大鼠慢性阻塞性肺疾病中磷酸二酯酶4活性的变化及其选择性抑制剂的作用
摘要
研究背景
慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)是具有气流受限不完全可逆特征并呈进行性发展的肺部慢性炎症性疾病。其主要病因为吸烟和空气污染。COPD由于患病人数多,死亡率高,社会经济负担重,已成为一个重要的公共卫生问题。在美国,COPD居当前慢性疾病死亡原因的第4位。近年来COPD在我国也日益引起重视,已成为严重危害我国人民健康的重要慢性呼吸系统疾病。COPD涉及小气道和肺实质的慢性炎症,而中性粒细胞,巨噬细胞,CD8+的T淋巴细胞参与了炎症过程。但是,现在还没有特效药物能遏制COPD病情的进行性进展,目前临床上常把治疗哮喘的药物用于治疗COPD,疗效不尽理想。PDE4是嗜中性粒细胞、CD8+T淋巴细胞和巨噬细胞上表达的优势PDE类型,所以其抑制剂对控制COPD的炎症反应可能有效。另外,在一些临床研究中也发现PDE4抑制剂能够有效地抑制COPD肺部炎症,比如:第二代选择性PDE4抑制剂西洛司特在对治疗中至重度COPD患者的为期6周的研究中取得了很好的临床效果,从气道组织的活检标本中可反映出其抗炎效果。
磷酸二酯酶(Phosphodiesterase,PDE)是特异的cAMP和cGMP水解酶,能将细胞内的cAMP和cGMP水解生成相应的无活性的单核苷酸,因此PDE及其抑制剂可通过调节细胞内cAMP和cGMP水平从而影响生物体的各种代谢功能。PDE是一个庞大的同工酶超家族,现已确定的同工酶达11种,共30余种。PDE4
BackgroundChronic obstructive pulmonary disease (COPD) is a chronic inflammatory progressive disease in which airflow limitation is reversible incompletely. Phosphodiesterase 4 (PDE4), the specific of cAMP and cGMP hydrolase, can hydrolyze cAMP and cGMP in cell into unactive mononucleotide. Therefore PDE and its inhibitor could have effects on metabolism of organies by regulating the intracellular level of cAMP and cGMP. PDE4 is the main regulator of metabolism of cAMP. In addition, it is the main PDE isoenzyme in inflammatory and immunologic cells and the main PDE isoenzyme in lung tissue. PDE4 is the main type of PDEs located in neutrophil, CD8+ lynphocyte and macrophage, therefore the PDE4 inhibitors could be effective in controling the inflammatory reaction of COPD.The key to the question that how the PDE4 activity changes in COPD is uncertain. The antiinflammatory effect of PDE4 inhibitors is generally accepted, but we should do more research to study their effects on other pathophysicologic changes of COPD. MethodsMale SD rats were divided into 6 groups: control group, model group, RP001 1 mg/kg group, RP001 3 mg/kg group, RP001 10 mg/kg group and DXM group. We established the COPD rat model by exposure to cigarette smoking and intratracheal instillation of lipopolysaccharide, which lasted 3 monthes (90d). The rats have
received drug interventions from the 15th day of the experimental period. The rats in control group didn' receive any intervention. We observed dynamic changes of body weight and measured airway resistance, dynamic lung compliance. The lower of left lungs were stained with hematoxylin and eosin after fixed with 10% formalin. We used light microscope to observe pathologic changes. The PDE4 activity in lung homogenate were measured by HPLC. The activity of SOD and the amount of MDA , two common indexes in estimating oxidation and antioxidation functions of lung, were measured. Results:1. In comparison with control group, the body weight of rats in model group decreased significantly since the 3th week to the 12th week. In comparison with model group, the body weight in RPOOl lmg/kg group increased significantly in the 3th, 4th and 6th week. Meanwhile, in RPOOl 3mg/kg group the significant difference was in the 3th, 4th, 6th, 7th, 8th, 9th, 11th and 12th week. In RPOOl lOmg/kg group the body weight increased significantly from the 3th week to the end, compared with model group. There was no significant diffrernce between DXM group and model group all of the progression.2. In model group, the airway resistance increased significantly and the dynamic lung compliance decreased significantly. All of the drug intervention groups showed amelioration for the changes of airway resistances and dynamic lung compliance.3. In model group, we observed that columnar epithelial cells in bronchuses had partially fallen off. Intensive hyperplasia of goblet cells and muscular layer under mucous membrane in bronchuses and serious infiltration of inflammatory cells underneath mucous membrane were also observed. Thinness, swelling, disruption, destruction of alveolar wall and elastic net, formation of pneumatocele in lungs, which were significant characteristics in COPD, were observed. The pathologic changes of drug intervention groups ameliorated in some extent.4. The activity of PDE4 in model group increased significantly. The activity of PDE4 in all the drug intervention groups, especially in RPOOl lOmg/kg group and DXM group, displayed significant decreases compared with model group.
