杀菌/中和内毒素多肽（BNEP）对小鼠内毒素性急性肺损伤的保护作用
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摘要
目的:革兰氏阴性菌外膜内毒素(LPS)是引起严重创伤、大面积烧伤患者并发全身炎症反应综合征(SIRS)、脓毒症以及多器官功能障碍综合征(MODS)的重要因素。目前尚无有效的LPS拮抗剂应用于临床,已经发现杀菌性/通透性增加蛋白(BPI)对革兰氏阴性菌具有杀菌及中和内毒素的作用。笔者所在单位根据BPI的分子结构,设计并合成出了具有BPI生物学活性的模拟肽BNEP。体内外实验表明BNEP能够中和内毒素,对内毒素血症动物具有明显的保护作用。鉴于肺脏是LPS所致MODS中最易受累的器官,本实验将对BNEP在LPS所致急性肺损伤(ALI)中的保护作用进行研究。为探索可用于临床的内毒素拮抗剂提供一定的实验依据。
方法:选用BALB/C小鼠,采取经鼻滴注LPS的方式复制ALI模型,LPS滴注后取不同时相点检测小鼠肺湿重增加率、肺病理学变化,以验证动物模型是否可行、可靠。然后将小鼠随机分为对照组、LPS组、BNEP组,对照组滴注生理盐水、LPS组滴注LPS(500μg/kg体重),BNEP组滴注LPS后立即尾静脉注射BNEP(10mg/kg体重),各组小鼠处理后48h取肺检测湿干重比、肺血管通透性(肺内Evans蓝含量)、肺病理学变化:另在上述方法处理后3h再取各组小鼠用免疫组织化学的方法检测肺内LPS受体TLR2、TLR4的表达情况。对比上述检测结果,以观察BNEP对小鼠ALI的影响。
结果:LPS滴注后4h小鼠肺湿重开始增加,8h、12h、24h肺湿重呈渐增趋势,48h肺湿重增加达高峰(51.9%),随后开始下降,72h湿重仅增加14.3%;病理切片中可见小鼠肺充血、肺泡及肺间质水肿、出血、以中性粒细胞为主的炎性细胞浸润,肺泡壁增厚,肺泡内透明膜形成,这些病理改变亦随时间呈渐进性加重,在48h达到高峰。对照组小鼠在经鼻滴入生理盐水后48h,肺组织结构清晰完整,无炎症反应等ALI典型病理变化:LPS组小鼠肺病理学检查则显示上述急性炎症反应的特征,湿干重比以及肺内Evans蓝含量与对照组相比明显增加,BNEP处理后小鼠肺湿干重比减小、肺血管通透性降低、病理变化减轻,各项指标与LPS组比较有显著改善。免疫组化发现对照组小鼠肺内有少量TLR2、TLR4表达,主要存在于肺泡巨噬细胞、肺泡Ⅱ型上
Objective: Lipopolysaccharides (LPS), usually called endotoxin, is a major component of the outer leaflet of gram-negative bacterial outer membrane and has been demonstrated to be a key molecule in the pathophysiology of systemic inflammatory response syndrome (SIRS), sepsis and multiple organ dysfunction syndrome (MODS). Despite of intensive efforts focused on the treatment of sepsis mediated by LPS and various inflammatory cytokines, none of the safe and potent LPS-neutralizing agents has been applied in clinical therapy so far. Bactericidal/permeability increasing protein (BPI),a kind of acid cationic protein found in the primary granules of human and mammalian neutrophils, exerts the bactericidal activities and the effects of neutralization of LPS. An 14-aminoacid peptide, named bactericidal/neutralizing-endotoxin peptide (BNEP), was designed and synthesized by the researchers in our institute based on the crystal structure of BPI. Many our previous experiments have demonstrated that BNEP exerted strong antibacterial and LPS-neutralizing activities in vivo and vitro. As we know, lung is one of early target organ in MODS induced by LPS, so to define the treatment role of BNEP on acute lung injury (ALI) induced by LPS is the aim of the study.Materials and methods: Healthy BALB/C mice were used to replication ALI models by intranasal instillation of LPS. At selected time points, the lungs were excised for lung weight and histopathology measurement. Pulmonary edema caused by LPS was determined by calculating the ratio increase of lung weight by use of normal lung weights (negative control) as the reference weight. All above scores were assigned to confirm the reliable of ALI induced by LPS. Then the mice were divided randomly into three groups: control, LPS and BNEP group. The control mice were administered intranasally with 60 μ l LPS-free saline, LPS group with 500ul/kg LPS. In BNEP group, each mouse received 10mg/kg BNEP via tail vein after instillation of LPS. After 48 hours, the lungs were excised for the
