核因子-κB介导小鼠ARDS中性粒细胞聚集调控机制及PDTC干预作用研究
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摘要
研究目的
急性呼吸窘迫综合征(acute respiratory distress syndrome, ARDS)本质是过度失控的炎症反应,众多效应细胞参与ARDS进程,而中性粒细胞(polymorphonuclear leukocyte, PMN)是最重要的效应细胞。ARDS早期单核-巨噬细胞被激活,释放肿瘤坏死因子-α(tumor necrosis factor-a, TNF-a)、白介素-1β(interleukin-1, IL-1β)、IL-6、IL-8等促炎细胞因子,这些因子可激活PMN和内皮细胞等效应细胞,使大量PMN在肺内聚集,释放髓过氧化物酶(myeloperoxidase, MPO)、氧自由基及溶酶体酶等多种炎症介质,导致肺毛细血管内皮细胞和肺泡上皮细胞的广泛损伤。
核因子-κB (nuclear factor-kappa B, NF-κB)是炎症反应的关键性核转录因子,通常状态下,NF-κB以二聚体形式与核因子-κB抑制蛋白(inhibitor κB protein, IκB)结合形成NF-κB-IκB三聚体复合物,以非活性状态存在于胞浆中。其中发挥主要生理功能的是NF-κB p50/p65二聚体,NF-κB p65为其主要亚单位。脂多糖(lipopolysaccharide, LPS)是革兰氏阴性菌外膜主要成分,可使IκB激酶激活致NF-κB磷酸化,IκB解离,NF-κB进入细胞核与DNA特定的κB序列结合后活化,高效诱导抗炎-促炎细胞因子、粘附分子及趋化因子的基因转录。而TNF-α、 IL-1β也可激活NF-κB。IL-10是一种主要的抗炎细胞因子,对NF-κB的活化产生抑制作用,减少促炎细胞因子的表达。
粘附分子家族中与PMN活化关系最为密切的是整合素CD11b/CD18。在趋化因子的作用下,CD11b/CD18在PMN表面表达增多,并与其配体血管内皮细胞表面的细胞间粘附分子-1(intercellular adhesion molecule-1, ICAM-1)相互作用,参与PMN及血管内皮细胞的粘附、跨越血管内皮等病理生理过程。中性粒细胞趋化因子(cytokine induced neutrophil chemoattractant,CINC)是PMN特异性趋化因子,也是趋化因子IL-8家族的同源物。LPS、TNF-α及IL-1β的刺激可诱导巨噬细胞、肺间质细胞等效应细胞释放CINC,对PMN向肺内募集起重要作用。另一种趋化因子内皮中性粒细胞激活肽-78(epithelial neutrophil activating pep-tide-78, ENA-78)也可趋化PMN。此外,ENA-78与CDllb/CD18受体结合,会使内皮细胞内游离的Ca2+增加,致肺毛细血管通透性增加。
吡咯烷二硫代氨基甲酸(pyrrolidine dithiocarbamate, PDTC)为一种抗氧化剂,是NF-κB的特异性抑制剂。PDTC通过稳定抑制蛋白IκB、增加IκB的合成、阻止IκB的磷酸化和后续降解,抑制NF-κB激活,使细胞因子、粘附分子、趋化因子的表达减少,抑制PMN等炎性细胞在肺内的聚集。
本研究探讨了小鼠ARDS时NF-κB活化、促炎-抗炎细胞因子表达与PMN肺内聚集的关系；阐述肺组织粘附分子(CD11b/CD18、ICAM-1)及趋化因子(CINC、ENA-78)表达在PMN活化中的作用；研究PDTC对NF-κB的干预作用,为临床治疗ARDS提供新的思路。
研究方法
一、动物模型的建立及分组
腹腔内注射LPS(20mg/kg)建立BALB/c小鼠ARDS模型,随机分对照组(N组)、脂多糖组(L组)、PDTC干预组(P+L组)。于LPS注射前半小时腹腔内注射PDTC (120mg/kg)来实现对小鼠ARDS的干预。NS组腹腔内注射20ml/kg的生理盐水。
二、标本的采集
每组小鼠于造模后2h、6h、12h及24h腹腔注射10%水合氯醛3.5ml/kg麻醉。因所测指标无法一次取材成功,分三部分取材。第一部分：眼眶采血,取血清-80℃保存,备查细胞因子及总蛋白。随后开胸,心尖采血行血气分析；留取肺组织,取右肺以备HE染色和免疫组化染色；取左肺计算肺湿/干重比值(wet/dry weight ratio, W/D)。第二部分：收集支气管肺胞灌洗液(bronchoalveolar lavage fluid,BALF),离心,取上清液行细胞因子及总蛋白水平测定,收集细胞沉渣,行白细胞计数及测PMN比例。第三部分：取肺组织放入-80℃液氮中保存,备Western blot.逆转录及实时定量PCR及髓过氧化物酶(myeloperoxidase, MPO)活性的检测。
三、检测方法及指标
1、HE染色评估肺组织病理损伤及评分；测肺通透指数(lung permeability index, LPI=BALF中蛋白/血清蛋白)及W/D;测P(A-a)02。
2、取BALF细胞沉渣,行Wright-Giemsa染色,计数白细胞总数及PMN的比例。
3、收集BALF上清液及取备用血清,ELISA法检测TNF-α、IL-1β、IL-8及IL-10的水平；应用试剂盒检测肺组织MPO活性。
4、提取肺组织总蛋白,Western blot法测定磷酸化NF-kB (phospho-nuclear fac-tor-kappa B, p-NF-κB)表达；提取胞浆及胞核蛋白,Western blot法测定胞浆及胞核NF-kBp65表达。
5、免疫组化法检测肺组织NF-kB、粘附分子(CDllb/CD18. ICAM-1)及趋化因子ENA-78表达。
6、采用逆转录及实时定量PCR (Real-time PCR)法测肺组织CINCmRNA表达强度。
7.、统计学分析：所有数据以平均数±标准差表示,采用SPSS19.0统计软件包分析。两两之间的比较采用t检验,P<0.05表明差异有统计学意义,P<0.01表明差异有显著统计学意义。
研究结果
一、一般情况
NS组小鼠呼吸平稳,解剖后肺部外观色泽粉红；L组小鼠呼吸急促,口唇发绀,解剖后肺体积增大,色泽暗红或紫红,脏层胸膜下点状、片状出血,切面淡红色液体渗出；P+L组小鼠呼吸急促减轻,解剖后肺体积无明显增大,色泽呈红色,表面见少许散在的出血点,包膜张力减轻。
二、HE染色评估肺组织病理损伤及评分
1、HE染色
NS组肺组织结构完整正常；L组部分肺泡壁不同程度的破坏或断裂,肺微血管充血、出血、微血栓形成,肺泡腔及肺间质内见大量炎性细胞浸润、Ⅰ型肺泡上皮细胞坏死；PDTC干预后肺组织损伤程度减轻。
2、肺病理评分
L组肺组织病理评分明显高于NS组(P<0.01)；PDTC干预后病理评分较L组下降(P<0.05),病理评分与HE染色肺组织损伤的动态变化趋势相似。
三、肺血管通透性变化的检测