5. The activity of SOD in lung of model group decreased significantly. While the amount of MDA in lung of model group increased significantly. But the two datas ameliorated significantly only in RPOOl 10 mg/kg group in comparison with model group. Conclusions1. The COPD rat model could be established by chronic exposure to cigarette smoking and intratracheal instillation of lipopolysaccharide.2. PDE4 activity in lung increases significantly during the development of COPD in rat. Body weight, pulmonary function, the balance between oxidation and antioxidation and pathologic form of bronchus and lung have significant changes.3. Selective PDE4 inhibitor RPOOl exhibits a significant amelioration for pathophysiologic changes of COPD rat model by reducing PDE4 activity in lung.
慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)是具有气流受限不完全可逆特征并呈进行性发展的肺部慢性炎症性疾病。其主要病因为吸烟和空气污染。COPD由于患病人数多,死亡率高,社会经济负担重,已成为一个重要的公共卫生问题。在美国,COPD居当前慢性疾病死亡原因的第4位。近年来COPD在我国也日益引起重视,已成为严重危害我国人民健康的重要慢性呼吸系统疾病。COPD涉及小气道和肺实质的慢性炎症,而中性粒细胞,巨噬细胞,CD8+的T淋巴细胞参与了炎症过程。但是,现在还没有特效药物能遏制COPD病情的进行性进展,目前临床上常把治疗哮喘的药物用于治疗COPD,疗效不尽理想。PDE4是嗜中性粒细胞、CD8+T淋巴细胞和巨噬细胞上表达的优势PDE类型,所以其抑制剂对控制COPD的炎症反应可能有效。另外,在一些临床研究中也发现PDE4抑制剂能够有效地抑制COPD肺部炎症,比如:第二代选择性PDE4抑制剂西洛司特在对治疗中至重度COPD患者的为期6周的研究中取得了很好的临床效果,从气道组织的活检标本中可反映出其抗炎效果。
磷酸二酯酶(Phosphodiesterase,PDE)是特异的cAMP和cGMP水解酶,能将细胞内的cAMP和cGMP水解生成相应的无活性的单核苷酸,因此PDE及其抑制剂可通过调节细胞内cAMP和cGMP水平从而影响生物体的各种代谢功能。PDE是一个庞大的同工酶超家族,现已确定的同工酶达11种,共30余种。PDE4
BackgroundChronic obstructive pulmonary disease (COPD) is a chronic inflammatory progressive disease in which airflow limitation is reversible incompletely. Phosphodiesterase 4 (PDE4), the specific of cAMP and cGMP hydrolase, can hydrolyze cAMP and cGMP in cell into unactive mononucleotide. Therefore PDE and its inhibitor could have effects on metabolism of organies by regulating the intracellular level of cAMP and cGMP. PDE4 is the main regulator of metabolism of cAMP. In addition, it is the main PDE isoenzyme in inflammatory and immunologic cells and the main PDE isoenzyme in lung tissue. PDE4 is the main type of PDEs located in neutrophil, CD8+ lynphocyte and macrophage, therefore the PDE4 inhibitors could be effective in controling the inflammatory reaction of COPD.The key to the question that how the PDE4 activity changes in COPD is uncertain. The antiinflammatory effect of PDE4 inhibitors is generally accepted, but we should do more research to study their effects on other pathophysicologic changes of COPD. MethodsMale SD rats were divided into 6 groups: control group, model group, RP001 1 mg/kg group, RP001 3 mg/kg group, RP001 10 mg/kg group and DXM group. We established the COPD rat model by exposure to cigarette smoking and intratracheal instillation of lipopolysaccharide, which lasted 3 monthes (90d). The rats have
