleomycin-administered experimental group (n=20). Each rat was anaesthetized with sodium pentobarbital (100mg/kg) and then subjected to a tracheostomy. The rats in the experimental groups received intratracheal instillation of 4% bleomycin (5mg/kg). Then upright spin was used to ensure a homogenous distribution of bleomycin to rat lungs. The control animals received intratracheal saline only.
2.Preparation of Lung SamPles
Five rats from experimental group and 5 from the control group were sacrificed at 3,7,14 and 28 days after bleomycin instillation. Left lung was inflation-fixed via tracheal cannula using 4%paraformaldehyde for morphology obverasion and immunohistochemistry study. Put the middle lobe of right lung inRNase-free Eppendorf tubes and stored at-80℃freezer for Quantitative Real-Time RT-PCR and Western blotting.
3.Cell culture
Human pulmonary fibroblasts (HPF) were obtained from Bioleaf Biotechnology Co., Ltd. (Shanghai, China). The cells were maintained in Dulbecco's modified Eagle medium (DMEM, GIBCO, Grand Island, NY, USA) supplemented with fetal bovine serum (FBS) and cultured at 37℃in a 5% CO2 incubator. The cells were routinely passaged and cells at logarithmic growth phase were used for experiments.
4.Experimental methods
(1) Morphology observation::histologicalstudy.
(2) Immunohistochemistry measurement the expression levels of FSAP.
(3)Quantitative Real-Time RT-PCR; measurement the expression levels of FSAP and collagenⅢmRNA.
(4)Western blotting:measurement the expression levels of FSAP and collagenⅢprotein.
(5) Bromodeoxyuridine (BrdU) incorporation.
(6) Cell Migration Assay.
(7) Phosphorylation of mitogen-activated protein kinase (MAPK) p42/p44.
5. Statistical analysis
Normally distributed data are expressed as the mean±SEM and were assessed for significance by Student's t test or ANOVA withPost-hoc continuity correction for multiple comparisons as indicated in the text. Non-normally distributed data were assessed for significance using the Wilcoxon rank sum test. Statistical calculations were Performed usingSPSS 13.0 software.Statistical difference was accepted at P<0.05.
Results
1.Lung morphology
(1)Changes of lung pathology. Prominent inflammatory reaction was noted from the third day, showing congestion of small blood vessels, minor hemorrhage in interstitial and alveolar space, and effusion of neutrophils as well as occasional widening of alveolar septum. On the day 7, thickening of alveolar walls and intraalveolar edema became prominent. More neutrophils and macrophages were present and inflammatory cell infiltration spread to the interstitial and alveolar spaces. Diminishing of alveolar space and proliferation of blood capillary were strongly induced. Additionally, the number of fibroblasts was markedly increased.The number of infiltrating macrophages peaked on the day 14. On the other hand, marked widening of alveolar septum was observed and the collagen fibers became apparent and widen, accompanied by increasing of lymphocyte and fibroblasts. On the day 28, the level of fibrosis further progressed. Abundant collagen fibers deposition and collapse of alveolar spaces were observed, along with marked thickening of capillary walls. In the saline-treated control animals, such morphological changes were not observed at any time point examined.
(2)Changes of FSAP expression.In the saline-treated control animals, positive signals of FSAP were observed weakly in a small number of alveolar macrophages. On the three days after instilled with bleomycin, positive signals for FSAP were observed prominently in alveolar epithelial cells and microvascular endothelial cells. At the later time points, no obvious changes of FSAP expression and localization was observed on day 7 and 14, whereas positive signals of FSAP were markedly diminished on day 28.
2. Quantitative Real-Time RT-PCR.
The mRNA expression of FSAP tended to increase at the beginning of bleomycin instillation, then peaked on day 7, and deceased thereafter. After incubated with PDGF-BB, the mRNA was markedly increased. FSAP and heparin alone have a slight inhibitory effect on the synthesis of collagenⅢ. Notably, when FSAP and PDGF-BB were incubated in the presence of heparin, PDGF-BB-mediated stimulus effect was also decreased by FSAP. In accordance with the above results, there is a loss of FSAP-mediated effect when in the presence of aprotinin.