1、肺组织湿/干重比值(W/D)
L组肺组织W/D值较NS组明显增加(P<0.01),随着造模时间延长W/D值逐渐增加；PDTC干预后肺水肿程度减轻,W/D值均较LS组明显下降(P<0.05),但高于NS组。
2、肺通透指数(LPI)
L组肺LPI较NS组明显升高(P<0.01),PDTC干预后肺泡腔及肺间质内蛋白渗出减少,与L组对比,LPI下降(P<0.05)。
四、BALF中白细胞总数及PMN比例
L组白细胞总数及PMN比例较NS组进行性增加(P<0.01)；PDTC干预后白细胞总数下降,各时相点均低于L组,2h及6h与L组对比,差异有统计学意义(P<0.05),12h及24h与L组对比,差异具有显著统计学意义(P<0.01)；但P+L组各时相点PMN比例与L组对比呈下降趋势,差异有统计学意义(P<0.05)。
五、血清及BALF中TNF-α、IL-1β、IL-8及IL-10的表达
L组血清及BALF中TNF-α、IL-1β及IL-8的浓度明显高于NS组(P<0.01)；PDTC干预后TNF-α、IL-1β及IL-8的浓度在2h、6h及12h低于L组(P<0.05),而在24h明显低于L组,差异有显著统计学意义(P<0.01),但均高于NS组。
与NS组对比,L组在最初6h内血清及BALF中IL-10的浓度升高(P<0.05),随着时间延长,IL-10的浓度明显升高(P<0.01)；PDTC干预后,与L组比较,2h IL-10的浓度略降低,差异无统计学意义(P>0.05),但6h、12h及24h IL-10的浓度低于L组,差异有统计学意义(P<0.05)。
六、肺组织中MPO的活性
L组肺组织MPO活性较NS组明显增加(P<0.01)；PDTC干预后与L组比较,MPO活性降低(P<0.05),但不能回到正常水平。
七、Western blot检测肺组织p-NF-κB、胞浆及胞核中NF-κBp65蛋白的表达
L组肺组织p-NF-κB蛋白的表达增强,与NS组相比(P<0.01)；PDTC干预后p-NF-κB蛋白的表达增强较L组降低(P<0.05)。
L组肺组织胞浆中P65蛋白的表达强度降低,而胞核中P65蛋白表达强度升高,与NS组比较(P<0.01)。PDTC干预后胞浆中P65蛋白的表达强度较L组升高(P<0.05)；在2h、6h及12h而胞核中P65蛋白表达强度较L组降低,差异有统计学意义(P<0.05),在24h胞核中P65蛋白表达强度较L组明显降低,差异有显著统计学意义(P<0.01)。
八、逆转录及实时定量PCR检测CINCmRNA的表达
NS组肺组织有极少量的CINCmRNA的表达；与NS组相比,L组CINCm RNA的表达明显增加(P<0.01)；PDTC干预后肺组织CINCmRNA的表达与L组对比表达明显减少(P<0.01)。
九、免疫组化检测肺组织NF-κB、CD11b/CD18及ICAM-1、ENA-78的表达
以细胞出现棕褐色或棕黄色着色为阳性表达。与NS组对比,L组可见较多的NF-κB阳性免疫反应的细胞,主要表达在肺间质PMN; PDTC干预后NF-κB阳性免疫反应的细胞减少。L组CDllb/CD18阳性表达的细胞较NS组明显增加,主要在PMN上表达；PDTC干预后CD11b/CD18阳性表达的细胞减少。ICAM-1主要表达在血管内皮细胞,与NS组对比,L组ICAM-1阳性免疫反应的细胞增加；PDTC干预后ICAM-1阳性免疫反应的细胞减少。与NS组对比,L组ENA-78阳性表达的细胞增加,以内皮细胞阳性表达为主；PDTC干预后ENA-78阳性表达的细胞减少。
研究结论
1、LPS诱导的ARDS中,NF-κB磷酸化降解,NF-KBp65由胞浆向胞核转移,NF-κB激活,细胞因子(IL-1β、IL-8、IL-10和TNF-a).粘附分子及趋化因子表达增加。
2、小鼠ARDS早期促炎-抗炎反应失衡,表现在促炎细胞因子表达增多,抗炎细胞因子相对不足,且高表达时间滞后。
3、在趋化因子(CINC. ENA-78)的作用下,PMN表面粘附分子CD11b/CD18表达增多,与其配体内皮细胞表面的粘附分子ICAM-1相互作用,使PMN粘附在血管壁,并跨越内皮向肺组织浸润。
4、PDTC通过抑制NF-κB的激活,下调细胞因子、粘附分子及趋化因子的表达,减少PMN聚集,改善促炎-抗炎介质的失衡,使PMN释放的MPO减少,肺组织损伤减轻。
创新性及意义
1、本研究系统地从肺组织p-NF-κB及胞浆、胞核p65蛋白表达阐述ADRS时NF-κB活化。
2、探讨粘附分子、趋化因子的高表达对ARDS肺组织PMN聚集的影响。
3、研究NF-κB的特异性抑制剂PDTC对ARDS小鼠的保护机制,为进一步研究其发病机制、拓展ARDS的治疗途径提供广阔的前景。
Objectives
The main pathogenesis of the acute respiratory distress syndrome (ARDS) was excessive uncontrolled inflammatory response mediated by various inflammatory cy-tokines, but the treatment was limited effective. And a large number of polymorpho-nuclear neutrophil (PMN) accumulated in lung tissue plays an important role in dam-age of pulmonary capillary endothelial cells and alveolar epithelial cells. Previous studies have demonstrated that a large number of polymorphonuclear neutrophil (PMN) accumulated in lung tissue and release inflammatory cytokines, such as Inter-leukin-1β(IL-1β),Il-6,IL-8and tumor necrosis factor-alpha (TNF-a) and so on when body against infection or trauma, which plays an important role in starting and main-taining the inflammatory response. And it could promote the excessive production and expression of inflammatory mediators including cytokines, chemokines, adhesion molecules, myeloperoxidase (MPO) and ROS.