received drug interventions from the 15th day of the experimental period. The rats in control group didn' receive any intervention. We observed dynamic changes of body weight and measured airway resistance, dynamic lung compliance. The lower of left lungs were stained with hematoxylin and eosin after fixed with 10% formalin. We used light microscope to observe pathologic changes. The PDE4 activity in lung homogenate were measured by HPLC. The activity of SOD and the amount of MDA , two common indexes in estimating oxidation and antioxidation functions of lung, were measured. Results:1. In comparison with control group, the body weight of rats in model group decreased significantly since the 3th week to the 12th week. In comparison with model group, the body weight in RPOOl lmg/kg group increased significantly in the 3th, 4th and 6th week. Meanwhile, in RPOOl 3mg/kg group the significant difference was in the 3th, 4th, 6th, 7th, 8th, 9th, 11th and 12th week. In RPOOl lOmg/kg group the body weight increased significantly from the 3th week to the end, compared with model group. There was no significant diffrernce between DXM group and model group all of the progression.2. In model group, the airway resistance increased significantly and the dynamic lung compliance decreased significantly. All of the drug intervention groups showed amelioration for the changes of airway resistances and dynamic lung compliance.3. In model group, we observed that columnar epithelial cells in bronchuses had partially fallen off. Intensive hyperplasia of goblet cells and muscular layer under mucous membrane in bronchuses and serious infiltration of inflammatory cells underneath mucous membrane were also observed. Thinness, swelling, disruption, destruction of alveolar wall and elastic net, formation of pneumatocele in lungs, which were significant characteristics in COPD, were observed. The pathologic changes of drug intervention groups ameliorated in some extent.4. The activity of PDE4 in model group increased significantly. The activity of PDE4 in all the drug intervention groups, especially in RPOOl lOmg/kg group and DXM group, displayed significant decreases compared with model group.
5. The activity of SOD in lung of model group decreased significantly. While the amount of MDA in lung of model group increased significantly. But the two datas ameliorated significantly only in RPOOl 10 mg/kg group in comparison with model group. Conclusions1. The COPD rat model could be established by chronic exposure to cigarette smoking and intratracheal instillation of lipopolysaccharide.2. PDE4 activity in lung increases significantly during the development of COPD in rat. Body weight, pulmonary function, the balance between oxidation and antioxidation and pathologic form of bronchus and lung have significant changes.3. Selective PDE4 inhibitor RPOOl exhibits a significant amelioration for pathophysiologic changes of COPD rat model by reducing PDE4 activity in lung.