3.Western blotting
The changes in the Western blotting were in accordance with the findings in the quantitative Real-Time RT-PCR study. These results confirmed that the expression level of FSAP was markedly increased before the progression of pulmonary fibrosis, but decreased during pulmonary fibrosis.
4. Bromodeoxyuridine (BrdU) incorporation
Cell proliferation was stimulated by both PDGF-BB and heparin. Although FSAP alone did not have a significant inhibitory effect on cell proliferation, when FSAP and PDGF-BB were incubated in the presence of heparin there was a loss of PDGF activity. In the presence of aprotinin, which inhibits the proteolytic activity of FSAP, there was no inhibition of PDGF-BB stimulation.
5.Cell migration
FSAP has an inhibitory effect on the migration of HPF. Especially when in the present of heparin, a strong inhibitory effect of FSAP was observed and this effect can be reversed by the present of FSAP enzymatic activity blocking aprotinin.
6.Effect of FSAP on PDGF-BB-stimulated phosphorylation in HPF
PDGF-BB is a stimulator of MAPK-p42/44 phosphorylation in HPF. FSAP itself did not inhibit the effect of PDGF-BB on MAPK phosphorylation, but when in the presence of heparin, a strong inhibitory effect of FSAP was induced. Similarly, this effect also can be reversed by the presence of aprotinin.
Conclusion
In summary, we demonstrated an inhibitory effect of FSAP on PDGF-stimulated proliferation and migration of HPF in vitro. In addition, the dynamic expression changes of FSAP in a bleomycin-induced pulmonary fibrosis rat model indicated that FSAP may modulate inflammation and exert a beneficial effect in ARDS, suggesting that exogenous administration of FSAP may serve as a potential strategy for therapeutic interventions of ARDS.
急性肺损伤/急性呼吸窘迫综合征(ALI/ARDS)是心源性以外的各种肺内外致病因素导致的急性进行性缺氧性呼吸衰竭,病死率高,存活下来的患者往往发生不同程度的肺纤维化。传统上将ARDS的病理变化分为三期：水肿出血期、机化和修复期以及纤维化期,一般认为机化和修复发生在病程第3-10天。研究显示急性肺损伤时炎症、抗炎和肺组织重塑及胶原的沉积是平行发生的,损伤的同时即伴有肺组织的修复,包括肺泡上皮细胞的更新、细胞外基质的修复。在这个过程中各种细胞因子如血小板源性生长因子(PDGF)、转化生长因子(TGF)等起着重要作用。过度的修复往往导致损伤的加重和纤维化的形成。。
Ⅶ因子活化蛋白酶(FactorⅦactivating protease, FSAP)是最近在血浆和组织中发现的一种丝氨酸蛋白酶,正常情况下以64kDa的单链形式存在,能通过自身活化的方式转化为具有蛋白水解活性的双链形式(tcFSAP)。后者是由一个46kDa的重链和一个具有活性结合区域的29kDa的轻链通过二硫键连接而成的,一旦形成,tcFSAP能很快自身降解。FSAP主要在肝脏内表达,但在其他器官如肺脏、肾脏、胎盘和胰腺中也有FSAP蛋白的表达。
