| Asthma is one of the most serious and common respiratory disease and its incidence has climbed markedly in the past two decades.Given that asthma results in an economic and social burden that is both substantial and increasing, the researches on the prevention and treatment of asthma have been one of the frontier fields in human health.While asthma is a disorder of the conducting airways characterized by Th2- directed inflammation,a second set of mechanisms is being increasingly recognized as fundamental to disease chronicity and severity,for which the term "remodeling" has been used.It is regarded as some structural changes in the airway walls,such as mucous gland hyperplasia,reticular basement membrane thickening,vascular proliferation,airway smooth muscle hypertrophy and hyper- plasia.As a significant pathological basis of irreversible airway obstruction and persistent airway hyperresponsiveness,airway remodeling is also a major change responsible for corticosteroid refractoriness in the treatment of asthma. Recently, airway remodeling has become one of the main goals in asthma research.1,25-dihydroxyvitaminD3(1,25-(OH)2D3),the active metabolite of vitamin D3,is a lypophilic molecule that exerts its activities mainly via the interaction with the vitamin D receptor(VDR).Although 1,25-(OH)2D3 has traditionally been associated with the regulation of calcium homeostasis, a multitude of other sites of action for this steroid hormone have been discovered.A major advance occurred to the study of genetics asthma with the identification of VDR gene as an asthma susceptibility gene,suggesting that the 1,25-(OH)2D3 and VDR system may function as a regulator of asthma. In recent years,the general knowledge regarding the regulation of asthma by 1,25-(OH)2D3 has rapidly increased.Existing data have showed its sophisticated regulation on the disturbed immune responses and chronic inflammation in asthma.All of these published observations have suggested promising effects of 1,25-(OH)2D3 on controling many responses that lead to pathological and physiological symptoms of asthma.However,to date, there are no data on the regulatory effect of 1,25-(OH)2D3 on asthmatic airway remodeling.And the idea that it may play a beneficial role in the alleviation of airway remodeling is relatively new .The studies presented in this paper were performed in vivo and in vitro to elusive the contribution of 1,25-(OH)2D3 to asthmatic airway remodeling and further the potential mechanisms underlying this effect.The addition of its involvement in the understanding of asthma pathogenesis can shed light on better control and treatment in asthma.The studies consisted of three parts.Part One Effects of 1,25-(OH)2D3 on airway remodeling and expression of MMP-9 in murine model of asthmaObjective:to investigate the effects of 1,25-(OH)2D3 both on the regulation of airway remodeling and the expression of matrix metalloprotease-9(MMP-9) in a murine model of asthma,and to explore its potential role in the treatment of asthma.Methods:BALB/c mice were sensitized and challenged with ovalbumin to establish the asthmatic airway remodeling model.They were randomly divided into four groups:control group,asthma group, VD group and DEX (dexamethasone)group.The characteristic airway structure was detected by HE staining. Morphometric analysis of the stained sections was performed using computerized image analysis system.The expression of MMP-9 in both activity and mRNA level was detected by gelatin zymograph and RT-PCR, respectively. Results:(1)The infiltration of inflammatory cells, smooth muscle cell layer thickening and epithelial loss were decreased in VD group when compared with those in asthma group,but these changes were still more significant than those in the control group; (2)WAm/Pbm,WAi/Pbm and N/Pbm were decreased markly in VD group when compared with those in asthma group(P<0.05),but they were still higher than those in the control group (P<0.05); (3)The activity and mRNA level of MMP-9 in VD group were decreased when compared with those in asthma group(P<0.05),but it was still higher than those in the control group (P<0.05).Conclusions:Intervention with 1,25-(OH)2D3 could markly alleviate the asthmatic airway remodeling and partly restore the appropriate structure of airway wall.It could also inhibit the ASMC proliferation in vivo and lower the expression of MMP-9 in the