Effects Of The Vla-4 Antagonist Peptides Modified By Peg On Expression Of Eotaxin And Ccr3 In Mice Airway Epithelium

ObjectiveBronchial asthma (asthma) is a kind of disease characterized by reacting inflammation which is mainly caused by eosinophils (EOS) infiltration and hyperresponsiveness (AHR), but the pathogenesis has not been fully clarified. It is believed that the gas AHR airway inflammation is the basis of asthma, while the EOS is a key driving cell, and EOS aggregation in asthma airway plays an important role. In recent years, research on the pathogenesis of bronchial asthma has made great progress, researchers pay much attention to the important role of the chemokines on inflammatory, which is characterized by inflammatory cells of multiple chemotaxis and activation.Eosinophil activating chemokine (Eotaxin) and its CC chemokine receptor 3 (CCR3) has a selective chemotactic role on the EOS.In the allergic inflammatory response, eotaxin combined with CCR3-specific on the EOS help EOS activation in-situ and airway gathering, and adjust inflammation in asthma with the help of Th2 cytokines.IT is internationally recognized a new research field for anti-inflammatory drugs that the very late antigen -4 (VLA-4) of cell adhesion molecule leukocyte's adhesion and intermediate exudation mediated by inflammatory. VLA-4 involved in the migration of inflammatory cells and infiltration in inflammatory reaction, and VLA-4 antagonist peptide LDV (Leu-Asp-Val) has shown a definite anti-inflammatory effects on asthma in the experimental studies on early and late stages, KILDVA (Lys-Ieu-Leu-Asp -Val-Ala) is its derivative. This study aims to observe the effect by mouse asthma model on expression of lung tissue of asthmatic mice Eotaxin and CCR3, and the effect on content levels of Eotaxin and the number of EOS in lavage fluid (BALF) and serum. This study explores the mechanism of VLA-4 antagonist peptide KILDVA to bronchial asthma, select PEG-modified VLA-4 antagonist peptides with characteristics of good efficacy and pharmacokinetic, then this study gives experimental and theoretical support for the development of peptide drug formulations and clinical applications.Methods1. The author prepares 180 BALB/C mice weighted 18 g-20 g, and divide theminto six different group at random, namely normal control group, model group, dexamethasonegroup, KILDVA group,mPEG2000-KILDVAgroup,and mPEG5000-KILDVA group.The author uses ovalbumin (OVA) to establish a mouse model of allergic asthma. All animals are given drugs by intraperitoneal injection.2. The author detects the number of Leukocyte and EOS in each group by means of wright (Switzerland), and studys pathological feature changes of airway epithelium by means of HE staining.3. The author uses Enzyme-linked immunosorbent assay (ELISA) test to detect all the mice bronchoalveolar lavage fluid and serum levels of Eotaxin.4. The author uses immunohistochemistry to detect the Eotaxin and CCR3 protein expression change of mice airway epithelium in each group.5. The author uses situ hybridization to detect the Eotaxin and CCR3 mRNA expression change of mice airway epithelium in each group.Results1. The number of the absolute number of Leukocyte and EOS in BALF.The results showed that the absolute number of Leukocyte and EOS in BALF in model group significantly increase compared with normal control group with significantly difference (p<0.01); The absolute number of Leukocyte and EOS in BALF in dexamethasone group, KILDVA group, mPEG2000-KILDVA group, mPEG5000-KILDVA group are significantly lower than that in model group with significant differences (p<0.05); The absolute number of Leukocyte and EOS in BALF in KILDVA group and mPEG2000-KILDVA group are higher than that in dexamethasone group with significant difference (p<0.05); The absolute number of Leukocyte and EOS in BALF in KILDVA group are higher than that in mPEG2000-KILDVA group with significantly difference (p<0.05); mPEG5000-KILDVA group and dexamethasone group are similar with no significant difference (p>0.05).2. Mice pathological changes in bronchia and lung tissue HE stainingMice pathological changes in bronchia and lung tissue.HE staining showed that mice airway epithelium is steady in normal control group without ciliated epithelial shedding, without inflammatory exudates in alveolar cavity, and without inflammatory cell infiltration below submucosal as well, meanwhile, airway smooth muscle is not thickened. For model mice group, endobronchial is visible in mucus plug with ciliary epithelial shedding, lumen obstruction, and goblet cell hyperplasia. A large number of inflammatory cells infiltration can be seen