Expression And Significance Of Indoleamine 2,3-dioxygenase In The Lung Tissue Of Asthmatic Mice

Bronchial asthma (Bronchial asthma) is one of the most common chronic respiratory diseases in the world, and its incidence is increasing year by year [1-2], a serious threat to the health of children. The pathogenesis of asthma is complex, the previous studies suggested that reduced and Thl subsets, the subgroup Th2 enhancements, recent studies found that T helper lymphocytes which is independent of Thl and Th2 cells in the Th17 involved in the occurrence and development of asthma. In recent years, the status of immune tolerance in the pathogenesis of asthma has been paid more and more attention, the domestic and foreign literature shows that the formation of immune tolerance induced asthma may become a new target for prevention and treatment of asthma. Indoleamine 2,3-dioxygenase (Indoleamine2, 3-dioxygenase, IDO) is a kind of immune regulation of enzyme, immune mediated inhibition of body reaction, play an important role in the induction of immune tolerance. IDO is the rate limiting enzyme of tryptophan catabolism, formed a low tryptophan environment of [3] in local tissue. IDO along the kynurenine pathway of tryptophan degradation of the L-metabolite kynurenine, kynurenic acid,3-hydroxy-Nick acid and a series of [4] functions. IDO in autoimmune diseases, organ transplantation, cancer, pregnancy in the process of the protection of the fetus from maternal rejection and more research, and in the pathogenesis of bronchial asthma research is less, so we establish a mite asthma mouse model, to investigate the IDO role in the pathogenesis of asthma.In allergen selection, our former are using ovalbumin (OVA) as allergic asthma model was established, but there are some defects, and dust mite is one of the most common allergen of asthma, similar to the pathological process of asthma in asthmatic model and human development, a variety of inflammatory factors, organization structure model of asthma the pathogenesis of better in. This experiment through the establishment of a mouse model of asthma mice dust mites, pathology, bronchoalveolar lavage fluid (Bronchoalveolar Lavage fluid BALF) in the number of inflammatory cells, the expression of IDO protein in serum and BALF fluid IL-17 and IFN-ylevels in lung tissue, explore the role of IDO in asthma, pathogenesis and the relationship with IL-17, IFN-y, in order to proceed from the immune tolerance, broaden the train of thought for immunological therapy of bronchial asthma, to find a new target in the treatment of asthma.ObjectiveTo detect the expression of IDO in lung tissue of asthmatic mice, asthma mice lung tissue expression of IDO, BALF and serum IL-17, IFN-y levels and relationship between the effect of budesonide, and IDO in the pathogenesis of bronchial asthma effects on immune tolerance and its significance.Materials and methods1 grouping and model of asthma mice making methodSPF level, health, at the age of 6 weeks,21 female BALB/c mice (purchased from the animal experimental center, Henan Province certificate No. SCXK (%) 20100002). Before the experiment, weighing, according to random number table method, the experimental mice were divided into control group, asthma group, budesonide group,7rats in each group to prepare the animal model. The sensitization phase:asthma group and intervention group:subcutaneous injection of 2000 U/m for first days 1 dust mite extract 100uL,7 days third,5,2000 U/m 1 intraperitoneal injection of dust mite extract 100 uL A total of 3, ninth,11,13 days by intraperitoneal injection of 4000 U/m 150 dust mite extract 1 a total of 3 times, the control group with physiological saline with the same method, the same dose of sensitized instead of sensitized mice (Note:every ml 2% dust mite extract containing aluminum hydroxide solution); excitation stage:fifteenth,16,17 days, with 4000 U /m 1 dust mite extract 50uL/time, slowly the nose,1 times/d, nasal drops before 0.3m 1 with 0.3% pentobarbital anesthetized mice. The intervention group:before every challenge, given budesonide (2ml) inhalation of LMG for 30 minutes, more than with the asthma group; control group:induced with the same dose of saline nose drops.2 patients of lung tissue and bronchoalveolar lavage specimens treated miceThe mice were intranasally challenged in the last 0.3% 24h,0.3ml by intraperitoneal injection of pentobarbital anesthetized animal, immediately take the eye blood, blood was collected in EDTA tubes,3000r/min