| Chronic airway inflammation and airway remodeling are both two significant characters of Chronic Obstructive Pulmonary Disease (COPD). Airway smooth muscle cells( ASMC) have an important role not only on airway remodeling but also on chronic airway inflammation. Its hyperplasia is a crutial element of pathophysiology of COPD and is important to this disease's occurrence and development.Recently more and more importance are attatched to the discussion and research on the mechanism of the proliferation of ASMC of subjects suffered from COPD .Based the regular therapy of anti-infiammation,the reseach and development of the drugs inhibiting the proliferation of ASMC will become a new and more effective treatment of COPD.Platelet-activating factor(PAF) is a potent phospholipid mediator produced by most stimulated inflammatory cells, which plays an important role in COPD.The blood PAF levels in patients on the acute phase of COPD are increased obviously .Nowadays it is clear that PAF are comprehensivelyinvolved in the development of chronic inflammation of COPD: it can increase the permeability of capillary vessels; it can also enhance the adherence,accumulation and chemotaxis abilities of platelet and neutrophil; it is involved in the process of respiratory burst and the release of oxidant radical; the metabolism of arachidonic acid and phosphoinositide is related to PAF ; additionally PAF can regulate the balance between matrim metalloproteinase-1 and tissue inhabitor of metalloproteinase-1 expression in human body. At the same time,PAF is known for its ability to promote the synthesis and release of various cytokines,such as TNF- α, IL-1, IL-8 and IL-6,which can induce the synthesis and release of PAF,too.Thus,interactions between PAF and these cytokines form a complicated feedback cytokine network system.The role of the feedback system in the chronic inflammation are well documented.But so far, few results are available concerning the role of PAF on the proliferation of ASMC and the role of it on airway remodeling. In this study, PAF was used as stimulus.MTT assay, flow cytometry and immuneohis-tochemistry for proliferating cell nuclear antigen(PCNA) were used to analyse the function of PAF on proliferation of cultured rat airway smooth muscle cells(RASMC) and to explore its mechanism on airway remodeling.The transcription factor nuclear factor- kB (NF- kB) belongs to the family of various protein complexes of nuclear factor- k B /Rel.It is close relative to COPD and plays a particularly important role in the transcription and expression of a variety of genes, primarily the ones related to inflammation mediators, interleukins, adhesion molecules etc. By this way, NF- kB becomes the crucial factor of the regulation of chronic nonspecific inflammation of COPD. But nowadays there are few reports on the role ofNF- k B on the proliferation of ASMC, which plays a decisive role in airway remodeling.Recent research has revealed that PAF is the most proximal mediator involved in activating NF- kB and can activate NF- kB directly. But it is not clear whether the effect of PAF on the activation of NF- kB is related to the functiong of PAF on stimulation of the proliferation of AMSC or not.In order to explore the role of NF- kB on the proliferation of ASMC ,we used PAF as the stimulus and N-acetylcysteine (NAC,20mmol/L) as the intervenor in the experiment,then we chosed electrophoretic mobility shift assay (EMSA) to examine directly NF- kB activity of the ASMCs on different conditions.EMSA is a classical experiment method to detect the activity of NF- kB.The results of the experiment can offer us new theoretical base on which the role of NF- kB on the pathogenesis and treatment of COPD can be further clarified.1. Platelet-activating factor stimulates the proliferation of rat airway smooth muscle cellsThe cells were divided into control group and PAF groups.The cells in PAF group were subdivided into four small groups by concentration of PAF 106, 10-7, 10-8, 10-9mol/L,MTT assay was used not only to investigate the effects of PAF on proliferation of ASMC but also to confirm the optimal concentration.Flow cytometry and immuneohistochemistry for proliferating cell nuclear antigen(PCNA) were also used to analyse its function on proliferation of ASMC.In the results,PAF(10-610-9mol/L)could stimulate the cell proliferation and reached the maximal effect at 10-7mol/L.The cell percentage of the ASMCs of 10-7mol/L PAF subgroup at Gq/1 phase was much lower than that of control group(P<0.05).In this subgroup ,the percentage of expression of PCNA at 48h(71.05% ±1.22%) was significantlyincreased compared with the control group (53.27%±2.56%P<0.05). These data demonstrate PAF can stimulate the proliferaion of ASMCs and it is an important stimulus in airway remodeling.2. Effect of NAC on proliferation-inducing effects of PAF on rat airway smooth muscle cellsPAF(107mol/L)was used as stimulus and N-acetylcysteine (NAC,20mmol/L) was used as the intervenor.MTT assay, flow cytometry and immunohistochemistry for PCNA were used to investigate the proliferation of ASMC on the different conditions.In NAC group,A492nm of RASMC at 48h, proliferative index(PI) and the percentage of expression of PCNA at 48h were all lower than those of PAF group,but higher than those of control group,which implicated NAC (20mmol/L) partly inhibited the cell proliferation induced by PAF (P<0.05) .Summarily,these data indirectly explained that the function of PAF on ASMCs proliferation might be exerted partly through NF- kB activation pathway.3.Exspression of NF- kB activity of the ASMCsNF- kB activity of the ASMCs was examined by electrophoretic mobility shift assay (EMSA).As a result, NF- kB activity had existed to some extent before the stimulation of PAF.In all groups of PAF,the activity of NF- k. B of the ASMCs was increased differently during 0.5-4h,what's more,it reached the max at lh.Contrasted among all groups of PAF, PAF(10-7mol/L) had the maximal effect on stimulation of NF- kB activity of RASMC.Especially at lh,A492nm of the NF- kB activity of PAF group is as 7 times as that of control group[ (1.83 + 0.13) vs (0.27±0.08), n=3, P<0.05]. With the preatment of NAC (20mmol/L), A492nm of the NF- kB activity (1.15 ±0.21) were decreased obviously, which was between that of PAF group... |
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