引文
1 David M, Essayan MD. Molecular mechanisms in allergy and dinicul immunology eyelic nucleotide phoaphodiesterases[J]. Journal of Allergy and Clinical Immunology, 2001, 108: 671-680
2 Sndeding SH, Beavo JA. Regulation of cAMP and cGMP signaling: new phosphediesterases and new functioas [J]. Current Opinion in Cell Biology, 2000, 12: 174-179
3 Baraldo S, Turato G. Badin C, et al. Neutrophilic infiltration within the airway smooth muscle in patients with COPD [J]. Thorax, 2004, 59: 308-312
4 Tang H, Song Y, Chen J, et al. Upregulation of phosphodiesterase-4 in the lung of allergic rats[J]. American Journal of Respiratory and Critical Care Medicine, 2005, 171: 823-8
5 Fujishige K, Kotera J, MichibataH, et al. Cloningand characterization of a novel human phoaphediesterase that hydrolyzes both cAMP and cGMP (PDE10A) [J]. The Journal of Biological ChemisT, 1999, 274: 18438-18445
6 Souness JE, Aldous D, Sargent C. Immunosuppressive and anti-inflammatory efects of cyclic AMP phosphodiseterase (PDE) type 4 inhibitors[J], Immunopharmaeology, 2000, 47: 27
7 Martoin C, Gnggel R, Vergelli C, et al. Airway relaxant and anti-inflammatory properties of a PDFA inhibitor with low affinity for the high-affinity rolipram binding site[J]. Naunyn-Schmiedeberg's AreMves of Pharmacology, 2002, 365(4): 284-289
8 Muise ES, Chute LC, David Claveau, et al. Comparison of inhibition of ovalbumin-induced broncheconstriction in guinea pigs and in vitro inhibition of tumor necrosis factor-a formation with phoaphodiesterase 4 (PDE4) selective inhibitors [J]. Biochemical pharmacelogy, 2002, 63: 1527-1535
9 Grootendorst DC, Gauw SA, Baan R, et al. Does a Single dose ofthe phosphediesterase 4 inhibitor, cilomilast (15 mg), induce bronchndilation in patients with chronic obstructive pulmonary disease? [J] Pulmoar Pharmacology & Therapeutics, 2003, 16(2): 115-120
10 Juanita HV, Mehmet K, Jan AJ, et al. Local and systemic inflammation in patients with chronic obstructive pulmonary disease[J]. Am J Respir Crit Med, 2002, 166: 1281-1224
11 Vestbo J, Sorenson T, Lange P, et al. Long-term effect of inhaled budesonide in mild and moderate chronic obstructive pulmonary disease: a randomised controlled trial [J]. Lancet, 1999, 353: 1819-18
12 Wang P, Wu P, Ohleth KM, et al. Phosphodiesterase 4B2 is the predominant phosphodiesterase species and undergoes differential regulation of gene expression in human monocytes and neutrophils [J]. Mol Pharmacol, 1999, 56(1): 170-174
13 Santos S, Peinado Ⅵ, Ramirez J, et al. Enhance expression of vascular endothelial growth factor in pulmonary areries of smokers and patients with moderate chronic obstructive pulmonary disease [J]. Am J Respir Crit Care Med, 2003, 167(9): 1250-1256
14 Culpitt SV, Nightingale JA, Barnes PJ. Effect of fluticasone propionate on induced sputum matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with COPD [J]. Am J Respir Crit Care Med, 1999, 160: 1635-1639
15 Teixeira MM, Gristwood RW, Cooper N, et al. Phosphodiesterase (PDE) 4 inhibitors: anti-inflammatory drugs of the future? [J]. Trends Pharmacol Sci, 1997, 18: 164-170
16 Peter J, Barnes DS. Chronic obstructive pulmonary disease [J]. N Eng J Me, 2000, 343(26): 269-280
17 CaroI A, Pettersen M S, Kenneth B, et al. Airways inflammation and COPD: epithelial-ncutrophil interactions [J]. Chest, 2002, 121 (5): 142-150
18 Torphy TJ. Phosphodicstcrasc isozymes: molecular targets for novel antiasthma agents [J]. Am J Respir Crit Care Mcd, 1998, 157: 351-370
19 Dym O, Xcnarios I, Ke H, et al. Moleular docking of competitive phosphodicstcrasc inhibitors [J]. Mol Pharmacol, 2002, 61: 20—5
20 Ottoncllo L, Gonella R, Dapino P, et al. Prostagladin E2 inhibits apoptosis in human neutrophilic polymorphonuclear Icukocytes: role of intracellular cyclic AMP levels [J]. Exp Hematol, 1998, 26: 895-902