FSAP在不同生理和病理生理状态下的准确作用目前还不十分清楚,最近发现其在凝血方面具有双重作用：首先FSAP是一种Ⅶ因子激活剂,能在不需要组织因子(tissue factor, TF)存在的情况下促进凝血系统激活；其次,FSAP还能通过激活组织型纤溶酶原激活物(uPA)来促进血凝块溶解。除了在凝血方面的作用外,与其他凝血途径中的丝氨酸蛋白酶一样,FSAP还具有与细胞迁移和增殖有关的细胞活性。FSAP能抑制血管平滑肌细胞和内皮细胞的迁移和增殖。研究发现局部应用FSAP能抑制股动脉损伤小鼠的血管新生内膜的增厚,这种保护作用是通过抑制新生血管内膜平滑肌细胞和炎性细胞的增殖来介导的,提示FSAP可能具有抗炎作用。除了上述作用外,还有研究发现在CCl4所致肝损伤早期FSAP表达增加,而在慢性肝纤维化小鼠的血浆中FSAP水平下降。体外实验中证实FSAP能抑制肝脏星状细胞的增殖和迁移,其作用机制与FSAP抑制血小板源性生长因子-BB(PDGF-BB)介导的p42/p44丝裂原活化蛋白激酶(p42/p44MAPK)磷酸化有关。提示FSAP可能是急慢性肝损伤导致肝纤维化过程中的重要调节因子,具有一定的抗纤维化活性。
在ARDS和肺纤维化中FSAP是否具有同样的调节作用目前尚不得而知。Wygrecka等发现在ARDS患者的血浆和支气管肺泡灌洗液(BAL)中均有FSAP蛋白水平的增加和活性增强,并且在ARDS患者的肺泡上皮细胞、肺血管内皮细胞和巨噬细胞内均有FSAP表达,提示FSAP与ARDS的发生发展可能有密切关系。有研究表明bFGF和PDGF-BB等生长因子在ARDS的发病过程中有诱导内皮细胞活化、刺激纤维母细胞增殖并可能影响ARDS后期的肺纤维化形成。而FSAP对上述生长因子的抑制作用提示FSAP在ALI/ARDS的发生及发展中可能扮演重要角色。本研究中我们试图应用博莱霉素导致的大鼠肺损伤和肺纤维化动物模型在细胞和分子水平来探讨FSAP在肺损伤和肺纤维化中的作用。
材料与方法
一、动物模型
健康清洁级雄性SD大鼠40只,体重200±20g,随机分为对照组和实验组,每组20只。实验组动物用2%戊巴比妥钠(0.2ml/kg)腹腔内注射麻醉,行气管切开置管,缓慢注入0.4%博来霉素0.25ml(5mg/kg,4mg/ml),注药后立即将动物直立旋转,使药物在肺内分布均匀,制成肺损伤和肺间质纤维化模型。对照组动物同样方法向气管内注入等量生理盐水。术后于干燥、温暖环境饲养,自由饮水和进食。
二、标本的采集与处理
每组分别于实验后3d、7d、14d、28d随机选取5只动物称重,5%水合氯醛腹腔注射(0.6ml/100mg)麻醉后,分离气管,打开胸腔,暴露心肺,经左主支气管缓慢注入4%多聚甲醛,然后取左肺置于4%多聚甲醛中,4℃过夜,石蜡包埋,制作4μm组织切片,用于肺形态学观察、免疫组织化学检测。收集另侧肺组织,置于无RNase的Eppendorf管中,液氮速冻,-800C冰箱保存,用于荧光定量PCR及Western-blot检测。
三、细胞培养
人肺纤维母细胞(HPF)购于上海拜力生物科技公司。生长于含10%小牛血清的DMEM培养基中,置37℃、5%CO2饱和湿度培养箱内培养。1-2天换液1次,取指数生长期细胞用于检测。
四、实验方法
1、组织水平
(1)肺组织形态学观察：HE染色。
(2)免疫组织化学技术：肺组织FSAP检测。
(3)荧光定量PCR：肺组织FSAP mRNA检测。
(4) Wetern-blot:肺组织FSAP蛋白检测。
2、细胞水平
(1) BrDU法检测FSAP对肺纤维母细胞增殖的影响。
(2) Transwell小室法检测FSAP对肺纤维母细胞迁移的影响。
(3) Western blotting法检测FSAP肺纤维母细胞p42/p44丝裂原活化蛋白激酶(MAPK)活化的影响
(4)荧光定量PCR法检测FSAP对人肺纤维母细胞胶原ⅢmRNA表达的影响。
(5) Western blotting法检测FSAP对肺纤维母细胞胶原Ⅲ蛋白表达的影响
五、统计学分析
应用SPSS13.0统计软件进行统计学处理,数据以均值士标准差(x±s)表示,单因素多水平比较采用One way ANOVA,两样本均数间比较采用independent t test。相关分析采用Spearman分析。结果以p<0.05有意义。
结果
一、肺组织形态改变
1、肺组织病理改变
大鼠气管内注射博莱霉素后3天光镜下观察即有急性肺损伤表现,肺泡间隔增宽,小血管充血,肺泡和间质内少量出血,中性粒细胞渗出。7天后肺胞壁水肿、增厚比较明显,主要以巨噬细胞(AM)和中性粒细胞(PMN)为主的炎性细胞浸润,伴有毛细血管增生；部分肺胞腔变小或消失,成纤维细胞数量增多；14天后,肺胞间隔明显增宽,肺胞炎达高峰,淋巴细胞(LYM)及成纤维细胞明显增多,胶原纤维纵横交错排列；28天后肺间质纤维成分明显增多,肺纤维化最重,肺胞结构紊乱,部分塌陷和消失,毛细血管腔明显增厚。
2、免疫组化检测FSAP表达
免疫组化显示正常肺组织仅在巨噬细胞内有少量的FSAP表达,博莱霉素造成肺损伤后3天肺泡上皮细胞和微血管内皮细胞中有FSAP表达明显,7天和14天上述部位FSAP表达与3天时未见明显变化,而28天时FSAP表达明显减弱。
二、细胞培养
1、FSAP抑制HPF的DNA合成和细胞增殖
PDGF-BB能明显刺激HPF细胞增殖而肝素对HPF细胞增殖有轻度抑制作用,FSAP本身无论是否加用肝素对HPF增殖无太多影响,但能明显抑制PDGF-BB所引起的HPF细胞增殖。加用FSAP酶抑制剂Aprotitin后FSAP的抑制细胞增殖作用能被逆转。