in airway wall and lung tissue, airway smooth muscle is thickened; In dexamethasone group, KILDVA group, mPEG2000-KILDVA group, and mPEG5000-KILDVA group, inflammatory cells with eosinophil-based airway submucosal edema significantly reduce in bronchia and lung tissue, the symptom such as the exciliary epithelial shedding, lumen obstruction improved significantly; The result of concerning research in dexamethasone group and mPEG5000-KILDVA group is better than that in KILDVA group, mPEG2000-KILDVA group.3. Bronchia lavage fluid and serum levels of Eotaxin in each group with ELISA methods.The results showed that the Eotaxin in BALF and serum in model group is significantly higher than that in the normal control group with significant difference (p<0.01); The Eotaxin in BALF and serum in dexamethasone group, KILDVA group, mPEG2000-KILDVA group and mPEG5000 -KILDVA group are significantly lower than that in the model group with significant difference (p<0.05), but still higher than that in the normal control group (p<0.05); The Eotaxin in BALF and serum in KILDVA group and mPEG2000-KILDVA group are higher than that in dexamethasone group with significantly difference (p<0.05); The Eotaxin in BALF and serum in KILDVA group is higher than that in mPEG2000-KILDVA group with significantly difference (p<0.05); mPEG5000-KILDVA group and dexamethasone group are similar with no significant difference (p>0.05).4. Eotaxin and CCR3 expression with immunohistochemical staining method.The author prepares 2% in mice anaesthetized by intraperitoneal injection of 0.2 ml sodium pentobarbital, 4% paraformaldehyde perfusion-fixed and paraffin-embedded, coronal sections, uses SP immunohistochemical staining method to detect lung tissue Eotaxin and CCR3 expression. The results showed that Eotaxin and the CCR3 protein expression unit (PU) is significantly higher in the model group than that in the normal control group with significant difference (p<0.01); Eotaxin and the CCR3 protein expression unit (PU) in dexamethasone group, KILDVA group, mPEG2000 -KILDVA group and mPEG5000-KILDVA group are significantly lower than that in the model group, with significant difference (p<0.05), but still higher than that in the normal control group (p<0.05); Eotaxin and CCR3 protein expression in KILDVA group and mPEG2000-KILDVA group are higher than that in dexamethasone group with significant difference (p<0.05); Eotaxin and CCR3 protein expression in KILDVA group is higher than that in mPEG2000 -KILDVA with significant difference (p<0.05); mPEG5000 -KILDVA group and dexamethasone group are similar with no significant difference (p>0.05).5. Eotaxin and CCR3 mRNA expression with situ hybridization method.The author prepares 2% in mice anaesthetized by intraperitoneal injection of 0.2 ml sodium pentobarbital, 4% paraformaldehyde perfusion-fixed and paraffin-embedded, coronal sections, detects Eotaxin and CCR3 mRNA expression of lung tissue with in situ hybridization. The results showed that the positive expression of Eotaxin and CCR3 mRNA in model group is significantly higher than that of the normal control group with significant differences (p<0.01); The positive expression of Eotaxin and CCR3 mRNA in dexamethasone group, KILDVA group, mPEG2000- KILDVA group and mPEG5000-KILDVA group are significantly lower than that in model group with significant differences (p<0.05), but still higher than that in the normal control group with(p<0.05); Eotaxin and CCR3 mRNA expression in KILDVA group and mPEG2000-KILDVA group are higher than that in the mPEG5000–KILDVA group with significant different (p<0.05); mPEG5000 -KILDVA group and dexamethasone group are similar with no significant difference (p> 0.05).Conclusion1. The establishment of an experimental asthmatic mice model can simulate the pathogenesis of human asthma. It is an ideal animal model to study asthma and is feasible for in-depth study of asthma. It can cause pathological changes of bronchial and lung tissue of asthmatic mice.2. The increased expression of Eotaxin and CCR3 in asthmatic mice model is important factor to cause infiltration and activation of EOS in bronchial and lung tissue, the two has a significant positive correlation.3. VLA-4 antagonist peptides KILDVA reduce the protein content of Eotaxin and CCR3, reduce the expression of mRNA in airway epithelial cells and lung tissues of asthmatic mice, so as to reduce expression levels of Eotaxin, and reduce EOS number in BALF in asthmatic mice, to ultimately achieve the purpose of interfering with airway inflammation and cure bronchial asthma.4. mPEG5000-KILDVA showed good anti-asthma effect compared with mPEG2000-KILDVA and KILDVA.