following measurement^) the ratio of wet lung weight Vs dry lung weight;(2) the permeability of pulmonary capillary vessel(Ewans Blue); (3) histopathology. Immunohistochemistry were used for detecting the expression of TLR2 and TLR4 in the lung tissue.Results: The wet lung weight, which indicate the lung edema, increased initially at 4h(0.8%) after LPS instillation and increase continuously at 8h, 12h and 24h, and decrease to 14.3% at 72h after reached peak at 48h(51.9%). The histopathology results showed obviously lung hyperemia, edema, and hemorrhage in a time manner dependent. Instillation of LPS in mice increased in inflammatory cell count, mainly neutrophils. Aggregation of inflammatory cells was found in alveolar and alveolar walls. In control group, the lung structures are integral and there were no inflammatory reaction appeared at 48h after saline administration. In LPS group, the classic histopathology of ALI was observed, include the increased ratio of wet lung weight Vs dry lung weight, and the permeability of pulmonary capillary vessel. In BNEP group, the lung injuries were attenuated significantly compared with that of LPS group. The immunohistochemistry experiments showed that there were a few of Toll-like receptor-2(TLR2) and TLR4 in the lung of control group mice, and usually major expressed in alveolar macrophage, alveolar type II epithelial cell, endothelial cell and alveolar type I epithelial cell. In LPS group, the TLR2 and TLR4 up-regulated and can also been seen on neutrophil. In BNEP group, down-regulated expression was determined significantly compared with that of LPS group.Conclusions: (1) the murine model of ALI by intranasal instillation of LPS is feasible and reliable. The time-dependently change of lung edema, Hyaline membrane forming and inflammatory cells influx into the lung tissue were obvious and familiar with pathology of classic ALI. So this ALI model is well suited for study of pathogenesis of ALI and preliminary pharmacological trial for therapeutic agents. (2) BNEP can relieve lung edema, decrease the permeability of pulmonary capillary vessel and reduce the amount of neutrophil influx into the lung tissue, which induced by LPS instillation. (3) BNEP can down-regulate the the expression of TLR2 and TLR4 in the lung tissue, which were stimulated by LPS. So, it suggested that BNEP could block the inflammatory response and protect the lung from inflammatory reaction-induced damage. Based on the previous testimony and these research results, BNEP, as a artificial peptide derived from BPI, would
have the possibility to develop as a new drug used for block biological activities of endotoxin.