2、FSAP抑制HPF迁移
PDGF-BB能明显刺激HPF的迁移,FSAP单独应用无论有无肝素存在对HPF迁移并无明显抑制作用；但在肝素存在的情况下FSAP能明显抑制PDGF-BB所刺激的HPF迁移,该抑制作用能被酶抑制剂Aprotitin逆转。提示FSAP能通过与PDGF-BB的作用来抑制细胞迁移。
3、FSAP对HPFp42/44MAPK磷酸化的影响
PDGF-BB能刺激HPF的p42/44 MAPK磷酸化,FSAP本身不能抑制PDGF-BB对MAPK的磷酸化作用,但当肝素与FSAP共同作用时,PDGF-BB的活性受到明显的抑制,加用酶抑制剂Aprotitin后FSAP的抑制作用被逆转。
4、FSAP对HPF胶原Ⅲ合成的影响
PDGF-BB作用72h后,HPF胶原ⅢmRNA和蛋白水平均显著增加,FSAP和肝素单独应用对胶原Ⅲ合成有轻度抑制作用,但当PDGF-BB与FSAP和肝素共同作用时,PDGF-BB刺激胶原合成的作用受到明显的抑制,加用Aprotitin则能逆转FSAP的抑制作用。
结论
本实验首先应用博莱霉素气管内注射的方法制备大鼠的肺损伤和肺纤维化动物模型,研究了肺损伤各个时期的病理特点,并运用免疫组化法和分子生物学方法比较了大鼠肺损伤各个时期FSAP表达的变化；在体外实验部分观察了FSAP对肺纤维母细胞迁移、增殖以及胶原合成的影响,得出以下结论：
1、博莱霉素引起的大鼠肺组织损伤包括炎性反应和纤维化病理发展过程,早期损伤以急性炎性渗出和出血为主,后期以慢性炎症和纤维组织增生为主。
2、在博莱霉素所致大鼠肺损伤早期,肺组织内FSAP表达明显增加,但在以慢性炎症和纤维增生为主的纤维化过程中FSAP水平下降。
3、FSAP能抑制PDGF-BB介导的人肺纤维母细胞增殖和迁移,并能抑制人肺纤维母细胞胶原Ⅲ的合成。
4、FSAP对人肺纤维母细胞活化的抑制作用与其抑制PDGF-BB介导的人肺纤维母细胞p42/P44MAPK磷酸化有关。
5、FSAP是肺损伤和肺纤维化过程HPF活化的重要调节因子,FSAP的表达不足可能与肺损伤加重和肺纤维化形成有关。
Acute lung injury (ALI) and its more severe form acute respiratory distress syndrome (ARDS) are common, devastating clinical syndromes which characterized by non-cardiogenic pulmonary edema, respiratory distress and hypoxemia (Gao & Barnes, 2009). Although the mortality rate associated with ARDS has improved in the last decade,40-70% of patients still die from this syndrome and survivors encounter significant physical and psychological impairments (Rubenfeld et al.,2005). ALI/ARDS are caused by a number of processes that direct or indirect injure to lung (Pietropaoli & Georas,2009). Traditional model of ARDS suggested that lung injury follows an orderly, sequential pattern in which damage to the alveolar capillary membrane accompanied by edema formation was followed by active clearance of edema fluid and subsequent repair of the alveolar-capillary membrane with a varying degree of fibrosis (Marshall et al.,1998). It is now recognized that these processes occur simultaneously in the lung of patient with ARDS (Chesnutt et al.,1997). Previous studies indicated that the changes of various cytokine such as platelet derived growth factor (PDGF) and transforming growth factor (TGF) are involved in these processes (Zagai et al.,2003, Budinger et al.,2005). Therefore, understanding the mechanisms of cytokine changes in these processes will provide possible opportunities for therapeutic intervention of ARDS.