centrifugal 10min, serum at -20 deg.c for refrigerator spare; lung quickly stripping, ligation of the left lung, left lung specimens were taken as, at -80 DEG C for Western refrigerator, bolt blot,24G venous indwelling trocar right bronchial intubation and lavage, injected into the trachea with 1mL syringe pump 0.8mL physiological saline, stay after 30 seconds back, repeated 3 times irrigation, saline finally back out of the 1.0-1.8mL, the recovery rate is more than 80%, transfer to EP, to 3000r/min in bronchoalveolar lavage fluid in centrifugal supernatant 10min, for detection of cytokines, cell pellet by 100ul physiological saline by cell counting plate calculation of the total number of white blood cells, and the right lung tissue, double distilled water washing of formaldehyde fixed paraffin embedded sections, respectively, for HE staining and immunohistochemistry staining.3 HE stainingThe right lung tissue formalin fixed paraffin block over, preparation, sectioning, toast, and dewaxing, hydration, hematoxylin eosin staining, HE staining slides were prepared, the same level of bronchial microscope to observe the inflammatory cell infiltration and morphological changes of the bronchial wall, (the production process in strict accordance with the HE staining steps).4 immunohistochemistry stainingThe mice lung tissue paraffin section, baking, cooling water, were fixed, paraffin embedded, frozen sections, made of 3 m thick slices.In the repeated dewaxing xylene solution 2 times, dehydration, adding antigen repairing liquid repair, endogenous peroxidase activity was blocked with 1:100, nIDO antibody, incubation,4 degrees for the night; two anti dropping, incubation, and then dehydrated mounting, production strictly in accordance with the immunohistochemical staining procedure. The application of computer pathological image analysis system, under the microscope at high magnification (10 * 40) expression and determination of protein positive cells were observed under the. Each slice randomly selected 5 high magnification range, the average unit area integrated optical density (IOD) value.5 IL-17, IFN-? levels in serum and BALF fluid detection in ELISA methodSerum and supernatant of BALF mice will be saved in-20 C refrigerator out, in strict accordance with the IL-17, IFN-? ELISA kit operation.6 The detection of IDO protein expression of Western bolt protein blotting-80 C refrigerator frozen mice left lung. Lung tissue weight, says 1g, adding tissue protein extraction reagent to extract protein, precooling homogenate grinder processing of 1-2min, the number of samples were placed on ice, and incubated for 30min, centrifugal 15-20 minutes, the supernatant was collected, stored at -20. Kaumas Coomassie brilliant blue G-250, bovine serum albumin (BSA) protein quantification, packaging,-20 degrees refrigerator, polyacrylamide gel preparation 15%, the solution was injected into the glass sandwich board, at least 100 DEG C water bath polymerization of 30min,5min,5min some kind of centrifugal, hole Marker and protein electrophoresis, then. Kaumas, Li Chunhong staining of Coomassie brilliant blue R-250 dyeing, bleaching in the table to the protein bands were clear, will transfer buffer glue into the prepared for transfer film, transfer film after the completion of skim milk powder closed, slowly shaking table at room temperature 2 h, and then placed in 10mL 1:1000 skimmed milk powder diluted antibody,4 degrees overnight, discard a anti blocking buffer, two antibody incubation, then the exposure and development.7 statistical analysisApplication SPSS 17.0 statistical software for data analysis. The analysis of data to mean+standard deviation (x±s) said, the sample mean difference between groups compared with single factor analysis of variance, LSD-t method two two, using the Pearson correlation between the two factors analysis, a=0.05 as the test standard.Results1 The experimental animal to observe the general conditionsMice in asthma model group after the dust mite extract repeated intranasal challenge, significant shortness of breath, head and facial itching, forelimb lift, back shrinkage, irritability asthma acute attack period, non stimulating water increased, defecate increase phenomenon. Budesonide intervention group also appeared similar to the symptoms of asthma group compared with asthma group, but the performance of light. The control group of mice breathing, exercise and diet showed no abnormality.2 Pathological changes in lung tissue of mice in each group wereMice