| Background:Bronchial asthma is one of the most common chronic diseases, it is a chronic inflammatory disease of the airways involved by many cells and cellular components such as neutrophils, plasma cells, monocytes, and a large number of acidophilic granulocyte infiltration and so on, chronic airway inflammation is thought to be the essence of asthma. Bronchial asthma airway inflammatory make human body have higher reactivity to all sorts of motivating factor, produce the airway constriction and reversible airflow limitation, appear cough, shortness of breath and breathing wait for a symptom, seriously affecting the patients’work and lives, therefore, the important measure for the treatment of asthma is reducing airway inflammation and cell infiltration. Glucocorticoids exert potent anti-inflammatory and immunosuppressive effects with multipath inhibition on the inflammatory medium, inflammation cells and inflammatory reaction. However, the mechanism of infiltration of inflammatory cells in patients with asthma is unclear, to explore the pathogenesis of asthma cell infiltration, looking for new treatment and drug targets is a major issue of concern.Along with the development of the researches about controlling asthma increasing, now there are more and more evidences that dysbacteriosis have close contact with allergies or asthma. Many epidemiological studies have found that obese have significant relationship with development of asthma. A previous epidemiological study found that dietary intake of development of asthma children and normal children in the past is totally different. Asthma group intaked more meat, less dietary fiber, weight of two groups is statistically significant. Dietary fiber is quite important on the body’s immune regulation with generated short chain fatty acids (SCFAs) which is including acetic acid, propionic acid, butyric acid by the intestinal anaerobic bacteria fermentation. In addition to provide5-10%of energy needs, SCFAs still play an important role on the intestinal immune function regulation through the expression of GPR43(G protein-coupled receptor43) on the surface of immune cell membrane.G protein coupled receptor (GPRs) is the largest superfamily of cell surface receptor, which is also called the7alpha helix transmembrane protein receptor, playing a very important role on the signal transduction process between cells. GPR43is a member of this family, which was discovered in the search for new GanBing peptide (Galanin) receptor subtypes cloning, the coding gene is located on the human chromosome19,19q13.1, arrangement with GPR40, GPR41and GPR42series in CD22gene downstream. As the specific receptors of SCFAs, it is also called Free fatty acid receptor2(FFAR2). Due to the receptor expressing on human various organizations such as immune cells, intestinal, fat, lung tissue, it has a variety of biological function including participating differentiation and migrationin of immune cells, lipid metabolism and mucosal immune process, confirming that it is related to the diseases such as IBD, asthma, rheumatoid arthritis, diabetes as well as cancer. In model of GPR43-/-mice, inflammatory of asthma, IBD was aggravated, proving that GPR43mediated anti-inflammatory effect which was caused by SCFAs. This receptor a large number of expression when leucocytes differentiate to all kinds of granulocyte precursor, proving GPR43play an important role on the process of activation and differentiation of leukocyte, Vinolo MA used SCFAs inducing directional migration of granulocyte in vitro, that was also dependent on activation of GPR43. Short chain fatty acids which are absored into the blood by intestinal tract are combinating with GPR43on the surface of immune cells, activating the intracellular ways such as MAPK/P38, PLC and ATF-2genes, making the intracellular signal Ca2+increasing, so as to produce all sorts of chemokines and ROS product, which will further induce the peripheral immune cell migrate to inflammation parts to play the role on immune regulation. Maslowski K M etc contributed asthma model with GPR43gene KO mice by OVA, and finally found airway inflammatory of KO group performanced more serious, more cell infiltration than wild type, the researchers believed that it was because the lack of GPR43immune cells induced high sensitivity (easier to cause allergic reaction) to various chemoattractants such as C5a and inflammatory chemokines. Secondly, Cox MA’s research confirmed that activation of the GPR43on mononuclear cells by SCFAs can release prostaglandin E2(PGE2), which could prevent the contraction effect on the airway in the past.Chronic inflammation and inflammatory cells infiltration is important pathophysiological characteristics of asthma, and GPR43is closely related to the migration of inflammatory cell and regulating function are, so this receptor may be involved in asthma, however, it has few reports about the relations between bronchial asthma and GPR43at present. This study established mice model of asthma to observe the expression of GPR43in lung tissue, to discusses the effect of GPR43on inflammatory cell infiltration on the airway, further treating asthma mice with GC to observe the regulation of GC on expression of GPR43.Objection:To investigate the Expression of GPR43in pulmonary tissue of asthmatic mice and the adjustment of glucocorticoids on the receptor.Methods:1.Thirty BALB/C6Mice were randomly divided into three groups:the control group, asthma group and dexamethasone group, n=10in each group. The asthma model was induced by classical method with ovalbumin(OVA). The mice in asthma group were intraperitoneally sensitized with OVA adsorbed on aluminum hydroxide [Al(OH)3] and nasally challenged with OVA. The group B of sensitization:all of the mice were injected pulsed liquid0.2ml(40μg OVA with lOmg Al(OH)3mixed). Challenge:All of the mice were covered (20cm×20cm×15cm) in the solution of5%OVA by aerosol inhalation three times a week for8weeks for40minutes every time. In the group A, same volume of physiological saline solution were sensitized and challenged instead of OVA. In the group C,2mg/ml concentration of dexamethasone was injected into enterocoelia30minutes before each atomization.2.Specimen collection and processing:broken neck to make mice death24h after the final atomization, open the chest, ligature the right side of bronchial, lavage bronchoalveolar with0.9%ice bath physiological saline0.3ml, recover lavage fluid, centrifuge BALF and take the supernatant, observe staining under the microscope, account the number of total cells,eosinophils, lymphocytes, macrophages. The right was put into liquid nitrogen tank immediately after the resection for extracting RNA. The general condition of all of the mice were observed, including respiration, activity,hair and so on.48h after the last antigen stimulated blood were decapitated, opening the mice, removing the left lung upper lobe tissue into4%parafomaldehyde fixative fixed for24hours, in order to maintain the original structure. The lung tissue was sliceed through paraffin embedded.3.Lung biopsy was going staining by the methods of HE, count the number of inflammatory cells of bronchoalveolar lavage fluid (BALF) and calculate the airway wall area per unit length basement membrane(WAt/Pbm), in order to check whether the success of the asthma model or not.4.The expression of GPR43mRNA was examined using RT-PCR analysis. Expression of GPR43in lung tissue was detected with immunohistochemical. The expression of GPR43protein was evaluated by Western Blot.5.The statistical data was analyzed by SPSS13.0. Datas were reported as means+SEM (χ±s). We compared the characteristics of subjects using single factor analysis of variance (one-way ANOVA), Levene method test homogeneity of variance. The variance between every two groups was compared with LSD comparison method. If the data of the expression of GPR43do not meet the normal distribution, thus we used Dunnet T3method, The significance level setted at P<0.05think difference have statistical significance.Results:1. The appearance of the three groups of miceNormal control group:All of the mice after OVA inhalation appeared normal activities、smooth breathing、no irritability and so on. Asthma model group:All of the mice after OVA inhalation appeared irritability, shortness of breath, nose incitement, abdominal spasm, incontinence and so on, more seriously, limbs limp, dull coat. Dexamethasone group:All of the mice frequently bended nozzle, increased activity, but the degree is lighter than asthma group. EOS(4.686±0.463) are most in asthmatic group(F=777.80, P<0.01); a lot of eosinophils(4.686±0.463), lymphocytes(30.436±1.775) and macrophage (43.454±4.521) infiltrated in the pulmonary tissue in asthmatic group compared with control group (13.556±1.210、0.081±0.006、0.248±0.092、11.283±1.399、83.712±4.390)and dexamethasone group (18.433±0.625、1.091±0.109、4.746±0.516、20.529±1.551、55.137±3.412)(F=110.99, P<0.01).2. HE staining of lung biopsyThe mice of asthma model group appeared shedding of airway epithelial cells. Lots of inflammatory cells infiltrating in airway and perivascular, mainly eosinophils, which can also be found in pulmonary interstitial and alveolar. The mice of nomal control group appeared complete airway epithelium, a very small amount of inflammatory cells can be found in airway and perivascular. The mice of dexamethasone group appeared complete bronchial mucosa epithelial, mucous membrane muscle layer structure, regular bronchial cavity, some still had slight damage, EOS under bronchial submucosal significantly reduced. HE dyeing image analysis results show that WAt/Pbm of asthma group (18.325+1.004) significantly increased more than the control group (9.311+0.463), comparison of two groups have significant difference (P<0.01), the above index of dexamethasone group (12.347+2.132) significantly decreased more than asthma group, with significant difference (P<0.01), WAt/Pbm of dexamethasone group is higher compared with control group,(P<0.05).3.Detection Of GPR43in lung tissue by RP-PCR:The level of GPR43mRNA of asthma model group (0.512±0.067) compared with nomal control group (1.031±0.266) and dexamethasone group (0.759±0.046) was significantly lower(p<0.01). Observation of GPR43in lung tissue by the immunohistochemistry: GPR43expressed mainly in cell membrane such as inflammatory cells, airway epithelial cells, endothelial cells and so on. The average optical density levels of lung tissue expression of GPR43of asthma model group(1.375±0.115) compared with nomal control group(2.109±0.135) were significantly lower(p<0.01).Detection of GPR43in lung tissue by Western Blot:In the asthma model group, the levels of GPR43(0.24±0.06) in lung tissue were lower than those in the control group(1.28±0.09), the levels of GPR43in dexamethasone group (0.77±0.09) obviously improved, but still lower than those in the control group.Conclusion:1. GPR43receptor widely distributed in lung tissue of mouse.2. The levels of GPR43of asthma model group compared with normal control group were significantly lower. After treatment of dexamethasone, GPR43recovered partially expression, it is shown that GPR43is involved in the process of the pathophysiology of asthma.3.The expression of GPR43in mice lung tissue of asthma model group was significantly decreased, mainly in inflammatory cells, airway epithelial cells, endothelial cells of asthmatic mice lung tissue which may play an important role in asthma as main target cells.4.The levels of GPR43of asthma model group compared with normal control group were significantly lower, asthma and the metabolic diseases involved in GPR43, GPR43may have some relationship with each other. |
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