lung tissue on asthmatic condition,thus delay the process of airway remodeling.Part Two Effects of 1,25-(OH)2D3 on passively sensitized human airway smooth muscle cellsObjective:to investigate the effects of 1,25-(OH)2D3 on passively sensitized human airway smooth muscle cells(HASMCs) proliferation and their MMP-9 and a disintegrin and metalloprotease 33(ADAM33) expressions.Methods: HASMCs were passively sensitized with 10% serum from asthmatic patients.1,25- (OH)2D3 was used as the interventor.MTT colorimetri assay was used to examine the effect of 1,25-(OH)2D3 on cell proliferation at different concentrations(10–10M,10–9M,10–8M,10–7M).By this way,its optimal concentration was determined. And then the effects of 1,25-(OH)2D3 at the optimal concentration on cell proliferation was examined by the same MTT assay;cell cycle analysis by flow cytometry and immunocytochemical staining for proliferating cell nuclear antigen (PCNA).The expressions of MMP-9 and ADAM33 in HASMCs were studied by real-time quantitative RT-PCR and Western blotting analysis. Results:(1)Inhibition of cell proliferation by 1,25-(OH)2D3 was barely detectable at 10–10M.But with the increasing concentration ranging from10–9M to 10–7M, 1,25-(OH)2D3 markly inhibited the cell proliferation concentration-dependently and reached the maximum effect at the concentration of 10–7 M.Accordingly,10–7M was chosed as the optimal concentration of 1,25-(OH)2D3 for the following study;(2)At the concentration of 10–7M,1,25-(OH)2D3 could inhibit the cell proliferation of passively sensitized HASMCs in a time-dependent manner;(3)1,25-(OH)2D3 markly suppressed the PCNA- positive rate and hampered the G1/S transition in HASMCs passively sensitized with asthmatic serum;(4)1,25-(OH)2D3 also markly down-regulated the expressions of protein for MMP-9 and ADAM33,as well as their mRNA levels in passively sensitized HASMCs. Conclusions: 1,25- (OH)2D3 has directly inhibitory effects on passively sensitized HASMCs in vitro,including the inhibition of the cell proliferation and the expressions of MMP-9 and ADAM33,which is maybe concerned with the beneficial role for 1,25-(OH)2D3 on the prevention and therapy of asthmatic airway remodeling.Part three Potential role of NF-κB in the multiple inhibition of 1,25-(OH)2D3 in passively sensitized HASMCsObjective: to evaluate the potential role of nuclear factor-κB(NF-κB) in the regulation of 1,25-(OH)2D3 on passively sensitized HASMCs.Methods:HASMCs were primarily cultured and randomly divided into three groups: control group, asthma group and VD group. NF-κB DNA-binding activity was assayed by electrophoretic mobility shift assay(EMSA),while the expression of NF-κB p65 in cell nuclear was observed by immunofluorescence staining.Real-time quanti- tative RT-PCR was used to determine the mRNA levels of VDR,CYP24 and the inhibitor protein IκBα.Additionally,the protein level of IκBαwas also detected by western blotting.Results:(1)The expression of VDR has been detected in the normal HASMCs. And passive sensitization with asthmatic serum alone did not alter its mRNA level compared with that in the control cultures,but 1,25-(OH)2D3 pretreatment increased its mRNA level. Moreover,the mRNA encoding CYP24 was also markly up-regulated in the VD group.(2)The NF-κB DNA-binding activity of HASMCs in VD group was significantly decreased as compared with that in asthma group(P<0.01),but it was still higher than that in the control group(P<0.01);(3)1,25-(OH)2D3 markly weakened the fluorescence intensity of NF-κB p65 in the nuclear of passively sensitized HASMCs(P<0.01);namely,it markly arrested the NF-κB p65 nuclear translocation in passively sensitized HASMCs;(4)Addition of 1,25-(OH)2D3 also markly up-regulated the expressions of protein for IκBα,as well as its mRNA levels in passively sensitized HASMCs. Conclusions:HASMCs express functional VDR and 1,25-(OH)2D3 could up- regulate its mRNA levle and evoke its functional response in passively sensitized HASMCs,leading to the up-regulation of the IκBαexpression.And by this way,1,25-(OH)2D3 could decrease the NF-κB activity.Given that NF-κB signaling pathway extensively participates in HASMC dysregulation,these results indicated that the suppression of 1,25-(OH)2D3 on NF-κB activity was maybe a relevant mechanism for its negative effects on passively sensitized HASMCs... |
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