Analysis For The Relevance Of Asthma Susceptibility With The Alteration And Expression Defect In Integrinβ4

Objectives:Integrins are a class of ubiquitous cell surface transmembrane glycoprotein. Bronchial epithelial cells (BECs) forms airway epithelial barrier. To maintain the stability of its structure is dependent on the expression of integrins and extracellular matrix proteins to form heterologous adhesion between cells. At the same time, during airway hyperresponsiveness (AHR) or airway inflammation, some integrin subunits and their ligands were also involved in inflammatory cell migration and activation, resulting in the destruction of airway structure and integrity and airway epithelial exfoliation. Previously studies in our lab have confirmed that multiple adhesion molecules were abnormally expressed in asthma, among which integrinβ4 were downregulated in all detected asthma samples. However, whether integrinβ4 expression imbalance is universal in asthma; what is its abnormal expression mechanism; whether integrinβ4 expression imbalance is closely related to the development of asthma remained unclear. This present study was designed to examine the relevance of integrinβ4 expression with asthma and responding mechanism from experimental animal model and asthma patients. Methods:1. Relevance of integrinβ4 expression with asthma in animal models.①12 of SPF class SD rats were randomly divided into the asthma group (n = 6) and the control group (n = 6). The asthma group was intraperitoneally injected a 1 ml of mixture including 10% OVA and 10% aluminum hydroxide at the 1^st day and the 13^th day. At the 14^th day 1% OVA aerosol was inhaled for 30min every day in successive 7 days to induce asthma. The solution was replaced with normal saline in control group;②The small animal ventilator and small animal respiratory function analysis system (Buxco) were used to measure airway resistance (R_L) and decreased dynamic lung compliance (Cdyn);③The lavage fluid was collected for cell counting and classification after Giemsa staining;④Lung tissue biopsy was examined by using lung tissue extraction from aseptic conditions;⑤The expression of integrinβ4 on bronchial epithelial cells, basal cells, alveolar epithelial cells, vascular endothelial cell in model group and control group was examined by immunohistochemistry;⑥Lung tissue RNA was extracted and real time PCR was used to assay the RNA expression of integrinβ4.2. Relevance of integrinβ4 expression and asthma in asthma patients and the corresponding mechanism.①RNA was extracted from peripheral blood leukocytes in asthma patients and reverse transcripted into cDNA by using random primers. Then the expression of integrinβ4 was assayed by real-time PCR with specific primers and fluorescent probe;②DNA was extracted from peripheral blood leukocytes in asthma patients and the 5' regulatory region of integrinβ4 was amplified and bi-directional sequencing for analysis of sequence variation.Results1. Relevance of integrinβ4 expression with asthma in animal models.①The percentage of increased R_L and decreased Cdyn were about 2 times compared asthma group to control group;②The total number of cells and the proportion of eosinophilic cells and neutrophils in asthma model group were higher than the control group;③There is an obvious sign of bronchial contraction and infiltration of inflammatory cells surrounding small blood vessels, the alveolar space, interstitial lung in asthma group. Bronchial epithelial cells deviation, pulmonary arteriovenous edema and bronchiole obstruction could be seen. These results confirmed the successful establishment of asthma animal model; ④The results of immunohistochemistry showed that integrinβ4 in bronchial epithelial cells, basal cells, alveolar epithelial cells, vascular endothelial cells have high expression, but decrease in asthmatic rats;⑤Real time PCR showed that integrinβ4 expression decreased in rat asthma model.2. Relevance of integrinβ4 expression and asthma in asthma patients and the corresponding mechanism.①Real time PCR detection showed that the expression of integrinβ4 mRNA decreased in peripheral blood leukocytes of asthma patients;②5'flanking region of integrinβ4 in peripheral blood leukocytes of asthma patients exists gene sequence variations. There were four variation sites in 5'flanking region of integrinβ4 at -nt1029,-nt 1051 from G to A, at - nt1151 from T to G, and at -nt1164 from G to C, which may be correlated with downregulated expression of integrinβ4.Conclusion:This present study demonstrated that down-regulated integrinβ4 is highly correlated with asthma. Down-regulated integrinβ4 expression is associated with the variation in 5' flanking region.