锌指蛋白A20对急性肺损伤炎症反应的调控及机制研究
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摘要
第一部分A20蛋白在静脉注射LPS所致大鼠急性肺损伤中的变化及意义
[摘要]目的本实验通过建立大鼠LPS所致急性肺损伤模型,旨在研究A20蛋白在LPS急性肺损伤的表达变化及意义。方法实验选用SPF级雄性SD大鼠54只,随机分为2个实验组：正常对照组(尾静脉注射PBS,1ml/只,定义为0h)；LPS组(尾静脉注射LPS,10mg/kg,浓度为2mg/ml,根据不同时间点1h,2h,4h,6h,8h,12h,24h,48h分为8个亚组)。所有动物在注射LPS相应时间点放血处死,收集标本,观察光镜(HE染色),检测肺湿干比(W/D),行支气管肺泡灌洗,检测支气管肺泡灌洗液(BALF)中蛋白浓度,BALF中有核细胞总数,BALF中TNF-α、IL-1β水平,肺组织NF-κB DNA结合活性及核蛋白p65水平,肺组织中A20蛋白及mRNA含量,A20定位。结果与正常对照相比,注射LPS24小时后肺W/D、BALF中有核细胞总数(×104/ml)及BALF中蛋白浓度(ug/ml)均明显升高(p<0.05),光镜下可见肺泡间隔增厚,有较多炎症细胞浸润,肺泡腔内可见红细胞和中性粒细胞渗出；注射LPS后,BALF液中TNF-α、IL-1β水平及肺组织中NF-κB DNA结合活性及核蛋白p65含量随时间升高,于注射后2h达高峰,之后逐渐下降,但仍高于注射前,且在24h又发生一过性升高(p<0.05)；肺组织中A20蛋白及mRNA水平也逐渐升高,于注射后2h达高峰,之后下降,但仍高于注射前(p<0.05)；免疫组化示A20在LPS注射后2小时主要表达于肺泡巨噬细胞胞浆。结论1.健康大鼠静脉注射LPS10ml/kg24h后可出现急性肺损伤。2.急性肺损伤时,A20蛋白表达升高,且高峰期与ALI早期细胞因子及NF-κB活性一致。3.A20在急性肺损伤早期明显升高,且表达于肺泡巨噬细胞,由此推测A20可能通过调控肺泡巨噬细胞炎症活性影响肺损伤。
第二部分A20对LPS刺激后肺泡巨噬细胞炎症反应的作用及机制
[摘要]目的本实验通过建立A20基因沉默及过表达肺泡巨噬细胞株研究A20对LPS刺激后肺泡巨噬细胞炎症活性影响,旨在探讨A20对LPS所致肺损伤的作用及机制。方法实验选用大鼠肺泡巨噬细胞株(NR8383),慢病毒法构建A20基因沉默及过表达细胞株,Western Bblot及RT-PCR鉴定A20表达,并行体外培养慢病毒包装后的A20干扰(A20-SH)、干扰空载(SCR)、过表达(A20)及过表达空载(VEC)细胞株。向培养基中加入LPS进行干预(lug LPS/ml培养基),于刺激后0.5、1、2、4小时收集细胞培养上清液及细胞,ELISA法检测上清液中TNF-α、IL-1β水平及细胞中NF-κB DNA结合活性；Western Blot法检测A20蛋白及核内p65含量；RT-PCR检测A20、TNF-a、IL-1βmRNA含量。结果构建的A20-SH肺泡巨噬细胞株中,A20蛋白及mRNA含量较SCR明显降低(p<0.05),干扰效率达80%；过表达肺泡巨噬细胞中A20蛋白及mRNA含量较VEC明显升高(p<0.05),表达增加10倍以上。LPS刺激后,SCR组及VEC组A20蛋白及mRNA含量升高,且于1h达高峰,之后逐渐下降,而A20-SH组较SCR组明显降低(p<0.05),且始终维持在较低水平,A20组较VEC明显升高(p<0.05)。LPS刺激后各组肺泡巨噬细胞中细胞因子(TNF-α、IL-1β)mRNA及培养上清液中含量、肺组织中NF-κB DNA结合活性及核内p65含量都随时间升高,于1h达高峰,之后逐渐下降；但与SCR相比,A20-SH组明显升高(p<0.05)；与VEC组相比,A20组水平明显降低(p<0.05)。结论1.慢病毒RNAi表达载体及A20过表达载体可分别有效地抑制大鼠肺泡巨噬细胞株中的A20基因表达或增加该基因表达。2.A20基因沉默导致肺泡巨噬细胞NF-κB活性升高,促进TNF-α、IL-1β表达及分泌；反之A20基因过表达能够抑制肺泡巨噬细胞NF-κB活性及TNF-α、IL-1β表达和分泌。3.A20蛋白能够抑制肺泡巨噬细胞炎症反应活性,进而减轻肺损伤。
Part I:Change and significance of A20protein in acute lung injury induced by intravenous injection of LPS in rats
Abstract Objective To build the model of acute lung injury (ALI) induced by intravenous injection of LPS in vivo and to investigate the A20Protein Expression in ALI.
Methods54specified-pathogens free male Sprague-Dawley rats were randomized into two groups:Group control (intravenous injection of PBS,1ml per rat, defined as Oh); group ALI (intravenous injection of LPS,10mg/kg, and divided into eight subgroups according to the different time points1h,2h,4h,6h,8h,12h,24h,48h after injection of LPS). All animals were sacrificed at the corresponding time. The pathological change of lung was observed under light microscope. The wet/dry weight ratio of lung(W/D), the levels of total proteins and the total cell count in bronchoalveolar lavage fluid (BALF) were examined. TNF-alpha, IL-1β levels in BALF and NF-κB DNA binding activity in lung tissue were tested by ELISA. The levels of nuclear protein p65and A20protein were examined by Western blot. A20mRNA levels in lung tissue were analysis by RT-PCT.