As an important nuclear transcription factor, Nuclear factor-κB(NF-κB) is the intersection of multiple signaling pathways. The protein exists in the form of inactive in the cytoplasm by the form of dimer and directly combines with inhibitory protein IκB to form the trimeric complex at usually state. P50/P65heterodimer plays a major physiological function during inflammation, and NF-κB P65was as its main subunit. Lipopolysaccharide (LPS) is the major component of the outer membrane of gram-negative bacteria and is a common trigger of sepsis, which is the important init-iation factor to activate NF-κB. The Rel protein localization signal (NLS) exposed under the stimulation of LPS, then NF-κB bound to specific κB sequences of DNA in the nucleus in order to regulate the transcription and expression of gene. IL-10is a major anti-inflammatory cytokines, and could inhibit activation of NF-κB and reduce the expression of pro-inflammatory cytokines.
The expression of CD11b/CD18plays an important role in PMN aggregation and activation. CD11b/CD18is a heterodimer of the aM(CD11b) and β2(CD18) subunits, which is a key factor in inflammatory response to combine with the ICAM-1and me-diate the adhesion between PMN and microvascular endothelial cells, then transmit the intracellular signal. CINC, one of the IL-8family, is the specific chemokines of PMN, which plays an important role in the aggregation process of PMN in lung tissue. CINC-1and CINC-3played important role in PMN recruitment to the lung in LPS-induced ARDS.Another chemokine neutrophil activating peptide (ENA-78) also could chemotaxis PMN. In addition, ENA-78could bind to CD11b/CD18receptor, increase the free Ca2+in endothelial cells and the permeability pulmonary capillary.
Pyrrolidine dithiocarbamate(PDTC) is a metal chelating agent and an antioxidant, which is the specific inhibitor of NF-κB. The study has confirmed that PDTC could directly reduce binding capacity of NF-κB to DNA, and increase the synthesis of IκB in order to prevent the phosphorylation of IκB and the subsequent degradation. The process could prevent NF-κB move into the nucleus and NF-κB signaling pathway, thus that would inhibit the function of NF-κB. In conclusion, the response above could reduce the gene expression of inflammatory mediators and inflammation and reduce the PMN adhesion and the expression of molecules and chemokines in endo-thelial cell surface.
This study investigated the role of pro-inflammatory-anti-inflammatory cyto-kine imbalance and NF-κB activation in PMN accumulation in the lungs of ARDS mice. At the same time, the expression of CD11b/CD18, its ligand ICAM-1and CINCmRNA and the expression of ENA-78were measured, in order to clarify the relationship between adhesion molecule (CD11b/CD18and its ligand ICAM-1) and chemokine (CINC,ENA-78) and PMN aggregation in lung tissue of ARDS. The present study is aimed at evaluating direct effects of PDTC on PMN activities charac- terized by the protein changes of NF-κBp65, infiltration of PMN and excessive re-lease of inflammatory cytokines.
Methods
Animals
BALB/c mice6-8-weeks of age, weighing20±2g, were maintained at24℃room temperature. To further study the protective effect of PDTC on mice with LPS, mice were randomly divided into3groups:Control group (saline,20ml/kg,i.p.), LPS group (LPS,20mg/kg,i.p.), and PDTC+LPS group (PDTC,120mg/kg,i.p.; LPS,20mg/kg,i.p.). The mice were sacrificed using aortic phlebotomy at2h,6h,12h and24h.
Specimen collection
The blood, lung tissue and BALF samples in each group of mice were collected at the same time after modeling2h,6h,12h and24h. The mice were anesthetized by intraperitoneal injection of10%chloral hydrate (3.5ml/kg). The first part of sampling: mouse orbital blood was collected and collected serum protein was stored at-80℃for cytokines and total protein. Subsequently apical blood was collected for blood gas analysis. Lungs were collected, and take the right lung to prepare for HE staining and immunohistochemistry; and take the left lung calculate lung wet/dry weight ratio (W/D). The second part of sampling:the trachea exposed in another mice, and the BALF was collected. After centrifuged, the supernatant of BALF was collected and the expression of IL-1β, IL-8, TNF-a, IL-10and protein were measured and count the number of PMN was measured. The third part of sampling:Take the mouse lung tis-sue was stored at-80℃liquid nitrogen to prepare for Western blot,Quantitative Real-time RT-PCR and the MPO activity.
Detections
1. The lung injury and scores were evaluated by histopathological analysis; the lung permeability index (LPI=BALF protein/serum protein),W/D and P(A-a)O2were measured.
2. After centrifugation, the cells in BALF sediment were smeared and count the num-ber of PMN by Wright-Giemsa staining.
3. The expression levels of TNF-a, IL-1β, IL-8and IL-10was measured in serum and BALF supernatant by ELISA. The MPO activity was detected by colorimetric.
4. Total protein of lung tissue was extracted and the phosphorylated NF-κB (p-NF-KB) was determined by Western blot in order to indicate that NF-κB was activated. The protein in nuclear and cytoplasm were extracted and the production of protein NF-κBp65in lung tissue was measured by Western blot.
5. The expression of NF-κB、CDllb/CD18、ICAM-1and ENA-78in lung tissue was measured by immunohistochemistry.
6. The expression of CINC mRNA in lung tissue was measured at each time by quan-titative-polymerase chain reaction (Quantitative Real-time, RT-PCR).
7. Statistical analysis
The results were expressed as means±standard deviations. Statistical analysis was performed with analysis of variance and t test was used for comparison among groups. P value less than0.05was considered statistically significant. The statistical analysis was conducted by SPSS19.0software.
Results
1. General information
The mice in control group breath smooth and the lung appearance was pink. The mice in LPS group were shortness of breath, oral cyanosis, listlessness, and hemorr-hagic secretions could be seen in the nose, less activities and eating. And the lung vo-lume increased when dissected, meanwhile, the reddish liquid was leaked on the sheet section. In the PDTC intervention group, the shortness of breath was reduced, and the lung volume has no obvious increase when dissected.