2 Sndeding SH, Beavo JA. Regulation of cAMP and cGMP signaling: new phosphediesterases and new functioas [J]. Current Opinion in Cell Biology, 2000, 12: 174-179
3 Baraldo S, Turato G. Badin C, et al. Neutrophilic infiltration within the airway smooth muscle in patients with COPD [J]. Thorax, 2004, 59: 308-312
4 Tang H, Song Y, Chen J, et al. Upregulation of phosphodiesterase-4 in the lung of allergic rats[J]. American Journal of Respiratory and Critical Care Medicine, 2005, 171: 823-8
5 Fujishige K, Kotera J, MichibataH, et al. Cloningand characterization of a novel human phoaphediesterase that hydrolyzes both cAMP and cGMP (PDE10A) [J]. The Journal of Biological ChemisT, 1999, 274: 18438-18445
6 Souness JE, Aldous D, Sargent C. Immunosuppressive and anti-inflammatory efects of cyclic AMP phosphodiseterase (PDE) type 4 inhibitors[J], Immunopharmaeology, 2000, 47: 27
7 Martoin C, Gnggel R, Vergelli C, et al. Airway relaxant and anti-inflammatory properties of a PDFA inhibitor with low affinity for the high-affinity rolipram binding site[J]. Naunyn-Schmiedeberg's AreMves of Pharmacology, 2002, 365(4): 284-289
8 Muise ES, Chute LC, David Claveau, et al. Comparison of inhibition of ovalbumin-induced broncheconstriction in guinea pigs and in vitro inhibition of tumor necrosis factor-a formation with phoaphodiesterase 4 (PDE4) selective inhibitors [J]. Biochemical pharmacelogy, 2002, 63: 1527-1535
9 Grootendorst DC, Gauw SA, Baan R, et al. Does a Single dose ofthe phosphediesterase 4 inhibitor, cilomilast (15 mg), induce bronchndilation in patients with chronic obstructive pulmonary disease? [J] Pulmoar Pharmacology & Therapeutics, 2003, 16(2): 115-120
10 Juanita HV, Mehmet K, Jan AJ, et al. Local and systemic inflammation in patients with chronic obstructive pulmonary disease[J]. Am J Respir Crit Med, 2002, 166: 1281-1224
11 Vestbo J, Sorenson T, Lange P, et al. Long-term effect of inhaled budesonide in mild and moderate chronic obstructive pulmonary disease: a randomised controlled trial [J]. Lancet, 1999, 353: 1819-18
12 Wang P, Wu P, Ohleth KM, et al. Phosphodiesterase 4B2 is the predominant phosphodiesterase species and undergoes differential regulation of gene expression in human monocytes and neutrophils [J]. Mol Pharmacol, 1999, 56(1): 170-174
13 Santos S, Peinado Ⅵ, Ramirez J, et al. Enhance expression of vascular endothelial growth factor in pulmonary areries of smokers and patients with moderate chronic obstructive pulmonary disease [J]. Am J Respir Crit Care Med, 2003, 167(9): 1250-1256
14 Culpitt SV, Nightingale JA, Barnes PJ. Effect of fluticasone propionate on induced sputum matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with COPD [J]. Am J Respir Crit Care Med, 1999, 160: 1635-1639
15 Teixeira MM, Gristwood RW, Cooper N, et al. Phosphodiesterase (PDE) 4 inhibitors: anti-inflammatory drugs of the future? [J]. Trends Pharmacol Sci, 1997, 18: 164-170
16 Peter J, Barnes DS. Chronic obstructive pulmonary disease [J]. N Eng J Me, 2000, 343(26): 269-280
17 CaroI A, Pettersen M S, Kenneth B, et al. Airways inflammation and COPD: epithelial-ncutrophil interactions [J]. Chest, 2002, 121 (5): 142-150
18 Torphy TJ. Phosphodicstcrasc isozymes: molecular targets for novel antiasthma agents [J]. Am J Respir Crit Care Mcd, 1998, 157: 351-370
19 Dym O, Xcnarios I, Ke H, et al. Moleular docking of competitive phosphodicstcrasc inhibitors [J]. Mol Pharmacol, 2002, 61: 20—5
20 Ottoncllo L, Gonella R, Dapino P, et al. Prostagladin E2 inhibits apoptosis in human neutrophilic polymorphonuclear Icukocytes: role of intracellular cyclic AMP levels [J]. Exp Hematol, 1998, 26: 895-902