Results Compared with controls,24hours after LPS injection, the W/D ratio of lung, the levels of total proteins, the number of total cells in BALF were significantly higher (p <0.05). There were widespread alveolar walls thickening, massive neutrophils infiltration and hemorrhage under light microscope in the ALI group. In addition, after injection of LPS, the levels of TNF-aand IL-1(3in BALF, the DNA binding activity of NF-kappa B and the nuclear protein p65content increased with time, reached a peak at2h, then decreased gradually, but still higher than group control (p<0.05). As well as NF-kappa B and cytokines, A20protein and mRNA levels in lung tissue also gradually increased and reached a peak at2h, then decreased, but still higher than group control (p<0.05). Furthermore, immunohistochemistry showed that2hours after LPS injection, the A20was mainly expressed in alveolar macrophages cytoplasm.
Conclusions In acute lung injury, the expression of A20was elevated, with a peak as high as early cytokines and NF-κB. In early phase of ALI, A20protein was mainly expressed in alveolar macrophages cytoplasm, which indicated that A20may regulate inflammatory responses in ALI through alveolar macrophage.
Part II:The effects and mechanisms of A20on the inflammation responses of alveolar macrophage
Abstract Objective To establish the alveolar macrophage A20gene silencing and over-expression in vitro and to investigate the effects and mechanisms of A20on LPS induced lung injury.
Methods Constructed lentivirus-mediated iRNA vector and expression vector of A20gene, then transfected into rat alveolar macrophage cell lines (NR8383) respectively. The cells were treated with LPS (lug LPS per ml medium), and the culture supernatants and cells were collected at different time points:0.5,1,2,4hours after simulation of LPS. The levels of TNF-a and IL-1(3in supernatant levels, the DNA binding activity of NF-κB were analysis by ELISA. The levels of A20protein and nuclear p65were detected by Western Blot. The mRNA levels of A20, TNF-alpha and IL-1(3were detected by RT-PCR.
Results Compared with control, the levels of A20protein and mRNA in A20gene silencing group (A20-SH group) were significantly lower (p<0.05), with an interference efficiency of80%, while the levels in A20gene overexpression cells (A20group) were dramatically increased (p<0.05), by more than10times. After stimulation of LPS, the A20protein and mRNA content in control groups increased and reached a peak at1h, and then gradually decreased, while the levels in A20-SH group significantly lower (p<0.05), and remained at a low level all the time. On the contrary, compared with control group, the A20protein and mRNA content in A20group were significantly increased (p<0.05). In addition, after LPS simulation, in the control groups, the levels of TNF-alpha and IL-1β in supernatants and mRNA in cells increased with time, and reached a peak at1h, and then gradually decreased, as well as DNA binding activity NF-κB and nuclear p65content. Compared with control, the levels of TNF-alpha and IL-1β protein in supernatants and mRNA in cells, the DNA binding activity NF-κB and nuclear p65content in A20-SH group significantly increased (p<0.05), while A20group decreased (p<0.05).
Conclusions A20gene silencing led to increase in NF-κB activity, the promotion of TNF-alpha, IL-1β expression and secretion. On the contrary, A20gene overexpression can inhibit NF-κB activity and TNF-alpha, IL-1β expression and secretion of alveolar macrophage. A20protein can inhibit the inflammation activity of alveolar macrophages, thereby reducing the lung injury.
[摘要]目的本实验通过建立大鼠LPS所致急性肺损伤模型,旨在研究A20蛋白在LPS急性肺损伤的表达变化及意义。方法实验选用SPF级雄性SD大鼠54只,随机分为2个实验组：正常对照组(尾静脉注射PBS,1ml/只,定义为0h)；LPS组(尾静脉注射LPS,10mg/kg,浓度为2mg/ml,根据不同时间点1h,2h,4h,6h,8h,12h,24h,48h分为8个亚组)。所有动物在注射LPS相应时间点放血处死,收集标本,观察光镜(HE染色),检测肺湿干比(W/D),行支气管肺泡灌洗,检测支气管肺泡灌洗液(BALF)中蛋白浓度,BALF中有核细胞总数,BALF中TNF-α、IL-1β水平,肺组织NF-κB DNA结合活性及核蛋白p65水平,肺组织中A20蛋白及mRNA含量,A20定位。结果与正常对照相比,注射LPS24小时后肺W/D、BALF中有核细胞总数(×104/ml)及BALF中蛋白浓度(ug/ml)均明显升高(p<0.05),光镜下可见肺泡间隔增厚,有较多炎症细胞浸润,肺泡腔内可见红细胞和中性粒细胞渗出；注射LPS后,BALF液中TNF-α、IL-1β水平及肺组织中NF-κB DNA结合活性及核蛋白p65含量随时间升高,于注射后2h达高峰,之后逐渐下降,但仍高于注射前,且在24h又发生一过性升高(p<0.05)；肺组织中A20蛋白及mRNA水平也逐渐升高,于注射后2h达高峰,之后下降,但仍高于注射前(p<0.05)；免疫组化示A20在LPS注射后2小时主要表达于肺泡巨噬细胞胞浆。结论1.健康大鼠静脉注射LPS10ml/kg24h后可出现急性肺损伤。2.急性肺损伤时,A20蛋白表达升高,且高峰期与ALI早期细胞因子及NF-κB活性一致。3.A20在急性肺损伤早期明显升高,且表达于肺泡巨噬细胞,由此推测A20可能通过调控肺泡巨噬细胞炎症活性影响肺损伤。