2. Pathology results of lung tissue and scores
2.1HE staining
The structure of lung tissue was integrated and there were no inflammatory cells in control group. But in the LPS groups, we could observe the widened lung interval, highly congested pulmonary interstitial, fracture alveolar wall and a large number of infiltrative inflammatory cells, and inflammatory cell infiltration increased and lung tissue damage aggravated as the time went on. There were similar symptoms with ARDS groups in PDTC intervention group, but the lung tissue injury was milder than that in ARDS group.
2.2The pathology scores
The pathology score was obviously higher LPS group than that in control group (P<0.01), while the score was decreased after PDTC intervention than that in the LPS group (P<0.05), and the score was similar to lung tissue damage.
3. The changes of pulmonary vascular permeability
3.1lung wet/dry weight ratio (W/D)
The lung W/D values were significantly increased in mice of intraperitoneal in-jection LPS compared with the control group at2h,12h(P<0.01), and W/D values gradually increased as time extended. Compared with LPS group, W/D values was significantly decreased in mice of intraperitoneal injection of PDTC (P<0.05) but higher than that in control group.
3.2lung permeability index, LPI
LPI was significantly higher in LPS groups than that in the control group (P<0.01), and PDTC could inhibit LPI increased (P<0.05).
4. The total cells and PMN ratio in BALF
The total number of cells were obviously increased in LPS group compared with the control group(P<0.01); after the PDTC intervention the cells were decreased at2h and6h compared with LPS group (P<0.05) and significantly decreased at12h and24h(P<0.01).The cells were mainly PMN in BALF of LPS group and PDTC group, and the number was obviously higher than the control group (P<0.01), but that was lower in PDTC group than LPS group (P<0.05).
5. Expression of TNF-a, IL-1β, IL-8and IL-10in serum and BALF
The protective effect of PDTC on the overproduction of proinflammatory cyto-kines induced by LPS was observed in our study. The expression of TNF-a, IL-1β, and IL-8in serum and BALF in LPS group were markedly higher than that in control group (P<0.01). But in the group treated with PDTC(120mg/kg) before induced by LPS, the expression of TNF-α, IL-1β and IL-8were decreased compared with LPS group at2h,6h and12h (P<0.05) and dramatically decreased compared with LPS group at24h (P<0.01). The expression of IL-10in serum and BALF of LPS group was higher than that in control group in the initial6h (P<0.05). But as the time went on, the levels of IL-10was markedly increased (P<0.01). In the group treated with PDTC, the level of IL-10was slightly lower than that in the LPS group at2h, there was no significant difference (P>0.05), but it was lower than the LPS group at6h,12h and24h (P<0.05).
6. MPO activity in lung tissue
MPO activity is an important index to evaluate the accumulation of neutrophil in lung tissues. The activity of MPO in LPS group was markedly increased compared with control group (P<0.01). However, this change was blocked significantly in the group treated with PDTC before challenged by LPS (P<0.05).
7. Changes of p-NF-KB and NF-κB P65protein expression in cytoplasm and nucleus of lung tissue
The p-NF-KB was increased in LPS lung tissue compared with the control group (P<0.01), and after the intervention of PDTC, the p-NF-KB was decreased compared with LPS group (P<0.05).NF-κB P65protein expression was significantly lower cy-toplasm of LPS group than control group, but P65protein expression was significant-ly higher in pulmonary nucleus than control group (P<0.01). However, P65protein in the cytoplasm of lung tissue expression intensity was increased in PDTC+LPS group compared with LPS group (P<0.05), while P65protein expression was decreased in nucleus of PDTC+LPS group compared with LPS group at2h,6h and12h(P<0.05) and significantly decreased at24h(P<0.01).
8. The expression of CINCmRNA
The CINCmRNA expression was obviously increased in LPS group compared with control group (P<0.01), while the CINCmRNA expression was significantly re-duced when intervened by PDTC at each phase points compared with LPS group (P<0.01).
9. The expression of NF-κB、CD11b/CD18and ICAM-1in the lung tissue
NF-κB was mainly expressed in PMN, and the positive cells were stained brown. NF-κB positive cells were increased in LPS group and the positive cells were de- creased in PDTC intervention group compared with the control group. CD11b/CD18was mainly expressed in PMN of lung tissue and these cells were stained brown. CD11b/CD18positive cells were increased in LPS group and the cells were decreased after treated with PDTC compared with the control group. ICAM1was mainly ex-pressed in vascular endothelial cells. ICAM1positive cells were increased in LPS group and the cells were reduced after intervened by PDTC compared with the control group. The results of immunohistochemistry showed that ENA-78was highly ex-pressed in endothelial cells in LPS-induced ARDS mice than that in the control group. The expression of ENA-78in PDTC+LPS group was markedly lower than that in LPS group.
Conclusions
1. The NF-κB inhibitory protein IκB phosphorylation degradation and NF-κBp65ac-tivated in lung tissue from cytoplasm to nucleus which initiate the inflammation in ARDS. The activation of NF-κB induced by LPS could regulate the expression and release of cytokines such as TNF-a,IL-1β, IL-8and IL-10.
2. The pro-inflammatory-anti-inflammatory response was imbalanced in the early stage of ARDS, and the expression of pro-inflammatory cytokines was increased. Therefore, the production of anti-inflammatory cytokines was relative lack, and the high expression time was delayed.
3. The interaction between CD11b/CD18on PMN surface and its ligand ICAM-1is involved in ARDS induced by LPS. Meanwhile, CINC and ENA-78were highly ex-pressed in the lung tissue which prompting a large number of PMN aggregation in the lungs.
4. The expression of cytokines, adhesion molecules and chemokines and PMN aggre-gation were reduced by the inhibition of PDTC on NF-κB, in order to improve the pro-inflammatory-anti-inflammatory imbalance, reduce the release of MPO from PMN and the lung injury.
Innovation and Significance
1. In this study, we clarify the relationship among the p-NF-κB, p65protein in cytop-lasmic and nuclear and inflammatory cytokines in lung tissue in order to investigate the role of PMN infiltration in ARDS.
2. We study the mechanism between high expression of adhesion molecule and che-mokine in lung tissue of ARDS in order to study the mechanism of PMN aggregation in ARDS.
3. We investigate the protective mechanism about PDTC, a specific inhibitor of NF-κB, in ARDS mice induced by LPS. That would provide broad prospects and the-rapeutic approaches for the study of the pathogenesis and development of ARDS.
