The Effect Of Oral Administration Of BCG-PSN On Immunoloregulation In The Asthmatic Rat

ObjectiveTo explore the feasibility and the possible mechanism of immunoloregulation of prevention and treatment of asthma by bserving the changes of the airway inflammation, the number of the CD4+CD25+regulatory T cells in spleen, concentration of IL-4and IFN-y in blood serum and concentration of sIgA in intestinal mucosa after oral administration of BCG-PSN intervention.Method1. Fifty adult male Sprague-Dawley (SD) rats (weight160±20g) were divided randomly into five groups:Model group, Control group, oral Bacille Calmette-Guerin vaccine(BCG)group,oral BCG-PSN group [BCG-PSN(A)g-roup] and intramuscular injection of BCG-PSN group [BCG-PSN(B)group],10rats in each group.2. On the first day and fifteenth day, the other four groups except the control group were sensitized to be injected intraperitioneally with10%OVA (lml) plus10%aluminium hydroxide; After the21th day, they were challenged with2%OVA through Nebulized Pump, twenty minutes for each time, three times every week, lasting nine weeks to built the chronic rat asthma model. The control group were sensitized and challenged by normal saline.3. From the first day of experiment, the BCG and BCG-PSN were gived to rats. The BCG group were treated with Bacillus Calmette-Guerin Vaccine administered intragastrically,50ug for each time, two times for every weeks, lasting twelve weeks. The BCG-PSN(A) group were treated with polys accharide nucleic acid fraction of BCG administered intragastrically (250ug/kg-d), two times for every weeks, lasting twelve weeks.The BCG-PSN(B) group were injected intramuscularlly with polys accharide nucleic acid fraction of BCG(45ug/kg-d), two times for every weeks, lasting twelve week.4. All rats were anesthetized within24hours after the last challenge. The airway resistances of rats were assessed after intubation through provocated by histamine hydrochloric acid. Some parts of the spleen tissue were cut off to prepare the cell suspension and incubate with fluorescent antibody for the detection to the the percentage of the CD4+CD25+Treg/CD4+T cells.The peripheral blood were collected to detect the levels of IL-4and IFN-y by ELISA tests contributing to know the effect of BCG-PSN on TH1/TH2balance. To explore the effect of BCG-PSN on mucosal immune, intestinal mucosa were collected to detect the levels of sIgA by ELISA tests. Bronchial alveolar lavage fluid (BALF) of rats were acquired to count the number of cells and the percentage of eosinophil.The lung tissues were removed to detect the lung histomorphology.Results1. Establishment of rat model of asthma:In the challenge process, the Model group rats displayed the symptoms of the dysphoria, scratching the nose, shortness of breath, breathing nodding, cyanosis, eating little, quiescence, piloerection even depilation; The airway responsiveness airway hyperactivity of the Model group is higher than that of the Control groups(P <0.05); The histopathology of lung tissue manifested significantly inflammation around the airway, bronchial wall thickening, stenosis, airway epithelial cell shedding, mucus secretion increased significantly; their percentage of eosinophil was obviously increased (P<0.05),which indicated that the rat Model of asthma was established successfully.2. Determination of airway hyper responsiveness:Rats in each group were stimulated with histamine hydrochloride. When the concentration of histamine was0.32mg/ml, the airway resistances of the Model group、the BCG group、the BCG-PSN(A) group and the BCG-PSN(B) group were significantly higher than those of Control group (P<0.05). When the concentration of histamine was0.64mg/ml, the airway resistances of the BCG-PSN(A) group was significantly less than those of Model group0.05), but was significantly higher than those of BCG-PSN(B) group0.05).There was no statistical difference between the BCG group and the BCG-PSN(A) group(P>0.05).3. The histopathology of lung tissue:The histopathology of lung tissue of the Model group manifested significantly inflammation around the airway, bronchial wall thickening, stenosis, airway epithelial cell shedding, mucus secretion increased significantly; however, significantly reduced inflammation around the airway, airway inflammatory cell infiltration around the airway secretions decrease, bronchial wall thickening and epithelial cell detachment behavior improved significantly after rat treated by BCG or BCG-PSN.4. The assessment of cytological in bronchoalveolar lavage fluids (BALF) suggested that the number of inflammatory cells and percentage of eosinophil in BALF of the BCG-PSN(A) group were significantly less than the Model group (P<0.05),but were obviously higher than those of BCG-PSN(B) group (P<0.05). However, those were not statistical different between the BCG group and the (P>0.05).5. The level of interleukin4(IL-4) and gamma interferon(IFN-y) in peripheral blood serum:After sensitization and challenge, the levels of IL-4of Model group were obviously increased compared with the Control group (P<0.05). However, the levels of IFN-y of Model group were significantly lesser than the Control group (P<0.05). The levels of IL-4in BCG-PSN(A) group was obviously decreased than the Model group (P<0.05). The levels of IFN-y of BCG-PSN(A) was significantly higher than the Model group(P<0.05), but was obviously lower than BCG-PSN(B) group(P<0.05). The levels of However, those were not statistical different between the BCG group and the BCG-PSN(A) group (P>0.05).The levels of IL-4in BCG group, BCG-PSN(A) group and BCG-PSN(B)group were obviously increased respectively than the Model group (P<0.05).6. The detection of the number of the CD4+CD25+regulatory T cells:The percentage of the CD4+CD25+regulatory T cells accounted for the CD4+T cells in the model group was increasingly lower than Control group,(P<0.05); The percentage of BCG-PSN(A) group was prominently lower than than of BCG-PSN(B) group(P<0.05). However, those were not statistical different between the BCG group and the BCG-PSN(A) group (P>0.05).7. The level of secreting type immunoglobulin A(sIgA) of intestinal mucosa:There were not statistical differen among the control group, the Model group,the BCG group, the BCG-PSN(A) group and BCG-PSN(B) group(P>0.05).Conclusion1. The sensitization and challenge with OVA for nine weeks can construct successfully the chronic asthma rat model,2. Oral BCG-PSN can reduce airway hyperresponsiveness of the asthmatic rat, partly inhibited airway inflammation and inhibit recruitment of EOS. 3. Oral BCG-PSN can increase the levels of IFN-γ and reduce the levels of IL-4of peripheral blood serum regulating the TH1/TH2balance. The number of the CD4+CD25+regulatory T cells were lowered in the asthmatic rat. The number of the CD4+CD25+regulatory T cells accounted for the CD4+T cells were increased in the asthmatic rat after the intervention of the BCG-PSN.4. The effect of immunoloregulation of BCG-PSN intervention by oral administration or intramuslar injection was similar, but the effect of oral administration was weeker than that of the intramuslar injection.