第二部分A20对LPS刺激后肺泡巨噬细胞炎症反应的作用及机制
[摘要]目的本实验通过建立A20基因沉默及过表达肺泡巨噬细胞株研究A20对LPS刺激后肺泡巨噬细胞炎症活性影响,旨在探讨A20对LPS所致肺损伤的作用及机制。方法实验选用大鼠肺泡巨噬细胞株(NR8383),慢病毒法构建A20基因沉默及过表达细胞株,Western Bblot及RT-PCR鉴定A20表达,并行体外培养慢病毒包装后的A20干扰(A20-SH)、干扰空载(SCR)、过表达(A20)及过表达空载(VEC)细胞株。向培养基中加入LPS进行干预(lug LPS/ml培养基),于刺激后0.5、1、2、4小时收集细胞培养上清液及细胞,ELISA法检测上清液中TNF-α、IL-1β水平及细胞中NF-κB DNA结合活性；Western Blot法检测A20蛋白及核内p65含量；RT-PCR检测A20、TNF-a、IL-1βmRNA含量。结果构建的A20-SH肺泡巨噬细胞株中,A20蛋白及mRNA含量较SCR明显降低(p<0.05),干扰效率达80%；过表达肺泡巨噬细胞中A20蛋白及mRNA含量较VEC明显升高(p<0.05),表达增加10倍以上。LPS刺激后,SCR组及VEC组A20蛋白及mRNA含量升高,且于1h达高峰,之后逐渐下降,而A20-SH组较SCR组明显降低(p<0.05),且始终维持在较低水平,A20组较VEC明显升高(p<0.05)。LPS刺激后各组肺泡巨噬细胞中细胞因子(TNF-α、IL-1β)mRNA及培养上清液中含量、肺组织中NF-κB DNA结合活性及核内p65含量都随时间升高,于1h达高峰,之后逐渐下降；但与SCR相比,A20-SH组明显升高(p<0.05)；与VEC组相比,A20组水平明显降低(p<0.05)。结论1.慢病毒RNAi表达载体及A20过表达载体可分别有效地抑制大鼠肺泡巨噬细胞株中的A20基因表达或增加该基因表达。2.A20基因沉默导致肺泡巨噬细胞NF-κB活性升高,促进TNF-α、IL-1β表达及分泌；反之A20基因过表达能够抑制肺泡巨噬细胞NF-κB活性及TNF-α、IL-1β表达和分泌。3.A20蛋白能够抑制肺泡巨噬细胞炎症反应活性,进而减轻肺损伤。
Part I:Change and significance of A20protein in acute lung injury induced by intravenous injection of LPS in rats
Abstract Objective To build the model of acute lung injury (ALI) induced by intravenous injection of LPS in vivo and to investigate the A20Protein Expression in ALI.
Methods54specified-pathogens free male Sprague-Dawley rats were randomized into two groups:Group control (intravenous injection of PBS,1ml per rat, defined as Oh); group ALI (intravenous injection of LPS,10mg/kg, and divided into eight subgroups according to the different time points1h,2h,4h,6h,8h,12h,24h,48h after injection of LPS). All animals were sacrificed at the corresponding time. The pathological change of lung was observed under light microscope. The wet/dry weight ratio of lung(W/D), the levels of total proteins and the total cell count in bronchoalveolar lavage fluid (BALF) were examined. TNF-alpha, IL-1β levels in BALF and NF-κB DNA binding activity in lung tissue were tested by ELISA. The levels of nuclear protein p65and A20protein were examined by Western blot. A20mRNA levels in lung tissue were analysis by RT-PCT.
Results Compared with controls,24hours after LPS injection, the W/D ratio of lung, the levels of total proteins, the number of total cells in BALF were significantly higher (p <0.05). There were widespread alveolar walls thickening, massive neutrophils infiltration and hemorrhage under light microscope in the ALI group. In addition, after injection of LPS, the levels of TNF-aand IL-1(3in BALF, the DNA binding activity of NF-kappa B and the nuclear protein p65content increased with time, reached a peak at2h, then decreased gradually, but still higher than group control (p<0.05). As well as NF-kappa B and cytokines, A20protein and mRNA levels in lung tissue also gradually increased and reached a peak at2h, then decreased, but still higher than group control (p<0.05). Furthermore, immunohistochemistry showed that2hours after LPS injection, the A20was mainly expressed in alveolar macrophages cytoplasm.
Conclusions In acute lung injury, the expression of A20was elevated, with a peak as high as early cytokines and NF-κB. In early phase of ALI, A20protein was mainly expressed in alveolar macrophages cytoplasm, which indicated that A20may regulate inflammatory responses in ALI through alveolar macrophage.
Part II:The effects and mechanisms of A20on the inflammation responses of alveolar macrophage
Abstract Objective To establish the alveolar macrophage A20gene silencing and over-expression in vitro and to investigate the effects and mechanisms of A20on LPS induced lung injury.
Methods Constructed lentivirus-mediated iRNA vector and expression vector of A20gene, then transfected into rat alveolar macrophage cell lines (NR8383) respectively. The cells were treated with LPS (lug LPS per ml medium), and the culture supernatants and cells were collected at different time points:0.5,1,2,4hours after simulation of LPS. The levels of TNF-a and IL-1(3in supernatant levels, the DNA binding activity of NF-κB were analysis by ELISA. The levels of A20protein and nuclear p65were detected by Western Blot. The mRNA levels of A20, TNF-alpha and IL-1(3were detected by RT-PCR.