FactorⅦactivating protease (FSAP) is a plasma-derived protease structurally homologous to members of the haemostasis family (Romisch et al.,1999, Romisch, 2002). It serves to active pro-urokinase as well as factorⅦand might play a role in the regulation of both coagulation and fibrinolysis (Parahuleva et al.,2008). Recently, Wygrecka et al reported that FSAP protein level and activity were markedly increased in the plasma and BAL fluid of patients with ARDS, suggesting a role for FSAP in the development of ARDS (Wygrecka et al.,2007). Nonetheless, the precise role of FSAP in ARDS needs to be further elucidated. Accumulating evidences implicated that basic fibroblast growth factor (bFGF) and platelet-derived growth factor-BB (PDGF-BB) participate in the pathogenesis of ARDS by induction of endothelial cells activation, stimulation of fibroblasts proliferation as well as promotion pulmonary fibrosis (Henke et al.,1993, Madtes et al.,1994). Moreover, FSAP can inhibit hepatic stellate cells and vascular smooth muscle cells proliferation and migration by cleavage of PDGF-BB, thereby functions as a suppressor of fibrosis and inflammation response (Roderfeld et al.,2009). These findings led us to explore whether similar mechanisms of FSAP also play a role in the progression of ARDS. Additionally, since the pulmonary fibrosis is a long-term dynamic and progressive disease, the investigation of changing levels of the FSAP along the time course may have significance in understanding the real pathophysiology of the condition. To this end, in the present study we investigated the expression of FSAP in bleomycin-induced acute lung injury and fibrosis in rat model. The influence of FSAP on cultured fibroblasts was also further examined.
Materials and Methods
1.Animals models
Pathogen free male Sprague-Dawley rats (200±20g) were purchased from the Experimental Animal Center of China Medical University. Forty rats were randomly divided into control group (n=20) and bleomycin-administered experimental group (n=20). Each rat was anaesthetized with sodium pentobarbital (100mg/kg) and then subjected to a tracheostomy. The rats in the experimental groups received intratracheal instillation of 4% bleomycin (5mg/kg). Then upright spin was used to ensure a homogenous distribution of bleomycin to rat lungs. The control animals received intratracheal saline only.
2.Preparation of Lung SamPles
Five rats from experimental group and 5 from the control group were sacrificed at 3,7,14 and 28 days after bleomycin instillation. Left lung was inflation-fixed via tracheal cannula using 4%paraformaldehyde for morphology obverasion and immunohistochemistry study. Put the middle lobe of right lung inRNase-free Eppendorf tubes and stored at-80℃freezer for Quantitative Real-Time RT-PCR and Western blotting.
3.Cell culture
Human pulmonary fibroblasts (HPF) were obtained from Bioleaf Biotechnology Co., Ltd. (Shanghai, China). The cells were maintained in Dulbecco's modified Eagle medium (DMEM, GIBCO, Grand Island, NY, USA) supplemented with fetal bovine serum (FBS) and cultured at 37℃in a 5% CO2 incubator. The cells were routinely passaged and cells at logarithmic growth phase were used for experiments.
4.Experimental methods
(1) Morphology observation::histologicalstudy.
(2) Immunohistochemistry measurement the expression levels of FSAP.
(3)Quantitative Real-Time RT-PCR; measurement the expression levels of FSAP and collagenⅢmRNA.
(4)Western blotting:measurement the expression levels of FSAP and collagenⅢprotein.
(5) Bromodeoxyuridine (BrdU) incorporation.
