Study Of The Activation Mechanism Of Eosinophils During The Stage Of Exacerbation In Asthma Patients

BACKGROUND AND AIM:Bronchial asthma is a chronic respiratory disease characterized by chronic airway inflammation, mucus hypersecretion, airway hyperresponsiveness, airway remolding,variable airway obstruction. which has a great impact on a person's ability to lead a normal life and perform in. work or school. . The increasing incidence and prevalence of asthma in many parts of the world continue to make it a global health concern. There are several closely related phenotypes in asthma, of which allergic inflammation depends on regulation by many genes, that can decide lymphocyte subsets, immunoreactivity, migration and activation of eosinophils(EOS) and mast cells, adhesion molecule expression, cytokine synthesis, and tissue fibrosis and reparative process. Though rapid progress has been made in asthma basic research, asthma treatment may still depends on several traditional medicines such as glucocorticosteroid. The index for asthma inflammation evaluation and control mostly based on pulmonary function, FeNO and induced sputumn EOS. So there is still a long way to go before asthma can be well treated.Strong evidences show that EOS play an important role in epithelial cells injury, airway inflammation, airway remolding and immunoregulation of lung micro -environment and pulmonary function decline and airway obstruction. Clinical trials also showed that it could greatly improve asthma control, reduce asthma exacerbation and improve quality of life if induced sputum eosinophils was used as a marker for asthma control . Also the 2006 Global Initiative for Asthma (GINA) emphasized that eosinophils play a role in airway remolding.For EOS's importmant role in asthma, there are several studies on the role of EOS and its activation mechanism, whcih can obtain some information but mostly came from in vitro and focused on a single pathway in EOS activation. But the definite activation mechanism is unclear. Acorrding to all the above and the complexity of asthma, it may be useful to find the genes truly related to the disease if we study the differential expression gene based on different disease conditions. So, we screen the differential expression genes of EOS at different stages of asthma by suppression subtractive hybridization(SSH), and after that, since oxidative stress and EOS migration play a key role in asthma, we choose Vitamin D3 Up-regulated Protein 1(VDUP1) related to oxidative stress, slingshot-2L associated with cystoskeleton remolding for further identification and function analysis, which may help to elucidate the basic characteristics of asthma and lay some theoretic foundation for future targeting therapy.METHODS:1. Construction and identification of cDNA subtractive library of peripheral blood EOS in a asthma patient.EOS were isolated from the asthmatic patients at the time of exacerbation and after improvement. Super SMART cDNA synthesis technology and SSH were performed to construct subtracted cDNA libraries. The positive clones were obtained by differential screening (DS) method. Then the differentially expressed cDNA fragments were sequenced. BLAST searching and literature review were done to analyze their characters related to asthma.2. Differential expression analysis of VDUP1 and SSH-2L in asthma patientswe choosed 10 normal volunteers and 15 asthmatic patients at attack and 15 asthmatic patients in remission for further verification of two genes VDUP1 and SSH-2L by semi-quantitive RT-PCR and Western Blotting.3. The correation between VDUP1 and EOS activation and its role in asthmaThe objects come from the second part. We collected and measured induced sputum EOS%, dectected serum eosinophil cationic protein(ECP) concentration by enzyme linked immunosorbent assay (ELISA).The relation between the expression of VDUP1 and induced sputum EOS%, pulmonary function index forced expiratory volume in one second to predicted value expressed as FEV1%, peak expiratory flow to predicted value expressed as PEF% and serum ECP were analyzed. 4. Influence of TRX oxydizing agent and TRX alkylating agent on EOS VDUP1IL-5 play a necessary role in EOS mitration and activation, so 3 pieces of normal peripheral blood EOS were cultured with IL-5 and diamide, a TRX oxydizing agent and Chloro-2-4-dinitrobenzene, an alkylating agent, which both can destroy the interacting catalytic active center of TRX, ECP concentration were measured by ELISA as an index of EOS activation and then we explored the possible regulation mechanisms of VDUP1 and its relation to ECP level in supernatant.Statistical calculations were performed using SPSS 11.0 software. All data are expressed as mean±SEM. An analysis of variance (ANOVA) and LSD methods were used to determine differences between all experimental groups. Pearson's correlation analysis was used to determine correlations between data. Significance was accepted when p< 0.05.RESULTS:1. Differentially expressed gene fragments were obtained after further differential screening of about 200 bacterial PCR fragments. The genes associated with Cytoskeletal remodeling include slingshot homolog 2 (Drosophila)(2D), protein phosphatase 1 catalytic subunit, beta isoform(PPP1cβ), protein tyrosine phosphatase, non-receptor type 6(PTPN6), protein tyrosine phosphatase, non-receptor type 12(PTPN12), DOCk8, myosin regulatory light chain interacting protein(MIR).The genes associated with oxidative stress and celluar signal transduction include VDUP1, high-mobility group box 2(HMGB2), Aquaporin 9(AQP9), dual specificity phosphatase l(MKP-l), human ras -related protein , transmembrane protein BRI, transcriptional intermediary factor 2(TIF2), SNF-1 related kinase(SNFRK), DICE1.2. Further verification results showed the relative gene expression SSH-2L are (1.648±0. 68) , (0.918±0.395) , (0.876±0.436) , respectively in patients with attack, asthma patients in remission and normal volunteer, and VDUP1 were (0.228±0.117),(0.674±0.171),(0.699±0.138) respectively, the protein expression of VDUP1 are (0.348±0.147), (0.402±0.121) , (0.422±0.163) respectively. And there was significantly increasing relative expression intensity of SSH-2L in asthma attack patients without treatments compared with normal volunteers (P=0.038),and patients in remission(P=0.047). There was a significantly decreasing relative expression intensity of VDUP1 in asthma attacks patients without treatments compared with normal volunteers and patients in remission(P=0.000), respectively.. But there were no significantly difference between patients in remission and normal volunteers.3. The serum concentration of ECP were (4.41±1.36) ng/ml, (14.3±4.29) ng/ml , (6.70±1.40) ng/ml in asthma patients with attack, asthma patients in remission and normal volunteers respectively. Induced sputum EOS% were (1.35%±0.35%),(11%±4.8%),(3.6%±0.89%) respectively/There was a negative relationship between relative expression intensity of VDUP1 and EOS% in induced sputum (r=-0.714, p=0.000) and serum ECP concentration(R=-0.735,P=0.000)4.The expression of VDUP1 in EOS was decreased by IL-5 stimulation(P= 0.000), simultaneously, the ECP in supernatant was increased(P=0.000). Diamide and Chloro-2-4-dinitrobenzene had no effect on IL-5 induced VDUP1 down-regulation, but down-regulated ECP concentration in supernatant was obseved.CONCLUSION:1. A subtracted cDNA library of differentially expressed genes in the EOS of asthma patients before and after treatment has been constructed successfully. We identified 6 differential expressed genes related to cystoskeleton remolding and 8 ones on oxidative stress and celluar signal transduction. There may exist difference in gene expression of EOS at different stages of asthma.2. SSH-2L may participate in EOS mirgration, chemotaxis pathway.3. VDUP1 participates in EOS activation and plays a role in asthma.4. IL-5 induced EOS activation is related to oxidoreduction of TRX,maybe to down-regualtion of VDUP1 as well.