Results Compared with control, the levels of A20protein and mRNA in A20gene silencing group (A20-SH group) were significantly lower (p<0.05), with an interference efficiency of80%, while the levels in A20gene overexpression cells (A20group) were dramatically increased (p<0.05), by more than10times. After stimulation of LPS, the A20protein and mRNA content in control groups increased and reached a peak at1h, and then gradually decreased, while the levels in A20-SH group significantly lower (p<0.05), and remained at a low level all the time. On the contrary, compared with control group, the A20protein and mRNA content in A20group were significantly increased (p<0.05). In addition, after LPS simulation, in the control groups, the levels of TNF-alpha and IL-1β in supernatants and mRNA in cells increased with time, and reached a peak at1h, and then gradually decreased, as well as DNA binding activity NF-κB and nuclear p65content. Compared with control, the levels of TNF-alpha and IL-1β protein in supernatants and mRNA in cells, the DNA binding activity NF-κB and nuclear p65content in A20-SH group significantly increased (p<0.05), while A20group decreased (p<0.05).
Conclusions A20gene silencing led to increase in NF-κB activity, the promotion of TNF-alpha, IL-1β expression and secretion. On the contrary, A20gene overexpression can inhibit NF-κB activity and TNF-alpha, IL-1β expression and secretion of alveolar macrophage. A20protein can inhibit the inflammation activity of alveolar macrophages, thereby reducing the lung injury.
引文
1. Rubenfeld GD, Caldwell E, Peabody E, et al. Incidence and outcomes of acute lung injury [J]. N Engl J Med.2005; 353(16):1685-1693.
2. Wheeler AP, Bernard GR. Acute lung injury and the acute respiratory distress syndrome: a clinical review [J]. Lancet:2007.369(9572):1553-1564.
3. Fujiwara N, Kobayashi K. Macrophages in inflammation [J]. Curr Drug Targets Inflamm Allergy2005; 4(3):281-286.
4. Dixit VM, Green S, Sarma V, et al. Tumor necrosis factor-alpha induction of novel gene products in human endothelial cells including a macrophage-specific chemotaxin [J]. J Biol Chem.1990; 265(5):2973-2978.
5. Coornaert B,Carpentier I,Beyaert R.A20:Central gatekeeper in inflammation and immunity [J]. J Biol Chem.2009; 284(13):8217-8221.
6. Vereecke L, Sze M, Me Guire. Enterocyte-specific A20 deficiency sensitizes to tumor necrosis factor-induced toxicity and experimental colitis [J]. J Exp Med.2010; 207(7):1513-1523.
7. Vereecke L, Beyaert R, van Loo G. Genetic relationships between A20/TNFAIP3, chronic inflammation and autoimmune disease [J]. Biochem Soc Trans.2011; 39(4):1086-1091.
8. Musone SL, Taylor KE, Lu TT, et al. Multiple polymorphisms in the TNFAIP3 region are independently associated with systemic lupus erythematosus [J]. Nat Genet.2008; 40(9):1062-1064.
9. Fan Y, Tao JH, Zhang LP, et al. The association between BANK1 and TNFAIP3 gene polymorphisms and systemic lupus erythematosus:a meta-analysis [J]. Int J Immunogenet.2011; 38(2):151-159.
10. Hou CL, Zhang W, Wei Y, et al. Zinc finger protein A20 overexpression inhibits monocyte homing and protects endothelial cells from injury induced by high glucose [J]. Genet Mol Res.2011; 10(2):1050-1059.
11. Kang NI, Yoon HY, Lee YR, et al. A20 attenuates allergic airway inflammation in mice [J]. J Immunol.2009; 183(2):1488-1495.
12. Gon Y, Asai Y, Hashimoto S, et al. A20 inhibits toll-like receptor 2-and 4-mediated interleukin-8 synthesis in airway epithelial cells [J]. Am J Respir Cell Mol Biol.2004; 31(3):330-336.
13. Verstrepen L, Verhelst K, van Loo G, et al. Expression, biological activities and mechanisms of action of A20 (TNFAIP3) [J]. Biochem Pharmacol.2010; 80(12):2009-2020.
14. Harhaj EW, Dixit VM. Deubiquitinases in the regulation of NF-κB signaling [J]. Cell Res.2011; 21(1):22-39.
15. Vereecke L, Beyaert R, van Loo G. The ubiquitin-editing enzyme A20(TNFAIP3) is a central regulator of immunopathology [J]. Trends Immunol. 2009; 30(8):383-391.
16. Amato MBP BC, Medeiros DM, et al. Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome [J]. N Engl J Med 1998;338(6):347-57.
17. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome.The Acute Respiratory Distress Syndrome Network [J]. N Engl J Med 2000;342(18):1302-308.
18. Borovlkova LV, Lvanova S, Zhang MH, et.al. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin [J]. Nature 2000;405(6785):458-462.
19. Chesuntt AN, Matthay MA, Tibayan FA,et al. Early detection of type III procollagen peptide in acute lung injury. Pathogenetic and prognostic significance [J]. Am J Respir Crit Care Med 1997; 156(3Ptl):840-845.
20. Tremblay LN, Miatto D, Hamid Q, et al. Injurious ventilation induces widespread pulmonary epithelial expression of tumor necrosis factor-alpha and interlenkin-6 messenger RNA [J]. Crit Care Med 2002; 30(8):1693-1700.
21.Roajas M, Woods CR, Mora AL. Endotoxin-induced lung injury in mice:structural, functional, and biochemical responses [J]. Am J Physiol Lung Cell Mol Physiol 2005; 288(2):L333-L341.
22. Kabir K, Gelinas JP, Chen DF, et al. Characterization of a murine model of endotoxin-induced acute lung injury [J]. Shock 2002; 17(4):300-303.
23. Shenkar R, Abraham E. Mechanisms of lung neutrophil activation after hemorrhage or endotoxemia:roles of reactive oxygen intermediates, NF-κB, and cyclic AMP response element binding protein [J]. J Immunol 1999; 163(2):954-962.
24. Carter AB, Monick MM, Hunninghake GW. Lipopolysaccharide-induced NF-κB Activation and Cytokine Release in Human Alveolar Macrophages Is PKC-independent and TK- and PC-PLC-dependent [J]. Am. J. Respir. Cell Mol. Biol.1998; 18(3): 384-391.