急性呼吸窘迫综合征(acute respiratory distress syndrome, ARDS)本质是过度失控的炎症反应,众多效应细胞参与ARDS进程,而中性粒细胞(polymorphonuclear leukocyte, PMN)是最重要的效应细胞。ARDS早期单核-巨噬细胞被激活,释放肿瘤坏死因子-α(tumor necrosis factor-a, TNF-a)、白介素-1β(interleukin-1, IL-1β)、IL-6、IL-8等促炎细胞因子,这些因子可激活PMN和内皮细胞等效应细胞,使大量PMN在肺内聚集,释放髓过氧化物酶(myeloperoxidase, MPO)、氧自由基及溶酶体酶等多种炎症介质,导致肺毛细血管内皮细胞和肺泡上皮细胞的广泛损伤。
核因子-κB (nuclear factor-kappa B, NF-κB)是炎症反应的关键性核转录因子,通常状态下,NF-κB以二聚体形式与核因子-κB抑制蛋白(inhibitor κB protein, IκB)结合形成NF-κB-IκB三聚体复合物,以非活性状态存在于胞浆中。其中发挥主要生理功能的是NF-κB p50/p65二聚体,NF-κB p65为其主要亚单位。脂多糖(lipopolysaccharide, LPS)是革兰氏阴性菌外膜主要成分,可使IκB激酶激活致NF-κB磷酸化,IκB解离,NF-κB进入细胞核与DNA特定的κB序列结合后活化,高效诱导抗炎-促炎细胞因子、粘附分子及趋化因子的基因转录。而TNF-α、 IL-1β也可激活NF-κB。IL-10是一种主要的抗炎细胞因子,对NF-κB的活化产生抑制作用,减少促炎细胞因子的表达。
粘附分子家族中与PMN活化关系最为密切的是整合素CD11b/CD18。在趋化因子的作用下,CD11b/CD18在PMN表面表达增多,并与其配体血管内皮细胞表面的细胞间粘附分子-1(intercellular adhesion molecule-1, ICAM-1)相互作用,参与PMN及血管内皮细胞的粘附、跨越血管内皮等病理生理过程。中性粒细胞趋化因子(cytokine induced neutrophil chemoattractant,CINC)是PMN特异性趋化因子,也是趋化因子IL-8家族的同源物。LPS、TNF-α及IL-1β的刺激可诱导巨噬细胞、肺间质细胞等效应细胞释放CINC,对PMN向肺内募集起重要作用。另一种趋化因子内皮中性粒细胞激活肽-78(epithelial neutrophil activating pep-tide-78, ENA-78)也可趋化PMN。此外,ENA-78与CDllb/CD18受体结合,会使内皮细胞内游离的Ca2+增加,致肺毛细血管通透性增加。
吡咯烷二硫代氨基甲酸(pyrrolidine dithiocarbamate, PDTC)为一种抗氧化剂,是NF-κB的特异性抑制剂。PDTC通过稳定抑制蛋白IκB、增加IκB的合成、阻止IκB的磷酸化和后续降解,抑制NF-κB激活,使细胞因子、粘附分子、趋化因子的表达减少,抑制PMN等炎性细胞在肺内的聚集。
本研究探讨了小鼠ARDS时NF-κB活化、促炎-抗炎细胞因子表达与PMN肺内聚集的关系；阐述肺组织粘附分子(CD11b/CD18、ICAM-1)及趋化因子(CINC、ENA-78)表达在PMN活化中的作用；研究PDTC对NF-κB的干预作用,为临床治疗ARDS提供新的思路。
研究方法
一、动物模型的建立及分组
腹腔内注射LPS(20mg/kg)建立BALB/c小鼠ARDS模型,随机分对照组(N组)、脂多糖组(L组)、PDTC干预组(P+L组)。于LPS注射前半小时腹腔内注射PDTC (120mg/kg)来实现对小鼠ARDS的干预。NS组腹腔内注射20ml/kg的生理盐水。
二、标本的采集
每组小鼠于造模后2h、6h、12h及24h腹腔注射10%水合氯醛3.5ml/kg麻醉。因所测指标无法一次取材成功,分三部分取材。第一部分：眼眶采血,取血清-80℃保存,备查细胞因子及总蛋白。随后开胸,心尖采血行血气分析；留取肺组织,取右肺以备HE染色和免疫组化染色；取左肺计算肺湿/干重比值(wet/dry weight ratio, W/D)。第二部分：收集支气管肺胞灌洗液(bronchoalveolar lavage fluid,BALF),离心,取上清液行细胞因子及总蛋白水平测定,收集细胞沉渣,行白细胞计数及测PMN比例。第三部分：取肺组织放入-80℃液氮中保存,备Western blot.逆转录及实时定量PCR及髓过氧化物酶(myeloperoxidase, MPO)活性的检测。
三、检测方法及指标
1、HE染色评估肺组织病理损伤及评分；测肺通透指数(lung permeability index, LPI=BALF中蛋白/血清蛋白)及W/D;测P(A-a)02。
2、取BALF细胞沉渣,行Wright-Giemsa染色,计数白细胞总数及PMN的比例。
3、收集BALF上清液及取备用血清,ELISA法检测TNF-α、IL-1β、IL-8及IL-10的水平；应用试剂盒检测肺组织MPO活性。
4、提取肺组织总蛋白,Western blot法测定磷酸化NF-kB (phospho-nuclear fac-tor-kappa B, p-NF-κB)表达；提取胞浆及胞核蛋白,Western blot法测定胞浆及胞核NF-kBp65表达。
5、免疫组化法检测肺组织NF-kB、粘附分子(CDllb/CD18. ICAM-1)及趋化因子ENA-78表达。
6、采用逆转录及实时定量PCR (Real-time PCR)法测肺组织CINCmRNA表达强度。
7.、统计学分析：所有数据以平均数±标准差表示,采用SPSS19.0统计软件包分析。两两之间的比较采用t检验,P<0.05表明差异有统计学意义,P<0.01表明差异有显著统计学意义。
研究结果
一、一般情况
NS组小鼠呼吸平稳,解剖后肺部外观色泽粉红；L组小鼠呼吸急促,口唇发绀,解剖后肺体积增大,色泽暗红或紫红,脏层胸膜下点状、片状出血,切面淡红色液体渗出；P+L组小鼠呼吸急促减轻,解剖后肺体积无明显增大,色泽呈红色,表面见少许散在的出血点,包膜张力减轻。
二、HE染色评估肺组织病理损伤及评分
1、HE染色
NS组肺组织结构完整正常；L组部分肺泡壁不同程度的破坏或断裂,肺微血管充血、出血、微血栓形成,肺泡腔及肺间质内见大量炎性细胞浸润、Ⅰ型肺泡上皮细胞坏死；PDTC干预后肺组织损伤程度减轻。
2、肺病理评分
L组肺组织病理评分明显高于NS组(P<0.01)；PDTC干预后病理评分较L组下降(P<0.05),病理评分与HE染色肺组织损伤的动态变化趋势相似。
三、肺血管通透性变化的检测
1、肺组织湿/干重比值(W/D)
