| Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide and results in an economic and social burden that is both substantial and increasing. Many people suffer from this disease for years and die prematurely from it or its complications. None of the existing medications for COPD have been shown to modify the long-term decline in lung function that is the hallmark of this disease. Therefore, pharmacotherapy for COPD is used to decrease symptoms and/or complications. Cigarette smoking is by far the most important risk factor for COPD and the most important way that tobacco contributes to the risk of COPD. The protease-antiprotease imbalance hypothesis is thought to play a key role in cigarette smoke induced chronic lung disease, and has received considerable attention in recent years. However, neutrophil elastase is likely to be the major proteinase involved in lung destruction in alpha-1 antitrypsin deficiency. Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent endopeptidases that are responsible for the degradation and remodeling of the extracellular matrix (ECM). In recent years, the role of alveolar macrophages (AM) and MMPs in the pathogenesis of COPD is widely concerned. In the present study, the effects of cigarette smoke medium and TNF-α on MMPs expression in AM were investigated, and then the role of NF-κB signal pathway in the induction was further studied.Part 1 Effects of N-acetylcysteine on matrix metalloproteinases expression and gelatinases activity induced by cigarette smoke medium in alveolar macrophages of rats Chapter 1 Unbalance between matrix metalloproteinase-9and tissue inhibitor of metalloproteinase-1 induced by cigarette smoke medium in alveolar macrophages of ratsObjectiveTo investigate the effects of cigarette smoke medium (CSM) on the balance between matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in alveolar macrophages (AM) of rats, and then explore the role of them in the pathogenesis of claronic obstructive pulmonary disease (COPD).MethodsAM were obtained from bronchoalveolar lavage fluid from the rats which had smoked for 12 weeks. CSM was produced following the method of Wirtz and colleagues. The effects of CSM on the activity of AM was measured by MTT, and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of both MMP-9 and TIMP-1.ResultsIf the concentration of CSM exceeded 5%, CSM was toxic to AM. If the concentration of CSM was under 5%, the mRNA expression of MMP-9 and TIMP-1 induced by CSM was significantly increased in a dose-dependent manner (P<0.05), especially MMP-9, which leads to the unbalance between MMP-9 and TIMP-1. When the concentration of CSM exceeded 5%, and the mRNA expression of MMP-9 and TIMP-1 was correspondingly descended (P<0.05). If AM was stimulated by 5% CSM, MMP-9 mRNA expression was correspondingly increased in a time-dependent manner (P<0.05), but TIMP-1 mRNA expression was increased at first, and then was descendedConclusions CSM can induce the mRNA expression of MMP-9 and TIMP-1, which leads to the unbalance between MMP-9 and TIMP-1.The unbalance between MMP-9 and TIMP-1 induced by CSM possibly played an important role in the pathogenesis of COPD.Chapter 2 Inhibitory effects of N-acetylcysteine on gelatinases and macrophage metalloelastase expression in alveolar macrophages of rats induced by cigarette smoke mediumObjectiveTo study the effects of N-acetylcysteine (NAC) on the mRNA expression of gelatinases and macrophage metalloelastase from alveolar macrophages of rats induced by cigarette smoke medium (CSM), and then explore the changes of gelatinases activity.MethodsAM were obtained from bronchoalveolar lavage fluid from the rats which had smoked for 12 weeks. CSM was produced following the method of Wirtz and colleagues. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of gelatinase A (MMP-2), gelatinase B (MMP-9) and macrophage metalloelastase (MMP-12); Zymography was used to detect gelatinases activity.ResultsWhen the concentration of CSM was below 5%, the mRNA expression levels of MMP-9, MMP-2 and MMP-12 was signficantly increased with the concentration of CSM in a dose-dependent manner (P<0.05). While the concentration of CSM exceeded 5%, the genic expression levels of MMP-9, MMP-2 and MMP-12 were correspondingly decreased (P<0.05). If AM was stimulated by 5% CSM, MMP-9 and MMP-12 mRNA expression levels were correspondingly increased in a time-dependent manner (P<0.05), but MMP-2 mRNA expression was increased at first, and then was descended. When AM was stimulated by CSM, gelatinases activity was signficantly increased with the concentration of CSM in a dose-dependent manner (P<0.05). NAC can inhibit MMP-9, MMP-2 and MMP-12 expression induced by CSM from AM of the rats in a dose-dependent manner (P<0.05); when AM was pretreated using NAC, the activity levels of gelatinases induced by CSM were correspondingly decreased too (P<0.05).ConclusionsNAC does inhibit MMP-9, MMP-2 and MMP-12 expression induced by CSM in AM of rats. The mRNA over-expression of gelatinases and macrophage metalloelastase induced by CSM possibly played an important role in the pathogenesis of COPD.Part 2 Study of the signal pathway mediating matrix metalloproteinase-9 expression induced by TNF-α in alveolar macrophagesChapter 1 Induction of matrix metalloproteinase-9 in alveolar macrophages by TNF-α via NF-κB signal pathwayObjective To investigate the effects of pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC) on matrix metalloproteinase-9 (MMP-9) expression induced by tumor necrosis factor (TNF)-α, and to explore the role of NF-κB-mediated pathway in the induction.MethodsAlveolar macrophages (AM) were collected from bronchoalveolar lavage fluid from healthy subjects or patients with chronic obstructive pulmonary disease (COPD). The AM was incubated for 1.5 h with PDTC or NAC, and then stimulated for 24 h by TNF-α. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression levels of MMP-9, MMP-2 and TIMP-1; Zymography was used to detect gelatinases activity; MMP-9 protein expression was dectected by Western blotting; NF-κB activity was dectected using electrophoretic mobility shift assay (EMSA).ResultsWhen the AM from the patients and healthy subjects was stimulated by TNF-α, the mRNA levels of MMP-9 and MMP-2 were significantly increased in a dose dependent manner (P<0.05), so did the activity levels of them. But TIMP-1 mRNA expression was increased at first, and then was descended. Moreover, the mRNA levels of MMP-9, MMP-2 and TIMP-1 in the AM of patients with COPD were higher than those of healthy subjects. PDTC and NAC can significantly inhibit MMP-9 expression induced by TNF-α (P<0.05).When AM were stimulated by TNF-α at the concentration of 10 ng/mL, NF-κB activity was significantly increased (P<0.05). PDTC and NAC can significantly inhibit NF-κB activity induced by TNF-α (P<0.05).ConclusionTNF-α can up-regulate the mRNA expression of MMP-9, MMP-2 and TIMP-1 in AM; PDTC and NAC can inhibit MMP-9 expression induced by TNF-α; NF-κB-mediated pathway played an important role in the expression of MMP-9 induced by TNF-α. Chapter 2 Pyrrolidine dithiocarbamate and N-acetylcysteine inhibit nuclear factor-κB activation in alveolar macrophages by different mechanismsObjectiveTo study the effects of pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC) on the phosphorylation of IκB kinase (IKK)β, IKKα, and IκBα in alveolar macrophages (AM), and to explore the pharmacological mechanisms of NAC and PDTC as inhibitors of NF-κB activation.MethodsAM were collected from bronchoalveolar lavage fluid from the patients with chronic obstructive pulmonary disease. The AM were incubated for 1.5 h with NAC and PDTC, and then stimulated for 90 min by either tumor necrosis factor (TNF)-α or interleukin (IL)-1. Western blotting was used to detect the protein phosphorylation levels of IKKβ, IKKα, and IκBα NF-κB activity was analyzed by using electrophoretic mobility shift assay (EMSA).ResultsNAC significantly inhibited the phosphorylation of IKKβ, IKKα, and IκBα induced by TNF-α (P<0.05), but had no effects on the phosphorylation of IKKβ, IKKα and IκBα induced by IL-1 (P>0.05). PDTC did not inhibit the phosphorylation of IκBα induced by TNF-α and IL-1 (P>0.05). Similarly, NAC can significantly inhibit the activation of NF-κB induced by TNF-α (P<0.05), but had no effect on the activation of NF-κB induced by IL-1 (P>0.05), PDTC significantly inhibited the activation of NF-κB induced by TNF-α and IL-1 (P<0.05). The electrophoretic mobility shift assay also showed that PDTC and NAC do not directly inhibit NF-κB DNA binding activity in vitro (P>0.05).ConclusionPDTC prevents the degradation of IκBα via the ubiquitylation-proteasome proteolytic pathway. NAC can inhibit the processes upstream of IKK activation induced by TNF-α, which results in the decline of NF-κB activity.Part 3 Effects of NF-κB decoy oligonucleotides modified with locked nucleic acids on matrix metalloprotemase-9 expression induced by TNF-α in alveolar macrophagesObjectiveTo study the effects of NF-κB decoy oligonucleotides (ODNs) modified with locked nucleic acids (LNA) on gelatinases (MMP-9 and MMP-2) expression and NF-κB activity induced by TNF-α in alveolar macrophages (AM) from patients with chronic obstructive pulmonary disease (COPD).MethodsAM was collected from bronchoalveolar lavage fluid from patients with COPD. NF-κB decoy ODNs and mismatch ODNs were modified with LNA, and transfected into AM using Lipofectamine 2000. And then the AM were stimulated for 24 h by TNF-α. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression levels of MMP-9 and MMP-2; MMP-9 protein expression was dectected by Western blotting; NF-κB activity was dectected using electrophoretic mobility shift assay (EMSA).ResultsNF-κB decoy ODNs significantly inhibited MMP-9 and MMP-2 expression induced by TNF-α in AM (P<0.05), and mismatch ODNs had no effects on MMP-9 and MMP-2 expression induced by TNF-α. NF-κB decoy ODNs significantly inhibited the activation of NF-κB induced by TNF-α (P<0.05), and mismatch ODNs had no effects on the activation of NF-κB induced by TNF-α.ConclusionNF-κB decoy ODNs modified with LNA can inhibit gelatinases expression induced by TNF-α in AM, which cast new light for the treatment of COPD.
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