25. Zhao MQ, Fernandez LG, Doctor A, et al. Alveolar macrophage activation is a key initiation signal for acute lung ischemia-reperfusion injury [J]. Am J Physiol Lung Cell Mol Physiol.2006; 291(5):1018-1026.
26. GJ B. The pulmonary physician in critical care*6:The pathogenesis of ALI/ARDS [J]. Thorax 2002;57:540-46.
27. KH A. Histopathology of Pulmonary Edema and the Acute Respiratory Distress Syndrome:Pulmonary Edema [J]. New York:Marcel Dekker 1998:37-8.
28. Hogg JC DC. Leukocyte traffic in the lung [J].Amu Rev Physiol.1995;57:97-114.
29. Claude A PM, David A, et al. The Acute Respiratory Distress Syndrome [J]. Ann Intern .2004;141:460-70.
1 Bernard GR, Artigas A, Brigham KL, et al. The American-European Consensus Conferenceon ARDS:Definitions, mechanisms, relevantoutcomes, and clinical trial coordination[J]. Am J Respir Crit Care Med,1994,149:818-24.
2 Rubenfeld GD, Caldwel IE, PeabodyE, et al. Incidence and outcomes of acute lung injury[J]. N Engl J Med,2005,353:1685-93.
3 Ware LB, Matthay MA. The acute respiratory distress syndrome [J]. N Engl J Med, 2000,342:1334-49.
4 Hudson LD, Milberg JA, AnardiD, et al. Clinical risks for development of the acute respiratory distress syndrome[J]. Am J Respir Crit Care Med,1995,151:293-301.
5 Demling RH. Adult respiratory distress syndrome:current concepts[J]. Crit Care Med,1993,1:388-401.
6 Bellingan GJ. The pulmonary physician in critical care*6:The pathogenesis of ALI /ARDS[J]. Thorax,2002,57:540-46.
7 Lindauer ML, Wong J, Iwakura Y, et al. Pulmonary inflammation triggered by ricin toxin requires macrophages and IL-1 signaling[J]. J Immunol,2009,183(2):1419-26.
8 Zimmerman GA, Albertine KH, Carveth HJ, et al. Endothelial activation in ARDS[J]. Chest,1999,116:18-24.
9 Feletou and P.M. Vanhoutte. Endothelial dysfunction:a multifaceted disorder[J]. Am. J. Physiol. Heart Circ. Physiol.2006,291:H985-H1002.
10 T. Into, Y. Kanno, J. Dohkan,et al. Pathogen recognition by Toll-like receptor 2 activates Weibel-Palade body exocytosis in human aortic endothelial cells[J]. J. Biol. Chem,2007,282:8134-8141.
11 Maniatis NA, Kotanidou A, Catravas JD, et al. Endothelial pathomechanisms in acute lung injury[J]. Vascular Pharmacology,2008,49:119-133.
12 Claude A, Piantadosi MD, David A, et al. The Acute Respiratory Distress Syndrome[J]. Ann Intern Med,2004,141:460-70.
13 Fung YL, Silliman CC. The role of neutrophils in the pathogenesis of transfusion-related acute lung injury [J]. Transfus Med Rev,2009,23(4):266-83.
14 Looney MR, Nguyen JX, Hu Y, et al. Platelet depletion and aspirin treatment protect mice in a two-event model of transfusion-related acute lung injury [J]. J Clin Invest, 2009,119(11):3450-61.
15 Abraham E. Neutrophils and acute lung injury[J]. Crit Care Med,2003,31:S195-S9.
16 Matthay MA, Zimmerman GA. Acute Lung Injury and the Acute Respiratory Distress Syndrome[J]. Am J RespirCell Mol Biol,2005,33:319-27.
17 Kotani M, Kotani T, Ishizaka A, et al. Neutrophil depletion attenuates interleukin-8 production in mild-over stretch ventilated normal rabbit lung[J]. Crit Care Med,2004, 32:514-19.
18 Ginsburg I. The role of bacteriolysis in the pathophysiology of inflammation, infection and post -infectious sequelae[J]. APMIS,2002,110:753-70.
19 Power C, Wang JH, Sookhai S, et al. Proinflammatory effects of bacterial lipoprotein on human neutrophil activation status, function and cytotoxic potential in vitro [J]. Shock,2001,15:461-66.
20 Welsh CH, Lien DC. Endotoxin-pretreated neutrophils increase pulmonary vascular permeability in dogs[J].Appl Physiol,1989,66:112-19.
21 Brigham KL. Mechanisms of lung injury[J]. Clin Chest Med,1982,3:9-24.
22 Tasaka S, Amaya F, Hashimoto S, et al. Roles of oxidants and redox signaling in the pathogenesis of acute respiratory distress syndrome [J]. Antioxid Redox Signal,2008, 10(4):739-53.
23刘勇.中性粒细胞蛋白酶及其抑制剂与体外循环肺损伤[J].实用医学杂志,2006,22(20)：2443-45.
24 Thickett DR, Armstrong L, Christie SJ, et al. Vascular endothelial growth factor may contribute to increased vascular permeability in acute respiratory distress syndrome[J]. Am J Respir Crit Care Med,2001,164:1601-605.
25 Rosseau S, Hammer IP, Maus U, et al. Phenotypic characterization of alveolar monocyte recruitment in acute respiratory distress syndrome[J]. Physiol Lung Cell Mol Physiol,2000,279.
26 Zimmerman GA, Albertine KH, McIntyre TM, et al. Pathogenesis of sepsis and septic-induced lung inury in Acute respiratory distress syndrome. New York:Marcel Dekker,2003:245-87.
