| Objective(1)To investigate the effects of different doses of 1,25(OH)2D3 on airway inflammation and airway hyperresponsiveness in asthmatic mice;(2)To explore the effects of different doses of 1,25(OH)2D3 on the phenotype and function of lung dendritic cells(LDC)in asthmatic mice and the effects of TH2 and Th17 on the inflammatory response in asthma mice(3)To investigate the effect of different doses 1,25(OH)2D3 on the MKP1/p38MAPK/11?HSD2 signaling pathway in the lung tissues of asthmatic mice.Methods(1)Establishing the corresponding animal model accroding to the reference paper[1].30 wild type C57BL/6 mice at SPF level indoor environmental adaptability after 7 days,were divided into control group(Control),asthma group(PBS/asthma),low dose group(LVD/asthma),middle dose group(MVD/asthma)and high dose group(HVD/asthma);each group were 5 mice each.On days 1,8,15 after arrival,Each mice in PBS/asthma?LVD/asthma?MVD/asthma ? HVD/asthma were injected with 200ul of an antigen mixture in the abdominal cavity.To make them sensitize,the control group given 200ul PBS instead.Starting on day 22,mice in PBS/asthma group and 1,25(OH)2D3 intervention groups were treated by inhalation of 1%OVA.This treatment was conducted once a day,for a period of 30 minutes,and continued until day 35.Following inhalation of OVA,the mice in LVD/asthma group were injected in the abdominal cavity with 0.02?g 1,25(OH)2D3 dissolved in 0.5?l anhydrous ethanol and 20ul PBS.Mice in MVD/asthma group were injected with 0.08?g 1,25(OH)2D3 dissolved in 2.0?l anhydrous ethanol and 80ul PBS.Mice in HVD/asthma group were injected with 0.2?g 1,25(OH)2D3 dissolved in 5.0?l anhydrous ethanol and 200ul PBS.Mice in asthma group were injected with 200?l PBS in the abdominal following OVA inhalation.Mice in control group were treated by inhalation of atomized saline and injection of PBS.Airway resistance(RL)and dynamic lung compliance(Cdyn)in each group were measured by small animal lung function instrument(BUXCO)after 24 hours of stimulation in 35 days.After the airway hyperresponsiveness was measured,peripheral blood,bronchoalveolar lavage fluid(BALF)and lung tissue were obtained.Hematoxylin eosin(HE)staining and periodic acid Schififs staining(PAS)detect the degree of inflammatory cells infiltration and the hyperplasia of bronchial goblet cells respectively.Cells precipitated after bronchoalveolar lavage fluid centrifugation were staining by Wright-Giemsa and Classification count;Thl(IFN-y),Th2(IL-4,IL-5,IL-13),Th17(IL-17A)and Treg(IL-10)and other related cell factor level in the supernatant after centrifugation is detected by ELISA:The content of serum total immunoglobulin E(IgE)and OVA specific immunoglobulin E(OVA-IgE)in serum were also detected by ELISA.(2)An animal model of asthma with different doses of 1,25(OH)2D3 intervention was established above.The number and surface costimulatory molecules(CD40,CD80,CD86 and MHC?)expression of lung dendritic cell and the expression of Thl,Th2,Th17 and Treg cells in the lung tissues of each group were detected by Flow cytometry.The level of cell related transcrip-tion factors,such as Thl(T-bet),Th2(GATA-3),Th17(RORyt)and Treg(Foxp3),was detected by real-time quantitative PCR(qPCR).(3)An animal model of asthma with different doses of 1,25(OH)2D3 intervention was established above.Western blot and real-time quantitative PCR(qPCR)were used to detect MKP1,p38MAPK and 11?HSD21 the protein and mRNA expression in the lung tissue of each group.Results(1)Compared with PBS/asthma mice,increased lung compliance(Cdyn)and decreased airway resistance,decreased airway and pulmonary parenchyma inflammatory cell infiltration,reduced goblet cells and mucus secretion were found in LVD/asthma and MVD/asthma mice;The number of total cells,lymphocyte counts and eosinophils in BALF decreased,the total IgE and specific IgE(OVA-IgE)contents in serum decreased,the levels of IL-4,IL-5,IL-13 and IL-17A in BALF decreased,and the increase of IL-10 and IFN-y increased:on the contrary,decreased lung compliance(Cdyn)and increased airway resistance,increased airway and pulmonary parenchyma inflammatory cell infiltration,increased goblet cells and mucus secretion were found in HVD/asthma mice;The number of total cells,lympho cyte counts and eosinophils in BALF increased,the total IgE and specific IgE(OVA-IgE)contents in serum increased,the levels of IL-4,IL-5,IL-13 and IL-17A in BALF increased,and the increase of IL-10 and IFN-y decreased;(2)Compared with PBS/asthma group,the percentages of Th2 and Th17 cells in LVD/asthma and MVD/asthma groups were significantly reduced;the number and the expression of costimulatory molecules(CD40,CD80,CD86 and MHCII)of LDCs were significantly reduced;the expression of GATA-3 and RORyt in lung tissue of LVD/asthma and MVD/asthma groups were decreased,However,the expression and percentage of the above indexes in the lung tissue of HVD/asthma group were the opposite..(3)Low and Middle dose 1,25(OH)2D3 increased the protein and mRNA express ion of MKP-1,and reduced the protein and mRNA expression of p38MAPK and 11?HSD2 in the lung tissue of asthmatic mice.However,High dose 1,25(OH)2D3 reduced the expression of MKP-1 protein and mRNA,and increased the expression of p38MAPK and 11?HSD2 protein and mRNA in the lung tissue of asthmatic mice.Conclusion(1)Low and Middle dose 1,25(OH)2D3 can reduce airway inflammation and airway hyperresponsiveness in asthmatic mice,while high dose 1,25(OH)2D3 aggravates it.(2)low and middle dose of 1,25(OH)2D3 can inhibit Th2 and Thl7 responsesby inhibiting the function maturation of dendritic cells in lung tiusse of asthmatic mice,while high dose 1,25(OH)2D3 can activate Th2 and Thl7 responses by promoting the maturation of dendritic cells.(3)low and moderate dose of 1,25(OH)2D3 may promote MKP-1 expression,MKP-1 selectively inactivate p38MAPK signaling pathway,and then inactivate 11 beta HSD2 signaling pathway,thereby alleviating airway inflammation in asthma mice.High dose 1,25(OH)2D3 effect is just the opposite. |
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