The Relation Between IL-25 And Bronchial Asthma

ObjectiveBronchial asthma is a global serious health problem as a chronic inflammatory disease. It can effect everyone from children to the old.In morden theory of asthma, asthma is chronic airway inflammation with characteristics of intermittent and reversible airway obstruction, bronchial hyperresponsiveness, lymphocytes, eosinophil infiltration of airway submucosal. Asthma is also a kind of abnormal immune allergic disease with Th1/Th2 imbalance and Th2 cells are in advantages. IL-25 was discovered in 2001 as a new type of cytokine. IL-25 is also thinked a new member of the family of IL-17. IL-25 may come from various tissues. Most of tissues and cells could expressIL-25, and monocaryon/macrophage is main resource.The main functional ofIL-25 becomes one of the hot sports in recent years because of its complex biological characteristics and important role in the biological field. IL-25 was researched produced by Th2 cells. It can enhance the role of eosinophil infiltration of airway submucosal and play roles in many links of flare up in asthma. IL-25 can enhance the role of promotion pro-inflammatory cytokine secretion, such as IL-4,IL-13,Eotaxin. The function of IL-25 in the process of athma is complex. So there are more investigateions to do for IL-25 in the pathogenesis of asthma.RNA interference is a kind of highly conserved gene silencing technology. It has been founded that 21nt double-stranded RNA fragment can reduce the expression of targeting gene in different mammalian cell lines such as T lymphocytes, epithelial cells, liver cellsand so on. RNAi technology is the same as gene knockout and gene silencing in functions, and more convenient than Gene knockout technology. Therefore, the prospect that RNAi is becoming the target treatment is expancive.Our aim is to discuse the effect of IL-25 on asthma and the influence after interfered it.In our experiment we investigate the levels of IL-25 with asthma and offer the theoretical evidence and experimental support for new therapia, we measured the levels of IL-25 in serum of asthmatic children beforeand after glucocorticosteroid treatment and normal children with sandwich ELISA methods.we design 21nt base sequence according to principle of design of RNAi. To utilize pGCsi-IL-25 construction recombinant plasmid of pGCsi-IL-25 which can block the expression of IL-25mRNA in the lung and discuss the effection of IL-25-siRNA in the lung tissue on an asthma mouse model.Method1. The levels of IL-25 in serum of asthmatic children before and after glucocorticosteroid treatment and normal children was examined with sandwich ELISA methods.2. To establish the sequence of IL-25-siRNA that is only one, amply and equence identity which has synthetical expression plasmid pGCsi-IL-25.3. To establish an asthma mouse model in accordance with reference.Oberserve the change of ethology on mouse ,to determine the level of IL-4, INF-γand IgE in sera,the change of eosinophils counts in BALF,and the change of histopathology.4. Isolated culture spleen T lymphocyte of asthma mouse, and delivered difference ration of pGCsi-IL-25 and Lipofectamine2000 to T lymphocyte.We collected the cells after 72 hours, observed the results of transfected of T lymphocyte by fluorescent microscope, adopted RT-PCR to test the expression ofIL-4mRNA and IL-25mRNA, used ELISA to determine the change of IL-4 and IFN-γ, compared the effects of different ration of pGCsi-IL-25and Lipofectamine 2000.5. Based on the asthma mouse model, the RNAi group with reference.Among those:3days before sensitized and 1 day before stimulated was delivered the ration of pGCsi-IL-25and Lipofectamine 20001/2. At meantime, randomly get a part of fresh lung tissue and made frozen section to observe the respiratory transfected by confocal microscopy and IL-25-siRNA effected on correlation factors in lung tissue.We determined the level of IL-4, IL-25, IFN-γ, IgE in each group, detected the expression of IL-4mRNA and IL-25mRNA by RT-PCR, Observed the changes of histopathology in lung tissue and change of eosinophils cells counts in BALF.Results1. The results suggest that the levels of IL-25 in asthmatic children are higher than that in healthy children, and the IL-25 levels in asthmatic children after glucocorticosteroid treatment are lower than before glucocorticosteroid treatment.2. Identity construced the pGCsi-IL-25 was our need by amplification and sequencing.3. We observed the ethology change of mouse, the level of IL-4 on asthma mouse model was higer than that of normal control group,the level of INF-γwas lower than that of normal control group,at the same time, the level of IgE also increased obviously, the eosinophils cell counts in BALF higher compared to normal control group.Change of lung histopathology was typical that loss of bronchial mucosa epithelium and was hurt. Eosinophils, leukomonocyte and lymphom onocyte infiltrated, mucous formed. That established an mouse model successfully.4. pGCsi-IL-25-Lip interfered on T cell on asthma mouse model in vitro①After 48 hours observed that there had some green fluorescence proteinon asthma mouse T cell that indicate the succed in transfecting.②After transfected to T cell, the expression of IL-25 is significant lower in RNAi group than that of in asthma control group. It suggested that pGCsi-IL-25 was transfected into T cell successfully, and got the genesilencing.③The level of IL-4 and the expression of IL-4 mRNA were lower in RNAi group than that of asthma control group, no change on level of IFN-γ.④It could get the best specificity gene silence when the ration of pGCsi-IL-25 and Lipofectamine 2000.is1:2.5. pGCsi-IL-25-Lip interfered on asthma mouse model in vivo after respiratory transfection.①After transfected 24 hours,we could observed that there has some green fluorescent material on bronchial mucosa epithelialis and around lung tissue under the confocal microscopy.And that suggested that respiratory transfected succesfuly.②In asthma group eosinophils cell counts,as well as the level of?IL-25 was significant heighten.After delivered pGCsi-IL-25,it could descend eosinophils cell counts in RNAi group, especially after transfected 48-72 hours,and the level of IL-15 is positive correlation with eosinophils cellcounts.③The level of IgE in RNAi group is lower than asthma control group.At meantime, that of 48 hours and 72 hours transgected was significant lower than 24 hours transfected. The level of IgE got a stable level after respiratory transfected in vivo.④There is no change on levels of INF-γin sera in RNAi group, thel evel of IL-4 in RNAi group is lower than asthma control group.At meantime, that of 48 hours and 72 hours transgected was significant lower than 24 hours transfected. pGCsi-IL-25 effected on the level of IL-4 got a stable level after respiratory transfected invivo. ⑤Level of IL-25 in RNAi group is lower than asthma control group.At meantime, that of 48 hours and 72 hours transfected was significant lower than 24 hours transfected. pGCsi-IL-25–Lip effected on IL-25 got a table gene silencing level invivo.⑥pGCsi-IL-25–Lip could cut down the expression of IL-4mRNA in lung tissue after airway drop in it. That could got a stable the gene silencing level after tranfected 48-72 hours.Conclusion1. The serum IL-25 level in children with asthma exacerbation elevated and reduced in remission.IL-25 may involves in asthma pathogenesy and have the function of promotinginflammation development during the course of asthma. The results of our experiment indicate that IL-25 can induceinflammation development in asthma pathogenesy.2. Succeeded in identification on amplification and sequencing of pGCsi-IL-253. It could depress the expression of IL-25 when pGCsi-IL-25 transfected to T cell of mouse.4. pGCsi-IL-25–Lip could lessen eosinophils inflammation and correct the disbalance of Th1/Th2 on asthma mouse in lung tissue.5. Airway inhaled pGCsi-IL-25-Lip could be one of effective methods to cure asthma.