27 Fingan JH. The coagulation system and pulmonary endothlial function in acute lung injury[J]. Microvas Res,2009,77:35-38.
28温德良,刘卫江,谢长江,等.急性呼吸窘迫综合征患者凝血纤溶系统变化的临床研究[J].血栓与止血学,2008,14(3)：113-115.
29 Hoffmann JN, Vollmar B, Laschke MW, et al. Microhemodynamic and cellular mechanisms of activated protein C action during endotoxemia[J]. Crit Care Med,2004, 32:1011-1017.
30 Husari AW, Khayat A, Awdeh H, et al. Activated Protein C Attenuates Acute Lung Injury and Apoptosis in a Hyperoxic Animal Model[J]. Shock,2009; [Epub ahead of print].
31 Shi P, Chi HE. Acute lung injury/acute respiratory distress syndrome (ALI/ARDS):the mechanism, present strategies and future perspectives of therapies [J]. J Zhejiang Univ Sci B,2007,8:60-9.
32 Robert D, Kailash C, et al. Adult respiratory distress syndrome:A disorder in need of improved outcome [J]. Heart & Lung,1997,26:3-14.
33 Putensen C, Theuerkauf N, Zinserling J,et al. Meta-analysis:ventilation strategies and outcomes of the acute respiratory distress syndrome and acute lung injury [J]. Ann Intern Med,2009,151(8):566-76.
34 Amato MBP, Barbas CSV, Medeiros DM, et al. Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome [J]. N Engl J Med,1998, 338:347-57.
35 The Acute Respiratory Distress Syndrome Network. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome[J]. N Engl J Med,2000,342:1302-308.
36 Network. TNHLaBIACT. Higher versus lower positive end-expiratory pressure inpatients with the acute respiratory distress syndrome[J]. N Engl J Med,2004, 351:327-36.
37 Bollen CW, van Well GT, Sherry T, et al. High frequency oscillatory ventilation compared with conventional mechanical ventilation in adult respiratory distress syndrome:a randomized control trail[J]. Crit Care Med,2005,9:R430-R9.
38 Guerin C, Gaillard S, Lemasson S, et al. Effects of systematic prone positioning in hypoxemic acute respiratory failure:a randomized controlled trial[J]. JAMA,2004, 292:2397-387.
39邵敏,刘宝,王锦,等.肺复张策略在全麻术后发生急性呼吸窘迫综合征肥胖患者中的应用[J].中国急救医学,2009,29(2)：148-151.
40 Meduri GU, Golden E, Freire AX, et al. Methylprednisoloneinfusion in early severe ARDS:results of a randomized controlled trial [J]. Chest,2007,131:954-63.
41 Steinberg KP, Hudson LD, Goodman RB, et al. National Heart, Lung, and Blood Institute Acute Respiratory Distress Syndrome (ARDS) Clinical Trials Network. Efficacy and safety of corticosteroids for persistent acute respiratory distress syndrome[J]. N Engl J Med,2006,354:1671-84.
42 Peter JV, John P, Graham PL, et al. Corticosteroids in the prevention and treatment of acute respiratory distress syndrome (ARDS) in adults:meta-analysis[J]. BMJ,2008, 336:1006-9.
43 Bernard G, Vincent JL, Laterre PF, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis[J]. N Engl J Med,2001, 344:699-709.
44 Kotanidou A, Loutrati H, Papadomichelakis E, et al. Inhaled activated protein C attenuates lung injury induced by aerosolized endotoxin in mice[J]. Vascular pharmocology,2006,45:134-140.
2. Wheeler AP, Bernard GR. Acute lung injury and the acute respiratory distress syndrome: a clinical review [J]. Lancet:2007.369(9572):1553-1564.
3. Fujiwara N, Kobayashi K. Macrophages in inflammation [J]. Curr Drug Targets Inflamm Allergy2005; 4(3):281-286.
4. Dixit VM, Green S, Sarma V, et al. Tumor necrosis factor-alpha induction of novel gene products in human endothelial cells including a macrophage-specific chemotaxin [J]. J Biol Chem.1990; 265(5):2973-2978.
5. Coornaert B,Carpentier I,Beyaert R.A20:Central gatekeeper in inflammation and immunity [J]. J Biol Chem.2009; 284(13):8217-8221.
6. Vereecke L, Sze M, Me Guire. Enterocyte-specific A20 deficiency sensitizes to tumor necrosis factor-induced toxicity and experimental colitis [J]. J Exp Med.2010; 207(7):1513-1523.
7. Vereecke L, Beyaert R, van Loo G. Genetic relationships between A20/TNFAIP3, chronic inflammation and autoimmune disease [J]. Biochem Soc Trans.2011; 39(4):1086-1091.
8. Musone SL, Taylor KE, Lu TT, et al. Multiple polymorphisms in the TNFAIP3 region are independently associated with systemic lupus erythematosus [J]. Nat Genet.2008; 40(9):1062-1064.
9. Fan Y, Tao JH, Zhang LP, et al. The association between BANK1 and TNFAIP3 gene polymorphisms and systemic lupus erythematosus:a meta-analysis [J]. Int J Immunogenet.2011; 38(2):151-159.
10. Hou CL, Zhang W, Wei Y, et al. Zinc finger protein A20 overexpression inhibits monocyte homing and protects endothelial cells from injury induced by high glucose [J]. Genet Mol Res.2011; 10(2):1050-1059.
11. Kang NI, Yoon HY, Lee YR, et al. A20 attenuates allergic airway inflammation in mice [J]. J Immunol.2009; 183(2):1488-1495.
12. Gon Y, Asai Y, Hashimoto S, et al. A20 inhibits toll-like receptor 2-and 4-mediated interleukin-8 synthesis in airway epithelial cells [J]. Am J Respir Cell Mol Biol.2004; 31(3):330-336.