L组肺组织W/D值较NS组明显增加(P<0.01),随着造模时间延长W/D值逐渐增加；PDTC干预后肺水肿程度减轻,W/D值均较LS组明显下降(P<0.05),但高于NS组。
2、肺通透指数(LPI)
L组肺LPI较NS组明显升高(P<0.01),PDTC干预后肺泡腔及肺间质内蛋白渗出减少,与L组对比,LPI下降(P<0.05)。
四、BALF中白细胞总数及PMN比例
L组白细胞总数及PMN比例较NS组进行性增加(P<0.01)；PDTC干预后白细胞总数下降,各时相点均低于L组,2h及6h与L组对比,差异有统计学意义(P<0.05),12h及24h与L组对比,差异具有显著统计学意义(P<0.01)；但P+L组各时相点PMN比例与L组对比呈下降趋势,差异有统计学意义(P<0.05)。
五、血清及BALF中TNF-α、IL-1β、IL-8及IL-10的表达
L组血清及BALF中TNF-α、IL-1β及IL-8的浓度明显高于NS组(P<0.01)；PDTC干预后TNF-α、IL-1β及IL-8的浓度在2h、6h及12h低于L组(P<0.05),而在24h明显低于L组,差异有显著统计学意义(P<0.01),但均高于NS组。
与NS组对比,L组在最初6h内血清及BALF中IL-10的浓度升高(P<0.05),随着时间延长,IL-10的浓度明显升高(P<0.01)；PDTC干预后,与L组比较,2h IL-10的浓度略降低,差异无统计学意义(P>0.05),但6h、12h及24h IL-10的浓度低于L组,差异有统计学意义(P<0.05)。
六、肺组织中MPO的活性
L组肺组织MPO活性较NS组明显增加(P<0.01)；PDTC干预后与L组比较,MPO活性降低(P<0.05),但不能回到正常水平。
七、Western blot检测肺组织p-NF-κB、胞浆及胞核中NF-κBp65蛋白的表达
L组肺组织p-NF-κB蛋白的表达增强,与NS组相比(P<0.01)；PDTC干预后p-NF-κB蛋白的表达增强较L组降低(P<0.05)。
L组肺组织胞浆中P65蛋白的表达强度降低,而胞核中P65蛋白表达强度升高,与NS组比较(P<0.01)。PDTC干预后胞浆中P65蛋白的表达强度较L组升高(P<0.05)；在2h、6h及12h而胞核中P65蛋白表达强度较L组降低,差异有统计学意义(P<0.05),在24h胞核中P65蛋白表达强度较L组明显降低,差异有显著统计学意义(P<0.01)。
八、逆转录及实时定量PCR检测CINCmRNA的表达
NS组肺组织有极少量的CINCmRNA的表达；与NS组相比,L组CINCm RNA的表达明显增加(P<0.01)；PDTC干预后肺组织CINCmRNA的表达与L组对比表达明显减少(P<0.01)。
九、免疫组化检测肺组织NF-κB、CD11b/CD18及ICAM-1、ENA-78的表达
以细胞出现棕褐色或棕黄色着色为阳性表达。与NS组对比,L组可见较多的NF-κB阳性免疫反应的细胞,主要表达在肺间质PMN; PDTC干预后NF-κB阳性免疫反应的细胞减少。L组CDllb/CD18阳性表达的细胞较NS组明显增加,主要在PMN上表达；PDTC干预后CD11b/CD18阳性表达的细胞减少。ICAM-1主要表达在血管内皮细胞,与NS组对比,L组ICAM-1阳性免疫反应的细胞增加；PDTC干预后ICAM-1阳性免疫反应的细胞减少。与NS组对比,L组ENA-78阳性表达的细胞增加,以内皮细胞阳性表达为主；PDTC干预后ENA-78阳性表达的细胞减少。
研究结论
1、LPS诱导的ARDS中,NF-κB磷酸化降解,NF-KBp65由胞浆向胞核转移,NF-κB激活,细胞因子(IL-1β、IL-8、IL-10和TNF-a).粘附分子及趋化因子表达增加。
2、小鼠ARDS早期促炎-抗炎反应失衡,表现在促炎细胞因子表达增多,抗炎细胞因子相对不足,且高表达时间滞后。
3、在趋化因子(CINC. ENA-78)的作用下,PMN表面粘附分子CD11b/CD18表达增多,与其配体内皮细胞表面的粘附分子ICAM-1相互作用,使PMN粘附在血管壁,并跨越内皮向肺组织浸润。
4、PDTC通过抑制NF-κB的激活,下调细胞因子、粘附分子及趋化因子的表达,减少PMN聚集,改善促炎-抗炎介质的失衡,使PMN释放的MPO减少,肺组织损伤减轻。
创新性及意义
1、本研究系统地从肺组织p-NF-κB及胞浆、胞核p65蛋白表达阐述ADRS时NF-κB活化。
2、探讨粘附分子、趋化因子的高表达对ARDS肺组织PMN聚集的影响。
3、研究NF-κB的特异性抑制剂PDTC对ARDS小鼠的保护机制,为进一步研究其发病机制、拓展ARDS的治疗途径提供广阔的前景。
Objectives
The main pathogenesis of the acute respiratory distress syndrome (ARDS) was excessive uncontrolled inflammatory response mediated by various inflammatory cy-tokines, but the treatment was limited effective. And a large number of polymorpho-nuclear neutrophil (PMN) accumulated in lung tissue plays an important role in dam-age of pulmonary capillary endothelial cells and alveolar epithelial cells. Previous studies have demonstrated that a large number of polymorphonuclear neutrophil (PMN) accumulated in lung tissue and release inflammatory cytokines, such as Inter-leukin-1β(IL-1β),Il-6,IL-8and tumor necrosis factor-alpha (TNF-a) and so on when body against infection or trauma, which plays an important role in starting and main-taining the inflammatory response. And it could promote the excessive production and expression of inflammatory mediators including cytokines, chemokines, adhesion molecules, myeloperoxidase (MPO) and ROS.
