| Objective:Chronic obstructive pulmonary disease (COPD) is one of the most common disease of respiratory system.In recent years, with the deep research in the theory of COPD inflammation, various inflammatory cells and inflammatory mediators were involved in the development of COPD. It will be the key for treatment of COPD by weakening the activity of inflammatory cells and releasing of inflammatory mediators. Nuclear factor-κB(NF-κB) is an inducible transcription factor that plays the central role in the regulation of inflammatory cells activation and inflammatory mediators synthesis. Deregulation of of NF-κB could prevent the inflammatory cascade.Lipolysaccharide(LPS), as one of macromolecular substances on gram-negative bacterium cell's outer membrane, can activate the activity of transcription factors, induce proinflammatory factors secretion in the proinflammatory cells and enlarge inflammatory reaction。Vasoactive intestinal peptide(VIP) is one of the major peptide transmitters in the central and peripheral nervous systems involved in a wide range of biological functions. In an airway system where VIP-immunoreactive nerve fibers are present, VIP acts as neurotransmitter or neuromodulator of the inhibitory non-adrenergic and non-cholinergic airway nervous system and influences many aspects of pulmonary biology including modulation of inflammatory cells. Many studys have proved that VIP can restrain the secretion of inflammatory mediators.The possible mechanism is that VIP partially inhibited the NF-κB activation.But if VIPcan modulate COPD inflamation through the same mechanism still unknown .In this study,we separated peripheral blood mononuclear cells (PBMC)from AECOPD patiens or normal persons and stimulated PBMC from AECOPD with LPS in the presence or absence of VIP,then,detected NF-κB activation and TNF-αsecretion of PBMC,in order to study the effect of VIP on AECOPD inflammation,finally to provide a theory basis for the use of VIP in clinical treatment of COPD.Methods:The study recruited AECOPD patients and normal persons,gender, age, and smoking index of the two groups have good comparability. Under aseptic conditions, the blood(5 ml)are collected from the elbow vein and placed in the heparin anticoagulation tube than PBMC were separated with lymphocyte separation.1 NF-κB p65 was examined by immunocytochemical method in mononuclear cells. Cells(2.0×106 cells/ml ,0.5ml/well) were cultured in 48-well plate in RPMI 1640 medium. PBMC from AECOPD were divided randomly into four groups:①blank controller;②VIP group(VIP final concentration of 10-8 M) ;③LPS group( LPS final concentration of 1 mg/L);④VIP+LPS group(10-8 M VIP was added before LPS(1mg/L) stimulation for 20 min).PBMC from normal group were cultured in RPMI 1640 medium. All these cells were collected after being cultured separately for 1.5 h and centrifuged (800 rpm,4℃,10 min). The activation of NF-κB P65 in the cells were examined with immunocytochemical method.2 TNF-αwere measured by radioimmunoassay。Cells(2.0×106 cells/ml ,0.5ml/well) were cultured in 48-well plate in RPMI 1640 medium. PBMC from AECOPD were divided randomly into four groups:①blank controller;②VIP group (VIP final concentration of 0,10-10 M,10-9 M,10-8 M, 10-7 M,10-6 M) (VIP final concentration of 10-8 M);③LPS group (LPS final concentration of 1 mg/L);④VIP+LPS group(10-8 M VIP was added before LPS(1mg/L) stimulation for 20 min). PBMC from normal group were cultured in RPMI 1640 medium. They were collected after being cultured separately for 8 h and centrifuged (2000 rpm,4℃,10 min).The supernatant was collected and stored under -80℃to measure TNF-αcontent.3 Data are expressed as means±SEM.The deference between the normal and the control group of AECOPD were analyzed by two sample t-test.The group differences were analyzed by one-way analysis of variance(ANOVA)using SPSS13.0 software.If significant,the data were further analyzed by Student- Newman-Keuls(SNK-q) test and P <0.05 was considered statistically signifficant.Results:1①Immunocytochemistry results show that NF-κBp65 positive expression was lower than the blank controller of AECOPD(p<0.01);②The NF-κBp65 expression of four groups from AECOPD were statistically significant difference (p< 0.05),which were analyed by one-way analysis of variance (ANOVA);③The NF-κBp65 positive expression increased after LPS-stimulation (P<0.01);④Compared with blank controller, VIP group decreased the NF-κBp65 positive expression(P<0.05);⑤Compared with LPS group, VIP+LPS group decreased the NF-κBp65 positive expressin(P<0.01);⑥There was no statistical significant deference between blank controller and VIP+LPS group(p>0.05).2①The results of radioimmunoassay show that the TNF-αcontent of normal group was lower than the control of AECOPD(p<0.05);②The content of TNF-αfrom four groups was statistically significant difference (P<0.01) which were analyzed by one-way analysis of variance (ANOVA);③The content of TNF-αincreased after LPS- stimulation. There were statistical significant differences between blank controller and LPS group(P<0.01);④Compared with blank controller, VIP group decreased the positive the content of TNF-α(P<0.05);⑤Compared with LPS group, VIP+LPS group decreased the content of TNF-α(P<0.05);⑥There was no statistical significant deference between blank controller and VIP+LPS group(p>0.05).1 The relation of TNF-αlevels in PBMC and different contents of VIP. With the VIP concentration increasing, the TNF-αlevel decreased.Compared with 0 M group,10-6 M group 10-7 M group,10-8 M group decreased the content of TNF-α(P<0.05); Compared with 10-10M group,10-6 M group,10-7 M group and 10-8 M group decreased the content of TNF-α(P<0.05); Compared with 10-9M group,10-6 M group,10-7M group and 10-8M group decreased the content of TNF-α(P<0.05);Conclusion:1 NF-κB and TNF-αparticipated in the COPD inflammation.2 VIP could inhibit the activity of inflammation transcription factor and the secretion of TNF-αand the effect is dose- dependented.
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