13. Verstrepen L, Verhelst K, van Loo G, et al. Expression, biological activities and mechanisms of action of A20 (TNFAIP3) [J]. Biochem Pharmacol.2010; 80(12):2009-2020.
14. Harhaj EW, Dixit VM. Deubiquitinases in the regulation of NF-κB signaling [J]. Cell Res.2011; 21(1):22-39.
15. Vereecke L, Beyaert R, van Loo G. The ubiquitin-editing enzyme A20(TNFAIP3) is a central regulator of immunopathology [J]. Trends Immunol. 2009; 30(8):383-391.
16. Amato MBP BC, Medeiros DM, et al. Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome [J]. N Engl J Med 1998;338(6):347-57.
17. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome.The Acute Respiratory Distress Syndrome Network [J]. N Engl J Med 2000;342(18):1302-308.
18. Borovlkova LV, Lvanova S, Zhang MH, et.al. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin [J]. Nature 2000;405(6785):458-462.
19. Chesuntt AN, Matthay MA, Tibayan FA,et al. Early detection of type III procollagen peptide in acute lung injury. Pathogenetic and prognostic significance [J]. Am J Respir Crit Care Med 1997; 156(3Ptl):840-845.
20. Tremblay LN, Miatto D, Hamid Q, et al. Injurious ventilation induces widespread pulmonary epithelial expression of tumor necrosis factor-alpha and interlenkin-6 messenger RNA [J]. Crit Care Med 2002; 30(8):1693-1700.
21.Roajas M, Woods CR, Mora AL. Endotoxin-induced lung injury in mice:structural, functional, and biochemical responses [J]. Am J Physiol Lung Cell Mol Physiol 2005; 288(2):L333-L341.
22. Kabir K, Gelinas JP, Chen DF, et al. Characterization of a murine model of endotoxin-induced acute lung injury [J]. Shock 2002; 17(4):300-303.
23. Shenkar R, Abraham E. Mechanisms of lung neutrophil activation after hemorrhage or endotoxemia:roles of reactive oxygen intermediates, NF-κB, and cyclic AMP response element binding protein [J]. J Immunol 1999; 163(2):954-962.
24. Carter AB, Monick MM, Hunninghake GW. Lipopolysaccharide-induced NF-κB Activation and Cytokine Release in Human Alveolar Macrophages Is PKC-independent and TK- and PC-PLC-dependent [J]. Am. J. Respir. Cell Mol. Biol.1998; 18(3): 384-391.
25. Zhao MQ, Fernandez LG, Doctor A, et al. Alveolar macrophage activation is a key initiation signal for acute lung ischemia-reperfusion injury [J]. Am J Physiol Lung Cell Mol Physiol.2006; 291(5):1018-1026.
26. GJ B. The pulmonary physician in critical care*6:The pathogenesis of ALI/ARDS [J]. Thorax 2002;57:540-46.
27. KH A. Histopathology of Pulmonary Edema and the Acute Respiratory Distress Syndrome:Pulmonary Edema [J]. New York:Marcel Dekker 1998:37-8.
28. Hogg JC DC. Leukocyte traffic in the lung [J].Amu Rev Physiol.1995;57:97-114.
29. Claude A PM, David A, et al. The Acute Respiratory Distress Syndrome [J]. Ann Intern .2004;141:460-70.
1 Bernard GR, Artigas A, Brigham KL, et al. The American-European Consensus Conferenceon ARDS:Definitions, mechanisms, relevantoutcomes, and clinical trial coordination[J]. Am J Respir Crit Care Med,1994,149:818-24.
2 Rubenfeld GD, Caldwel IE, PeabodyE, et al. Incidence and outcomes of acute lung injury[J]. N Engl J Med,2005,353:1685-93.
3 Ware LB, Matthay MA. The acute respiratory distress syndrome [J]. N Engl J Med, 2000,342:1334-49.
4 Hudson LD, Milberg JA, AnardiD, et al. Clinical risks for development of the acute respiratory distress syndrome[J]. Am J Respir Crit Care Med,1995,151:293-301.
5 Demling RH. Adult respiratory distress syndrome:current concepts[J]. Crit Care Med,1993,1:388-401.
6 Bellingan GJ. The pulmonary physician in critical care*6:The pathogenesis of ALI /ARDS[J]. Thorax,2002,57:540-46.
7 Lindauer ML, Wong J, Iwakura Y, et al. Pulmonary inflammation triggered by ricin toxin requires macrophages and IL-1 signaling[J]. J Immunol,2009,183(2):1419-26.
8 Zimmerman GA, Albertine KH, Carveth HJ, et al. Endothelial activation in ARDS[J]. Chest,1999,116:18-24.
9 Feletou and P.M. Vanhoutte. Endothelial dysfunction:a multifaceted disorder[J]. Am. J. Physiol. Heart Circ. Physiol.2006,291:H985-H1002.
10 T. Into, Y. Kanno, J. Dohkan,et al. Pathogen recognition by Toll-like receptor 2 activates Weibel-Palade body exocytosis in human aortic endothelial cells[J]. J. Biol. Chem,2007,282:8134-8141.
11 Maniatis NA, Kotanidou A, Catravas JD, et al. Endothelial pathomechanisms in acute lung injury[J]. Vascular Pharmacology,2008,49:119-133.
12 Claude A, Piantadosi MD, David A, et al. The Acute Respiratory Distress Syndrome[J]. Ann Intern Med,2004,141:460-70.
13 Fung YL, Silliman CC. The role of neutrophils in the pathogenesis of transfusion-related acute lung injury [J]. Transfus Med Rev,2009,23(4):266-83.
14 Looney MR, Nguyen JX, Hu Y, et al. Platelet depletion and aspirin treatment protect mice in a two-event model of transfusion-related acute lung injury [J]. J Clin Invest, 2009,119(11):3450-61.