方法:选用BALB/C小鼠,采取经鼻滴注LPS的方式复制ALI模型,LPS滴注后取不同时相点检测小鼠肺湿重增加率、肺病理学变化,以验证动物模型是否可行、可靠。然后将小鼠随机分为对照组、LPS组、BNEP组,对照组滴注生理盐水、LPS组滴注LPS(500μg/kg体重),BNEP组滴注LPS后立即尾静脉注射BNEP(10mg/kg体重),各组小鼠处理后48h取肺检测湿干重比、肺血管通透性(肺内Evans蓝含量)、肺病理学变化:另在上述方法处理后3h再取各组小鼠用免疫组织化学的方法检测肺内LPS受体TLR2、TLR4的表达情况。对比上述检测结果,以观察BNEP对小鼠ALI的影响。
结果:LPS滴注后4h小鼠肺湿重开始增加,8h、12h、24h肺湿重呈渐增趋势,48h肺湿重增加达高峰(51.9%),随后开始下降,72h湿重仅增加14.3%;病理切片中可见小鼠肺充血、肺泡及肺间质水肿、出血、以中性粒细胞为主的炎性细胞浸润,肺泡壁增厚,肺泡内透明膜形成,这些病理改变亦随时间呈渐进性加重,在48h达到高峰。对照组小鼠在经鼻滴入生理盐水后48h,肺组织结构清晰完整,无炎症反应等ALI典型病理变化:LPS组小鼠肺病理学检查则显示上述急性炎症反应的特征,湿干重比以及肺内Evans蓝含量与对照组相比明显增加,BNEP处理后小鼠肺湿干重比减小、肺血管通透性降低、病理变化减轻,各项指标与LPS组比较有显著改善。免疫组化发现对照组小鼠肺内有少量TLR2、TLR4表达,主要存在于肺泡巨噬细胞、肺泡Ⅱ型上
Objective: Lipopolysaccharides (LPS), usually called endotoxin, is a major component of the outer leaflet of gram-negative bacterial outer membrane and has been demonstrated to be a key molecule in the pathophysiology of systemic inflammatory response syndrome (SIRS), sepsis and multiple organ dysfunction syndrome (MODS). Despite of intensive efforts focused on the treatment of sepsis mediated by LPS and various inflammatory cytokines, none of the safe and potent LPS-neutralizing agents has been applied in clinical therapy so far. Bactericidal/permeability increasing protein (BPI),a kind of acid cationic protein found in the primary granules of human and mammalian neutrophils, exerts the bactericidal activities and the effects of neutralization of LPS. An 14-aminoacid peptide, named bactericidal/neutralizing-endotoxin peptide (BNEP), was designed and synthesized by the researchers in our institute based on the crystal structure of BPI. Many our previous experiments have demonstrated that BNEP exerted strong antibacterial and LPS-neutralizing activities in vivo and vitro. As we know, lung is one of early target organ in MODS induced by LPS, so to define the treatment role of BNEP on acute lung injury (ALI) induced by LPS is the aim of the study.Materials and methods: Healthy BALB/C mice were used to replication ALI models by intranasal instillation of LPS. At selected time points, the lungs were excised for lung weight and histopathology measurement. Pulmonary edema caused by LPS was determined by calculating the ratio increase of lung weight by use of normal lung weights (negative control) as the reference weight. All above scores were assigned to confirm the reliable of ALI induced by LPS. Then the mice were divided randomly into three groups: control, LPS and BNEP group. The control mice were administered intranasally with 60 μ l LPS-free saline, LPS group with 500ul/kg LPS. In BNEP group, each mouse received 10mg/kg BNEP via tail vein after instillation of LPS. After 48 hours, the lungs were excised for the
following measurement^) the ratio of wet lung weight Vs dry lung weight;(2) the permeability of pulmonary capillary vessel(Ewans Blue); (3) histopathology. Immunohistochemistry were used for detecting the expression of TLR2 and TLR4 in the lung tissue.Results: The wet lung weight, which indicate the lung edema, increased initially at 4h(0.8%) after LPS instillation and increase continuously at 8h, 12h and 24h, and decrease to 14.3% at 72h after reached peak at 48h(51.9%). The histopathology results showed obviously lung hyperemia, edema, and hemorrhage in a time manner dependent. Instillation of LPS in mice increased in inflammatory cell count, mainly neutrophils. Aggregation of inflammatory cells was found in alveolar and alveolar walls. In control group, the lung structures are integral and there were no inflammatory reaction appeared at 48h after saline administration. In LPS group, the classic histopathology of ALI was observed, include the increased ratio of wet lung weight Vs dry lung weight, and the permeability of pulmonary capillary vessel. In BNEP group, the lung injuries were attenuated significantly compared with that of LPS group. The immunohistochemistry experiments showed that there were a few of Toll-like receptor-2(TLR2) and TLR4 in the lung of control group mice, and usually major expressed in alveolar macrophage, alveolar type II epithelial cell, endothelial cell and alveolar type I epithelial cell. In LPS group, the TLR2 and TLR4 up-regulated and can also been seen on neutrophil. In BNEP group, down-regulated expression was determined significantly compared with that of LPS group.Conclusions: (1) the murine model of ALI by intranasal instillation of LPS is feasible and reliable. The time-dependently change of lung edema, Hyaline membrane forming and inflammatory cells influx into the lung tissue were obvious and familiar with pathology of classic ALI. So this ALI model is well suited for study of pathogenesis of ALI and preliminary pharmacological trial for therapeutic agents. (2) BNEP can relieve lung edema, decrease the permeability of pulmonary capillary vessel and reduce the amount of neutrophil influx into the lung tissue, which induced by LPS instillation. (3) BNEP can down-regulate the the expression of TLR2 and TLR4 in the lung tissue, which were stimulated by LPS. So, it suggested that BNEP could block the inflammatory response and protect the lung from inflammatory reaction-induced damage. Based on the previous testimony and these research results, BNEP, as a artificial peptide derived from BPI, would
have the possibility to develop as a new drug used for block biological activities of endotoxin.