(6) Cell Migration Assay.
(7) Phosphorylation of mitogen-activated protein kinase (MAPK) p42/p44.
5. Statistical analysis
Normally distributed data are expressed as the mean±SEM and were assessed for significance by Student's t test or ANOVA withPost-hoc continuity correction for multiple comparisons as indicated in the text. Non-normally distributed data were assessed for significance using the Wilcoxon rank sum test. Statistical calculations were Performed usingSPSS 13.0 software.Statistical difference was accepted at P<0.05.
Results
1.Lung morphology
(1)Changes of lung pathology. Prominent inflammatory reaction was noted from the third day, showing congestion of small blood vessels, minor hemorrhage in interstitial and alveolar space, and effusion of neutrophils as well as occasional widening of alveolar septum. On the day 7, thickening of alveolar walls and intraalveolar edema became prominent. More neutrophils and macrophages were present and inflammatory cell infiltration spread to the interstitial and alveolar spaces. Diminishing of alveolar space and proliferation of blood capillary were strongly induced. Additionally, the number of fibroblasts was markedly increased.The number of infiltrating macrophages peaked on the day 14. On the other hand, marked widening of alveolar septum was observed and the collagen fibers became apparent and widen, accompanied by increasing of lymphocyte and fibroblasts. On the day 28, the level of fibrosis further progressed. Abundant collagen fibers deposition and collapse of alveolar spaces were observed, along with marked thickening of capillary walls. In the saline-treated control animals, such morphological changes were not observed at any time point examined.
(2)Changes of FSAP expression.In the saline-treated control animals, positive signals of FSAP were observed weakly in a small number of alveolar macrophages. On the three days after instilled with bleomycin, positive signals for FSAP were observed prominently in alveolar epithelial cells and microvascular endothelial cells. At the later time points, no obvious changes of FSAP expression and localization was observed on day 7 and 14, whereas positive signals of FSAP were markedly diminished on day 28.
2. Quantitative Real-Time RT-PCR.
The mRNA expression of FSAP tended to increase at the beginning of bleomycin instillation, then peaked on day 7, and deceased thereafter. After incubated with PDGF-BB, the mRNA was markedly increased. FSAP and heparin alone have a slight inhibitory effect on the synthesis of collagenⅢ. Notably, when FSAP and PDGF-BB were incubated in the presence of heparin, PDGF-BB-mediated stimulus effect was also decreased by FSAP. In accordance with the above results, there is a loss of FSAP-mediated effect when in the presence of aprotinin.
3.Western blotting
The changes in the Western blotting were in accordance with the findings in the quantitative Real-Time RT-PCR study. These results confirmed that the expression level of FSAP was markedly increased before the progression of pulmonary fibrosis, but decreased during pulmonary fibrosis.
4. Bromodeoxyuridine (BrdU) incorporation
Cell proliferation was stimulated by both PDGF-BB and heparin. Although FSAP alone did not have a significant inhibitory effect on cell proliferation, when FSAP and PDGF-BB were incubated in the presence of heparin there was a loss of PDGF activity. In the presence of aprotinin, which inhibits the proteolytic activity of FSAP, there was no inhibition of PDGF-BB stimulation.
5.Cell migration
FSAP has an inhibitory effect on the migration of HPF. Especially when in the present of heparin, a strong inhibitory effect of FSAP was observed and this effect can be reversed by the present of FSAP enzymatic activity blocking aprotinin.
6.Effect of FSAP on PDGF-BB-stimulated phosphorylation in HPF
PDGF-BB is a stimulator of MAPK-p42/44 phosphorylation in HPF. FSAP itself did not inhibit the effect of PDGF-BB on MAPK phosphorylation, but when in the presence of heparin, a strong inhibitory effect of FSAP was induced. Similarly, this effect also can be reversed by the presence of aprotinin.
Conclusion
In summary, we demonstrated an inhibitory effect of FSAP on PDGF-stimulated proliferation and migration of HPF in vitro. In addition, the dynamic expression changes of FSAP in a bleomycin-induced pulmonary fibrosis rat model indicated that FSAP may modulate inflammation and exert a beneficial effect in ARDS, suggesting that exogenous administration of FSAP may serve as a potential strategy for therapeutic interventions of ARDS.
引文
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