15 Abraham E. Neutrophils and acute lung injury[J]. Crit Care Med,2003,31:S195-S9.
16 Matthay MA, Zimmerman GA. Acute Lung Injury and the Acute Respiratory Distress Syndrome[J]. Am J RespirCell Mol Biol,2005,33:319-27.
17 Kotani M, Kotani T, Ishizaka A, et al. Neutrophil depletion attenuates interleukin-8 production in mild-over stretch ventilated normal rabbit lung[J]. Crit Care Med,2004, 32:514-19.
18 Ginsburg I. The role of bacteriolysis in the pathophysiology of inflammation, infection and post -infectious sequelae[J]. APMIS,2002,110:753-70.
19 Power C, Wang JH, Sookhai S, et al. Proinflammatory effects of bacterial lipoprotein on human neutrophil activation status, function and cytotoxic potential in vitro [J]. Shock,2001,15:461-66.
20 Welsh CH, Lien DC. Endotoxin-pretreated neutrophils increase pulmonary vascular permeability in dogs[J].Appl Physiol,1989,66:112-19.
21 Brigham KL. Mechanisms of lung injury[J]. Clin Chest Med,1982,3:9-24.
22 Tasaka S, Amaya F, Hashimoto S, et al. Roles of oxidants and redox signaling in the pathogenesis of acute respiratory distress syndrome [J]. Antioxid Redox Signal,2008, 10(4):739-53.
23刘勇.中性粒细胞蛋白酶及其抑制剂与体外循环肺损伤[J].实用医学杂志,2006,22(20)：2443-45.
24 Thickett DR, Armstrong L, Christie SJ, et al. Vascular endothelial growth factor may contribute to increased vascular permeability in acute respiratory distress syndrome[J]. Am J Respir Crit Care Med,2001,164:1601-605.
25 Rosseau S, Hammer IP, Maus U, et al. Phenotypic characterization of alveolar monocyte recruitment in acute respiratory distress syndrome[J]. Physiol Lung Cell Mol Physiol,2000,279.
26 Zimmerman GA, Albertine KH, McIntyre TM, et al. Pathogenesis of sepsis and septic-induced lung inury in Acute respiratory distress syndrome. New York:Marcel Dekker,2003:245-87.
27 Fingan JH. The coagulation system and pulmonary endothlial function in acute lung injury[J]. Microvas Res,2009,77:35-38.
28温德良,刘卫江,谢长江,等.急性呼吸窘迫综合征患者凝血纤溶系统变化的临床研究[J].血栓与止血学,2008,14(3)：113-115.
29 Hoffmann JN, Vollmar B, Laschke MW, et al. Microhemodynamic and cellular mechanisms of activated protein C action during endotoxemia[J]. Crit Care Med,2004, 32:1011-1017.
30 Husari AW, Khayat A, Awdeh H, et al. Activated Protein C Attenuates Acute Lung Injury and Apoptosis in a Hyperoxic Animal Model[J]. Shock,2009; [Epub ahead of print].
31 Shi P, Chi HE. Acute lung injury/acute respiratory distress syndrome (ALI/ARDS):the mechanism, present strategies and future perspectives of therapies [J]. J Zhejiang Univ Sci B,2007,8:60-9.
32 Robert D, Kailash C, et al. Adult respiratory distress syndrome:A disorder in need of improved outcome [J]. Heart & Lung,1997,26:3-14.
33 Putensen C, Theuerkauf N, Zinserling J,et al. Meta-analysis:ventilation strategies and outcomes of the acute respiratory distress syndrome and acute lung injury [J]. Ann Intern Med,2009,151(8):566-76.
34 Amato MBP, Barbas CSV, Medeiros DM, et al. Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome [J]. N Engl J Med,1998, 338:347-57.
35 The Acute Respiratory Distress Syndrome Network. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome[J]. N Engl J Med,2000,342:1302-308.
36 Network. TNHLaBIACT. Higher versus lower positive end-expiratory pressure inpatients with the acute respiratory distress syndrome[J]. N Engl J Med,2004, 351:327-36.
37 Bollen CW, van Well GT, Sherry T, et al. High frequency oscillatory ventilation compared with conventional mechanical ventilation in adult respiratory distress syndrome:a randomized control trail[J]. Crit Care Med,2005,9:R430-R9.
38 Guerin C, Gaillard S, Lemasson S, et al. Effects of systematic prone positioning in hypoxemic acute respiratory failure:a randomized controlled trial[J]. JAMA,2004, 292:2397-387.
39邵敏,刘宝,王锦,等.肺复张策略在全麻术后发生急性呼吸窘迫综合征肥胖患者中的应用[J].中国急救医学,2009,29(2)：148-151.
40 Meduri GU, Golden E, Freire AX, et al. Methylprednisoloneinfusion in early severe ARDS:results of a randomized controlled trial [J]. Chest,2007,131:954-63.
41 Steinberg KP, Hudson LD, Goodman RB, et al. National Heart, Lung, and Blood Institute Acute Respiratory Distress Syndrome (ARDS) Clinical Trials Network. Efficacy and safety of corticosteroids for persistent acute respiratory distress syndrome[J]. N Engl J Med,2006,354:1671-84.
42 Peter JV, John P, Graham PL, et al. Corticosteroids in the prevention and treatment of acute respiratory distress syndrome (ARDS) in adults:meta-analysis[J]. BMJ,2008, 336:1006-9.
43 Bernard G, Vincent JL, Laterre PF, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis[J]. N Engl J Med,2001, 344:699-709.
44 Kotanidou A, Loutrati H, Papadomichelakis E, et al. Inhaled activated protein C attenuates lung injury induced by aerosolized endotoxin in mice[J]. Vascular pharmocology,2006,45:134-140.
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