引文
1. Angus DC, Linde-Zwirble WT, Lidicker J, et al. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome and associated costs of care. Crit. Care Med, 2001, 29(7): 1303-1310.
2. Caroff M, Karibian D, Cavaillon JM, et al. Structural and function analyses of bacterial lipopolysaccharides. Microbes and Infection, 2002, 4: 915-926.
3. O'Reilly M, Newcomb DE, Remick D, et al. Endotoxin, sepsis, and the primrose path. Shock, 1999, 12: 411-420.
4. Luchi M, Morrison DC, Opal S, et al. A comparative trial of imipenem versus ceftazidime in the release of endotoxin and cytokine generation in patients with Gram-negative urosepsis. J. Endotoxin. Res, 2000, 6(1): 25-31.
5.徐能武,袁建成,肖光夏,等.抗生素诱导革兰氏阴性菌释放内毒素的实验研究(一).中华烧伤杂志,2001,17(1):75-79.
6.徐能武,袁建成,肖光夏,等.抗生素诱导革兰氏阴性菌释放内毒素的实验研究(二).中华烧伤杂志,2002,18(1):72-76.
7. Weiss J, Elsbach P, Olsson I, et al. Purification and characterization of a potent bacteria and membranes active protein from the granules of human polymorphonuclear leukocytes. J. Bio. Chem, 1978, 253: 2664-2672.
8. Levy O. Therapeutic potential of the bactericidal/permeability increasing protein. Expert Opin Investig Drugs, 2002 11(2): 159-167.
9. Capodici C, Weiss J. Both N-and C-terminal regions of the bioactive N-terminal fragment of the neutrophil granule bactericidal/permeability-increasing protein are required for stability and function. J. Immunol, 1996, 156: 4789-4796.
10. Levin M, Quint P A, Goldstein B, et al. Recombinant bactericidal/permeability increasing protein (Rbpi21)as adjunctive treatment for children with severe meningococcal sepsis: a randomized trial. Rbpi21 Meningococcal Sepsis Study Group. Lancet, 2000, 356(9234): 961-967.
11.郑江,肖光夏.杀菌性/通透性增加蛋白功能区结构模拟肽的分子设计、筛选及鉴定.第三军医大学学报,1998,20(6):190—194.
12.郑江,周红,鲁永玲,等.杀菌性/通透性增加蛋白(BPI)模拟肽中和内毒素作用的 研究.中华烧伤杂志,2002,18(2),95.-99.
13.郑江,袁建成,周红,等.杀菌性/通透性增加蛋白(BPI)模拟肽小鼠体内拮抗内毒素作用的研究.第三军医大学学报,1998,20(6):520-523.
14.程君涛,郑江,袁建成,等.杀菌性/通透性增加蛋白模拟肽对内毒素致血管内皮细胞损伤的保护作用.中华烧伤杂志,2002,8,18(8):485-488.
15.郑江,周红,秦孝建,等.杀菌性/通透性增加蛋白模拟肽(BPI)对铜绿假单胞菌形态学的影响。中国抗生素杂志,2002,27(8):474—477.