As an important nuclear transcription factor, Nuclear factor-κB(NF-κB) is the intersection of multiple signaling pathways. The protein exists in the form of inactive in the cytoplasm by the form of dimer and directly combines with inhibitory protein IκB to form the trimeric complex at usually state. P50/P65heterodimer plays a major physiological function during inflammation, and NF-κB P65was as its main subunit. Lipopolysaccharide (LPS) is the major component of the outer membrane of gram-negative bacteria and is a common trigger of sepsis, which is the important init-iation factor to activate NF-κB. The Rel protein localization signal (NLS) exposed under the stimulation of LPS, then NF-κB bound to specific κB sequences of DNA in the nucleus in order to regulate the transcription and expression of gene. IL-10is a major anti-inflammatory cytokines, and could inhibit activation of NF-κB and reduce the expression of pro-inflammatory cytokines.
The expression of CD11b/CD18plays an important role in PMN aggregation and activation. CD11b/CD18is a heterodimer of the aM(CD11b) and β2(CD18) subunits, which is a key factor in inflammatory response to combine with the ICAM-1and me-diate the adhesion between PMN and microvascular endothelial cells, then transmit the intracellular signal. CINC, one of the IL-8family, is the specific chemokines of PMN, which plays an important role in the aggregation process of PMN in lung tissue. CINC-1and CINC-3played important role in PMN recruitment to the lung in LPS-induced ARDS.Another chemokine neutrophil activating peptide (ENA-78) also could chemotaxis PMN. In addition, ENA-78could bind to CD11b/CD18receptor, increase the free Ca2+in endothelial cells and the permeability pulmonary capillary.
Pyrrolidine dithiocarbamate(PDTC) is a metal chelating agent and an antioxidant, which is the specific inhibitor of NF-κB. The study has confirmed that PDTC could directly reduce binding capacity of NF-κB to DNA, and increase the synthesis of IκB in order to prevent the phosphorylation of IκB and the subsequent degradation. The process could prevent NF-κB move into the nucleus and NF-κB signaling pathway, thus that would inhibit the function of NF-κB. In conclusion, the response above could reduce the gene expression of inflammatory mediators and inflammation and reduce the PMN adhesion and the expression of molecules and chemokines in endo-thelial cell surface.
This study investigated the role of pro-inflammatory-anti-inflammatory cyto-kine imbalance and NF-κB activation in PMN accumulation in the lungs of ARDS mice. At the same time, the expression of CD11b/CD18, its ligand ICAM-1and CINCmRNA and the expression of ENA-78were measured, in order to clarify the relationship between adhesion molecule (CD11b/CD18and its ligand ICAM-1) and chemokine (CINC,ENA-78) and PMN aggregation in lung tissue of ARDS. The present study is aimed at evaluating direct effects of PDTC on PMN activities charac- terized by the protein changes of NF-κBp65, infiltration of PMN and excessive re-lease of inflammatory cytokines.
Methods
Animals
BALB/c mice6-8-weeks of age, weighing20±2g, were maintained at24℃room temperature. To further study the protective effect of PDTC on mice with LPS, mice were randomly divided into3groups:Control group (saline,20ml/kg,i.p.), LPS group (LPS,20mg/kg,i.p.), and PDTC+LPS group (PDTC,120mg/kg,i.p.; LPS,20mg/kg,i.p.). The mice were sacrificed using aortic phlebotomy at2h,6h,12h and24h.
Specimen collection
The blood, lung tissue and BALF samples in each group of mice were collected at the same time after modeling2h,6h,12h and24h. The mice were anesthetized by intraperitoneal injection of10%chloral hydrate (3.5ml/kg). The first part of sampling: mouse orbital blood was collected and collected serum protein was stored at-80℃for cytokines and total protein. Subsequently apical blood was collected for blood gas analysis. Lungs were collected, and take the right lung to prepare for HE staining and immunohistochemistry; and take the left lung calculate lung wet/dry weight ratio (W/D). The second part of sampling:the trachea exposed in another mice, and the BALF was collected. After centrifuged, the supernatant of BALF was collected and the expression of IL-1β, IL-8, TNF-a, IL-10and protein were measured and count the number of PMN was measured. The third part of sampling:Take the mouse lung tis-sue was stored at-80℃liquid nitrogen to prepare for Western blot,Quantitative Real-time RT-PCR and the MPO activity.
Detections
1. The lung injury and scores were evaluated by histopathological analysis; the lung permeability index (LPI=BALF protein/serum protein),W/D and P(A-a)O2were measured.
2. After centrifugation, the cells in BALF sediment were smeared and count the num-ber of PMN by Wright-Giemsa staining.
3. The expression levels of TNF-a, IL-1β, IL-8and IL-10was measured in serum and BALF supernatant by ELISA. The MPO activity was detected by colorimetric.
4. Total protein of lung tissue was extracted and the phosphorylated NF-κB (p-NF-KB) was determined by Western blot in order to indicate that NF-κB was activated. The protein in nuclear and cytoplasm were extracted and the production of protein NF-κBp65in lung tissue was measured by Western blot.
5. The expression of NF-κB、CDllb/CD18、ICAM-1and ENA-78in lung tissue was measured by immunohistochemistry.
6. The expression of CINC mRNA in lung tissue was measured at each time by quan-titative-polymerase chain reaction (Quantitative Real-time, RT-PCR).
7. Statistical analysis
The results were expressed as means±standard deviations. Statistical analysis was performed with analysis of variance and t test was used for comparison among groups. P value less than0.05was considered statistically significant. The statistical analysis was conducted by SPSS19.0software.
Results
1. General information
The mice in control group breath smooth and the lung appearance was pink. The mice in LPS group were shortness of breath, oral cyanosis, listlessness, and hemorr-hagic secretions could be seen in the nose, less activities and eating. And the lung vo-lume increased when dissected, meanwhile, the reddish liquid was leaked on the sheet section. In the PDTC intervention group, the shortness of breath was reduced, and the lung volume has no obvious increase when dissected.
