| Objective: Bronchial asthma is an inflammatory disorder of the airways that is characterized by intermittent and partially reversible bronchial obstruction, as well as by airway hyper-responsiveness to a wide variety of stimuli. Many types of inflammatory mediators and inflammatory cells are involved in airway inflammation. The unbalance of lymphocyte Th1 and Th2 ratio is believed to be one of the reasons leading to asthma, although the exact pathogenesis of asthma remains obscure. The purpose of this study is to investigate the important roles of lymphocytes in pathophysiology of asthma. The individual therapies are expected to achieve by exploration of the specific diagnosis and therapeutic targets for asthma, and by development of the methods to prevent the asthma attacks.Methods: An ovalbumin (OVA)-induced allergic airway disease model of asthma was used with normal rats as control. (1) Changes in behavior, respiratory function curve, eosinophil count, levels of serum IgE, blood gas analysis, and pathological change in lung tissue were compared between asthmatic rats and normal rats. (2) The differentially expressed spleen lymphocyte proteins between asthmatic rats and normal rats were separated by two-dimensional gel electrophoresis (2-DE) and subsequently identified by MALDI-TOF-MS. (3) The real-time RT-PCR and in situ hybridization were used to measure the RNA levels of differentially expressed proteins at different stages of asthma attack and in different administration routes, as well as to confirm the vadility of proteins identified by MS.Results: (1) Wistar rats model of bronchial asthma challenged with ovalbumin were successfully established through a series of evidence, such as the changes of ethology, flow volume curve evaluation of respiratory function, blood eosinophile granulocyte (EOS) Count, the levels of serum IgE, the blood gas analysis of pro-treatment and post-treatment and pathological findings of lung. (2) 33 proteins expressed differentially between normal and asthmatic rats were analyzed and 23 proteins (score value > 59) were identified by using 2-DE and MALDI-TOF-MS. Further analysis verified that the expressions of 14 proteins were up-regulated, and 9 proteins spots were down-regulated. (3) Further analysis using bioinformatics tools suggested that the above-mentioned proteins participated in some process of energy metabolism, metabolism of cellular skeleton, synthesis of amino acids and transcriptional control. 6 proteins associated with cellular signal pathway such as annexin A3, GDI, HTRA2, and LMW-PTP, are likely important components involving differentiation of Th1 and Th2, the imbalance of Th1/Th2, and the airway chronic inflammation of asthmatic rat. HnRNPK and LIM are involved in the transcriptional regulation of cellular skeleton protein genes. However, there were energetic metabolism-relating protein such as CoQ10 and mitochondrial inner membrane protein. (4) Using real-time PCR, the mRNA levels of GDI, AnnexinA3, LMW-PTP, HTRA2 and HnRNPK in splenic lymphocytes were detected. The results are as follows.①Compared with normal Rat (Group A), the mRNA level of HTRA2 was up-regulated in 7-day asthmatic rats (Group B) and 14-day asthmatic rats (Group C), while down-regulated in therapeutic rats (Group D). and budesonide therapeutic rats (Group E).②Compared with Group A, the mRNA level of Annexin A3 was up-regulated in both asthmatic rats (Group B and C) and therapeutic rats (Group D and E).③Compared with Group A, the levels of mRNA of GDI and HnRNPK were up-regulated in all groups besides Group C.④Compared with Group A, there was no apparently distinct expression of mRNA of ACP1 between Group B and C, and between Group D and E.⑤In situ hybridization results of spleen and lung showed that Annexin A3 was expressed in cell membrane and endochylema of splenic lymphocytes. No expression was detected in bronchus, bronchiole, interstitial cell and endotheliocyte of lung. In splenic lymphocytes, the highest expression was appeared in the Group C, the higher in the Group B, and low expression in the Group D and Group E. However, no expression spleen was detected Group A.Conclusion: Some of proteins were expressed differentially in spleen lymphocyte of asthmatic rats. The proteins involving signal transduction pathway, including Annexin A3, HTRA2, LMW-PTP and HnRNPK. They probably function in two aspects. The first is to induce the change in cellular skeleton leading to airway remodeling. The second is to regulate differentiation and proliferation of T cell and smooth muscle cell, or control inflammatory reaction by participating in serin cellular signal pathway. The evidence that the mRNA levels of Annexin A3, HTRA2 and HnRNPK in asthmatic rats decreased after hormone treatment may suggest that the effects of corticosteroid on prevention and control of asthma might be achieve by depression of gene expression of Annexin A3, HTRA2 and HnRNPK, although they are not the direct inducers that cause an attack of asthma: Conclusively, Annexin A3, HTRA2 and HnRNPK may likely become novel diagnostic biomarkers and/or new drug targets for asthma. |
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