16.郑江,龚小云,吕根法,等.杀菌性/通透性增加蛋白模拟肽与LPS/LipidA的亲和力测定。解放军医学杂志,2003,28(3):197—199.
17.肖光夏.值得重视的潜在感染途径—肠源性感染。中国胃肠外科杂志,1998,1(1):7-10.
18.肖光夏.烧伤肠源性感染.见:黎鳌主编.黎鳌烧伤学.上海:上海科学出版社,2001,74—79
19. Roderick J. Szarka, Nandi Wang, Lyle Gordon, et al. A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation. Journal of Immunological Methods, 1997, 202(1): 49-57.
20. Marianne C, Noella G, Jorome L, et al. The Selective Phosphodiesterase 4 Inhibitor RP 73-401 Reduced Matrix Metalloproteinase 9Activity and Transforming Growth Factor-β Release During Acute Lung Injury in Mice: The Role of the Balance Between Tumor Necrosis Factor-α and Interleukin-10. Pharmacology, 301(1): 258-265.
21. Katsuhisa Oshikawa, Ken Yanagisawa, et al. ST2 protein induced by inflammatory stimuli can modulate acute lung inflammation. Biochemical and Biophysical Research Communications, 2002, 299(1): 18-24.
22. Katsuhisa Oshikawa, Yukihiko Sugiyama, Gene expression of Toll-like receptors and associated molecules induced by inflammatory stimuli in the primary alveolar macrophage. Biochemical and Biophysical Research Communications, 2003, 305(3): 649-655.
23. Mitsuhiro Yamada, Hiroshi Kubo, et al. Bone Marrow-Derived Progenitor Cells Are Important for Lung Repair after Lipopolysaccharide-Induced Lung Injury. The Journal of Immunology, 2004, 172: 1266-1272.
24.钱桂生.急性肺损伤和急性呼吸窘迫综合征研究现状与展望.解放军医学杂志, 2003,28(2):97-101.
25.鄢小建,姚咏明.内毒素受体研究新进展[J].中国危重病急救医学,2002,14(6):375-377.
26. Takeda K, Kaisho T, Akira S. Toll-like receptors. Ann Rev Immunol, 2003, 21: 335-376.
27.李红云,姚咏明.内毒素的胞内信号转导途径.见蒋建新主编.细菌内毒素基础与临床.北京:人民军医出版社,2004,116-131.
28. Sunil VR, Connor AJ, Guo Y, et al. Activation of type Ⅱ alveolar epithelial cells during acute endotoxemia. Am J Physiol Lung Cell Mol Physiol, 2002, 282: 872-880.
2. Caroff M, Karibian D, Cavaillon JM, et al. Structural and function analyses of bacterial lipopolysaccharides. Microbes and Infection, 2002, 4: 915-926.
3. O'Reilly M, Newcomb DE, Remick D, et al. Endotoxin, sepsis, and the primrose path. Shock, 1999, 12: 411-420.
4. Luchi M, Morrison DC, Opal S, et al. A comparative trial of imipenem versus ceftazidime in the release of endotoxin and cytokine generation in patients with Gram-negative urosepsis. J. Endotoxin. Res, 2000, 6(1): 25-31.
5.徐能武,袁建成,肖光夏,等.抗生素诱导革兰氏阴性菌释放内毒素的实验研究(一).中华烧伤杂志,2001,17(1):75-79.
6.徐能武,袁建成,肖光夏,等.抗生素诱导革兰氏阴性菌释放内毒素的实验研究(二).中华烧伤杂志,2002,18(1):72-76.
7. Weiss J, Elsbach P, Olsson I, et al. Purification and characterization of a potent bacteria and membranes active protein from the granules of human polymorphonuclear leukocytes. J. Bio. Chem, 1978, 253: 2664-2672.
8. Levy O. Therapeutic potential of the bactericidal/permeability increasing protein. Expert Opin Investig Drugs, 2002 11(2): 159-167.