2. Pathology results of lung tissue and scores
2.1HE staining
The structure of lung tissue was integrated and there were no inflammatory cells in control group. But in the LPS groups, we could observe the widened lung interval, highly congested pulmonary interstitial, fracture alveolar wall and a large number of infiltrative inflammatory cells, and inflammatory cell infiltration increased and lung tissue damage aggravated as the time went on. There were similar symptoms with ARDS groups in PDTC intervention group, but the lung tissue injury was milder than that in ARDS group.
2.2The pathology scores
The pathology score was obviously higher LPS group than that in control group (P<0.01), while the score was decreased after PDTC intervention than that in the LPS group (P<0.05), and the score was similar to lung tissue damage.
3. The changes of pulmonary vascular permeability
3.1lung wet/dry weight ratio (W/D)
The lung W/D values were significantly increased in mice of intraperitoneal in-jection LPS compared with the control group at2h,12h(P<0.01), and W/D values gradually increased as time extended. Compared with LPS group, W/D values was significantly decreased in mice of intraperitoneal injection of PDTC (P<0.05) but higher than that in control group.
3.2lung permeability index, LPI
LPI was significantly higher in LPS groups than that in the control group (P<0.01), and PDTC could inhibit LPI increased (P<0.05).
4. The total cells and PMN ratio in BALF
The total number of cells were obviously increased in LPS group compared with the control group(P<0.01); after the PDTC intervention the cells were decreased at2h and6h compared with LPS group (P<0.05) and significantly decreased at12h and24h(P<0.01).The cells were mainly PMN in BALF of LPS group and PDTC group, and the number was obviously higher than the control group (P<0.01), but that was lower in PDTC group than LPS group (P<0.05).
5. Expression of TNF-a, IL-1β, IL-8and IL-10in serum and BALF
The protective effect of PDTC on the overproduction of proinflammatory cyto-kines induced by LPS was observed in our study. The expression of TNF-a, IL-1β, and IL-8in serum and BALF in LPS group were markedly higher than that in control group (P<0.01). But in the group treated with PDTC(120mg/kg) before induced by LPS, the expression of TNF-α, IL-1β and IL-8were decreased compared with LPS group at2h,6h and12h (P<0.05) and dramatically decreased compared with LPS group at24h (P<0.01). The expression of IL-10in serum and BALF of LPS group was higher than that in control group in the initial6h (P<0.05). But as the time went on, the levels of IL-10was markedly increased (P<0.01). In the group treated with PDTC, the level of IL-10was slightly lower than that in the LPS group at2h, there was no significant difference (P>0.05), but it was lower than the LPS group at6h,12h and24h (P<0.05).
6. MPO activity in lung tissue
MPO activity is an important index to evaluate the accumulation of neutrophil in lung tissues. The activity of MPO in LPS group was markedly increased compared with control group (P<0.01). However, this change was blocked significantly in the group treated with PDTC before challenged by LPS (P<0.05).
7. Changes of p-NF-KB and NF-κB P65protein expression in cytoplasm and nucleus of lung tissue
The p-NF-KB was increased in LPS lung tissue compared with the control group (P<0.01), and after the intervention of PDTC, the p-NF-KB was decreased compared with LPS group (P<0.05).NF-κB P65protein expression was significantly lower cy-toplasm of LPS group than control group, but P65protein expression was significant-ly higher in pulmonary nucleus than control group (P<0.01). However, P65protein in the cytoplasm of lung tissue expression intensity was increased in PDTC+LPS group compared with LPS group (P<0.05), while P65protein expression was decreased in nucleus of PDTC+LPS group compared with LPS group at2h,6h and12h(P<0.05) and significantly decreased at24h(P<0.01).
8. The expression of CINCmRNA
The CINCmRNA expression was obviously increased in LPS group compared with control group (P<0.01), while the CINCmRNA expression was significantly re-duced when intervened by PDTC at each phase points compared with LPS group (P<0.01).
9. The expression of NF-κB、CD11b/CD18and ICAM-1in the lung tissue
NF-κB was mainly expressed in PMN, and the positive cells were stained brown. NF-κB positive cells were increased in LPS group and the positive cells were de- creased in PDTC intervention group compared with the control group. CD11b/CD18was mainly expressed in PMN of lung tissue and these cells were stained brown. CD11b/CD18positive cells were increased in LPS group and the cells were decreased after treated with PDTC compared with the control group. ICAM1was mainly ex-pressed in vascular endothelial cells. ICAM1positive cells were increased in LPS group and the cells were reduced after intervened by PDTC compared with the control group. The results of immunohistochemistry showed that ENA-78was highly ex-pressed in endothelial cells in LPS-induced ARDS mice than that in the control group. The expression of ENA-78in PDTC+LPS group was markedly lower than that in LPS group.
Conclusions
1. The NF-κB inhibitory protein IκB phosphorylation degradation and NF-κBp65ac-tivated in lung tissue from cytoplasm to nucleus which initiate the inflammation in ARDS. The activation of NF-κB induced by LPS could regulate the expression and release of cytokines such as TNF-a,IL-1β, IL-8and IL-10.
2. The pro-inflammatory-anti-inflammatory response was imbalanced in the early stage of ARDS, and the expression of pro-inflammatory cytokines was increased. Therefore, the production of anti-inflammatory cytokines was relative lack, and the high expression time was delayed.
3. The interaction between CD11b/CD18on PMN surface and its ligand ICAM-1is involved in ARDS induced by LPS. Meanwhile, CINC and ENA-78were highly ex-pressed in the lung tissue which prompting a large number of PMN aggregation in the lungs.
4. The expression of cytokines, adhesion molecules and chemokines and PMN aggre-gation were reduced by the inhibition of PDTC on NF-κB, in order to improve the pro-inflammatory-anti-inflammatory imbalance, reduce the release of MPO from PMN and the lung injury.
Innovation and Significance
1. In this study, we clarify the relationship among the p-NF-κB, p65protein in cytop-lasmic and nuclear and inflammatory cytokines in lung tissue in order to investigate the role of PMN infiltration in ARDS.
2. We study the mechanism between high expression of adhesion molecule and che-mokine in lung tissue of ARDS in order to study the mechanism of PMN aggregation in ARDS.
3. We investigate the protective mechanism about PDTC, a specific inhibitor of NF-κB, in ARDS mice induced by LPS. That would provide broad prospects and the-rapeutic approaches for the study of the pathogenesis and development of ARDS.
引文
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