in asthma model group:airway wall thickness increased, airway epithelial cell shedding, goblet cell metaplasia, collagen fiber hyperplasia, the infiltration of inflammatory cells, eosinophil, lymphocyte. Control group:airway inflammation and asthma were similar, but the degree was lighter. The mice of control group:airway wall thickness in normal, no goblet cell metaplasia, scattered inflammatory cells, luminal stenosis.3 groups of mice airway wall thickness in asthma group than in the intervention group and the control group, the intervention group was higher than that in the control group, the differences were statistically significant (P<0.05). (see Figure 4.1, table 4.1)3 Bronchoalveolar lavage cell count and classificationThe results showed that cells in group BALF, the total number of eosinophils, lymphocytes and intervention group compared with the control group, the intervention group was significantly higher than that in the control group, there was statistical significance difference, two two (P<0.05). (see table 4.2)4 Each group of lung tissue in mice with IDO immunohistochemical resultsImmunohistochemistry showed that the positive cells brown area area. The results showed that IDO mainly existed in the mouse around bronchial wall, the wall of small vessels, the positive area integral optical density average value of IDO was lower than that in asthma group showed that the intervention group and the control group, IDO expression in the intervention group was lower than that of the control group, there was significant difference between the three groups (P<0.05). (see table 4.3, Figure 4.2)5 BALF and in serum IL-17, IFN-? levelThe level of IL-17 in asthmatic mice serum group and intervention group compared with the control group, the intervention group increased, higher than that of the control group (F=65.15 P<0.05), the difference was statistically significant, the serum levels of IFN-? levels decreased with the intervention group and the control group, the intervention group was lower than that of the control group (F=107.31 P< 0.05), the difference was statistically significant; BALF mice the level of IL-17 in asthma group were higher than those in the intervention group and the control group, the intervention group was higher than that of control group, (F=22.23, P<0.05) difference was statistically significant; there was a significant difference in IFN-levels in 3 groups of mice in BALF two two, further comparison, the asthma group (1.53±0.30) compared with the intervention group (3.9±2.88) and the control group (11.92±4.19) decreased, the intervention group was lower than the control group.(see table 4.4, table 4.5)6 Results the expression of IDO protein in lung tissue of miceGray level ratio IDO/ control groups respectively:the asthma group, the intervention group the expression of protein was significantly lower than the control group (F=5.31 P<0.01), the intervention group was lower than the control group (F=5.31 P<0.01). (see table 4.6, figure 4.3).7 Correlation analysisPearson correlation analysis showed that:there was a positive correlation of IL-17 levels of IL-17 and BALF in serum (r=0.921, P<0.01) were positively correlated with IFN-?levels, IFN-?levels and BALF in serum (r=0.981, P<0.01); the level of IL-17 in serum and IFN-?levels were negatively correlated (r=-0.933, P<0.01); BALF IL-17 and IFN-? level was negatively correlated (r=-0.905, P<0.01); the expression of IL-17 protein in lung tissue and serum IDO in negative correlated (r=-0.916, P<0.01), and positively correlated with the IFN-? levels in serum (r=0.956, P<0.01); IL-17 BALF expression and IDO protein level in lung tissue in a negative correlation (r=-0.905, P<0.01), and IFN-? levels in BALF positive correlation (r=0.963, P<0.01), as shown in figure 4.4-4.11.Conclusions1. The low expression of IDO in lung tissue of asthmatic mice, the mice immune function decreased and tolerance of asthma (or lack of), conducive to the formation and development of bronchial asthma;2. Was negatively correlated with the level of IL-17 in serum and BALF IDO expression in lung tissue of mice, suggesting that IDO can inhibit the IL-17 response to reduce or inhibit airway inflammation;3. In serum and BALF in mice lung tissue IFN-? levels and IDO expression was positively correlated, suggesting that IFN-expression can be induced by IDO;4. Inhaled corticosteroids can inhibit the airway inflammation in asthmatic mice,and the expression level of IDO protein in lung tissue of mice increased.