9. Capodici C, Weiss J. Both N-and C-terminal regions of the bioactive N-terminal fragment of the neutrophil granule bactericidal/permeability-increasing protein are required for stability and function. J. Immunol, 1996, 156: 4789-4796.
10. Levin M, Quint P A, Goldstein B, et al. Recombinant bactericidal/permeability increasing protein (Rbpi21)as adjunctive treatment for children with severe meningococcal sepsis: a randomized trial. Rbpi21 Meningococcal Sepsis Study Group. Lancet, 2000, 356(9234): 961-967.
11.郑江,肖光夏.杀菌性/通透性增加蛋白功能区结构模拟肽的分子设计、筛选及鉴定.第三军医大学学报,1998,20(6):190—194.
12.郑江,周红,鲁永玲,等.杀菌性/通透性增加蛋白(BPI)模拟肽中和内毒素作用的 研究.中华烧伤杂志,2002,18(2),95.-99.
13.郑江,袁建成,周红,等.杀菌性/通透性增加蛋白(BPI)模拟肽小鼠体内拮抗内毒素作用的研究.第三军医大学学报,1998,20(6):520-523.
14.程君涛,郑江,袁建成,等.杀菌性/通透性增加蛋白模拟肽对内毒素致血管内皮细胞损伤的保护作用.中华烧伤杂志,2002,8,18(8):485-488.
15.郑江,周红,秦孝建,等.杀菌性/通透性增加蛋白模拟肽(BPI)对铜绿假单胞菌形态学的影响。中国抗生素杂志,2002,27(8):474—477.
16.郑江,龚小云,吕根法,等.杀菌性/通透性增加蛋白模拟肽与LPS/LipidA的亲和力测定。解放军医学杂志,2003,28(3):197—199.
17.肖光夏.值得重视的潜在感染途径—肠源性感染。中国胃肠外科杂志,1998,1(1):7-10.
18.肖光夏.烧伤肠源性感染.见:黎鳌主编.黎鳌烧伤学.上海:上海科学出版社,2001,74—79
19. Roderick J. Szarka, Nandi Wang, Lyle Gordon, et al. A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation. Journal of Immunological Methods, 1997, 202(1): 49-57.
20. Marianne C, Noella G, Jorome L, et al. The Selective Phosphodiesterase 4 Inhibitor RP 73-401 Reduced Matrix Metalloproteinase 9Activity and Transforming Growth Factor-β Release During Acute Lung Injury in Mice: The Role of the Balance Between Tumor Necrosis Factor-α and Interleukin-10. Pharmacology, 301(1): 258-265.
21. Katsuhisa Oshikawa, Ken Yanagisawa, et al. ST2 protein induced by inflammatory stimuli can modulate acute lung inflammation. Biochemical and Biophysical Research Communications, 2002, 299(1): 18-24.
22. Katsuhisa Oshikawa, Yukihiko Sugiyama, Gene expression of Toll-like receptors and associated molecules induced by inflammatory stimuli in the primary alveolar macrophage. Biochemical and Biophysical Research Communications, 2003, 305(3): 649-655.
23. Mitsuhiro Yamada, Hiroshi Kubo, et al. Bone Marrow-Derived Progenitor Cells Are Important for Lung Repair after Lipopolysaccharide-Induced Lung Injury. The Journal of Immunology, 2004, 172: 1266-1272.
24.钱桂生.急性肺损伤和急性呼吸窘迫综合征研究现状与展望.解放军医学杂志, 2003,28(2):97-101.
25.鄢小建,姚咏明.内毒素受体研究新进展[J].中国危重病急救医学,2002,14(6):375-377.
26. Takeda K, Kaisho T, Akira S. Toll-like receptors. Ann Rev Immunol, 2003, 21: 335-376.
27.李红云,姚咏明.内毒素的胞内信号转导途径.见蒋建新主编.细菌内毒素基础与临床.北京:人民军医出版社,2004,116-131.
28. Sunil VR, Connor AJ, Guo Y, et al. Activation of type Ⅱ alveolar epithelial cells during acute endotoxemia. Am J Physiol Lung Cell Mol Physiol, 2002, 282: 872-880.