Explore The Effects Of NKT Cells On The Different Degrees Of Bronchial Asthma

Objective: To explore the effect of Natural killer T cells on the different degreesof the mouse asthma by establishing the different degrees of the mouse asthma model.Thus to reveal the effect of NKT on the bronchial asthma and explore a new method of treatment for bronchial asthma.Methods: Establishing the different degrees of the mouse models of OVA-induce bronchial asthma and then the different severity of the mouse models were intervened by using α-galactosylceramide(α-Galcer) to activate NKT cells.The female BALB/c mice were randomly divided into five groups: NS group, 1% OVA group, 1% OVA + α-Galcer group,5%OVA group and 5%OVA+α-Galcer group.The mice in 1% OVA group, 1% OVA+α-Galcer group, 5%OVA group and 5%OVA+α-Galcer group were sensitized by intraperitoneal injection of OVA solution at 0d and 14 d,while mice in NS group were sensitized by intraperitoneal injection of NS solution as a control. One hour before the first challenged with aerosolized, the mice in NS group, 1% OVA group and 5%OVA group were given 20 ul of NS solution by the intranasal, 1% OVA +α-Galcer group and5%OVA+α-Galcer group were given 20 ul of α-Galcer solution. The mice in NS group were challenged with aerosolized NS solution, 1% OVA group and 1% OVA +α-Galcer group were challenged with aerosolized 1% OVA solution, 5% OVA group and 5%OVA+α-Galcer group were challenged with aerosolized 5% OVA solution on days 25-27,the behavioral changes of mice were observed during nebulization. Twenty-four hours after the last challenge, the AHR was measured by spirometer of mice.The mice were sacrificed,blood samples were collected and OVA-Specific Ig E were detected by ELISA and BALF were harvested for cell count and cytokines. The total number of cells were counted by automated cell counting instrument. The percentage of different cells( Lymphocytes, Netrophil, Macrophages and Eosinophil) were counted by flow cytometry. The cytokines(IFN-γ and IL-4,-5-13) in BALF were measured by CytometricBead Array. Pathological changes were observed by HE staining. The tissues can be assessed for general morphology and cellular infiltration, and the standard of this method discriminates around brochioles, blood vessels and the number of patchy cellular infiltration(Score0-3).Results: 1. The different degrees of the mouse models were established : After challenged with aerosolized NS solution for 10 min, the mice in NS group showed slight irritability, After challenged with aerosolized 1% OVA solution for 8min, the mice in 1%OVA group showed rather irritability, breathing deeply and fast, more often scratching,arched upright, forelimbs shrinkly lift, the above symptoms were obvious after challenged with aerosolized 5% OVA solution for 8 min in 5% OVA group.The scores of pathological inflammation, the total number of cells, percentages of Eosinophil and the level of cytokines(IL-4,-5,-13) in BALF, the level of OVA-specific Ig E in serum and airway resistance were significantly higher than the NS group(P<0.01 vs P<0.05), and 5% OVA group was much higher than 1% OVA group(P < 0.01 vs P < 0.05). 2.The behavioral changes of the different degrees of the mouse models after the intervention of α-Galcer :Only slight symtoms of irritability and scratching appeared in NS group after challenged with aerosolized NS solution for 10 min, the symptoms of irritability, breathing deeply and fast, more often scratching, arched upright, forelimbs shrinkly lift appeared after challenged with aerosolized 1% OVA for 8min, while the above symptoms appeared after challenged with aerosolized 1% OVA for 10 min and lessened in 1% OVA+α-Galcer group.Rather irritability, breathing deeply and fast, more often scratching, arched upright,forelimbs shrinkly lift,nodding breathing,incontinence and soon appeared after challenged with aerosolized 5% OVA for 5min in 5% OVA group, the symptoms appeared after challenged with aerosolized 5% OVA for 8min in 5% OVA+α-Galcer group. 3. The histopathological changes of the different degrees of the mouse models after the intervention of α-Galcer :Lung tissue around the bronchus,blood vessels,and pulmonary interstitial tissue showed very small amount of inflammatory cells in NS group; compared with NS group, the 1%OVA group histological results showed mild infiltration of inflammatory cells around bronchus, blood vessels and pulmonary interstitial tissue, some eosinophils, irregular bronchial lumen, alveolar fusion(P<0.05), compared with1%OVAgroup, 1% OVA+α-Galcer group show fewer inflammatory cells, little of eosinophils(P<0.01). 5%OVA group showed moderate infiltration of inflammatory cells around bronchus,blood vessels and pulmonary interstitial tissue, amounts of eosinophils, irregular and narrow bronchial lumen, alveolar fusion than NS group(P<0.01), but there no difference5% OVA+α-Galcer and 5%OVA group(P>0.05). 4. The changes of on total number cells and percentags of eosinophil in BALF of the different degrees of the mouse models after the intervention of α-Galcer : Relative to the NS group, the number of total cells in1%OVA group and 5%OVA group increased, while the 5%OVA group increased the most.The α-Galcer can reduced the number of total cells in BALF in different severity of mice asthma model(P < 0.05). The percentage of eosinophil in BALF of 5%OVA group significantly increased than NS group and 1%OVA group(P < 0.01),but there was no significant difference between 1%OVA group and NS group(P>0.05).The α-Galcer can reduced the percentages of eosinophil in BALF of 1%OVA +α-Galcergroup,but the same effect was not observed in 5%OVA +α-Galcer group. 5. The detection for cytokines in BALF of the different degreesof the mouse models after the intervention of α-Galcer :The secrection of IL-4 and IL-5 of BALF in 5%OVA group and 1%OVA group significantly increased than NS group, whlie secrection of IFN-γ in BALF decreased than NS group.The α-Galcer can decreased the level of IL-4 and IL-5 and increased the level of IFN-γin1%OVA +α-Galcer group, but there was no significant difference between 5%OVA+α-Galcer group and 5%OVA group.6. The influences on AHR of the different degrees of the mouse models after the intervention of α-Galcer : The airway resistance of 1% OVA group and 5% OVA group were significantly higher than the NS group at the concentration of 4mg/ml, 8mg/ml of histamine(P<0.01).The AHR in 5% OVA group was significantly higher compared with 1% OVA group at the low doses of histamine stimulation(P<0.05),while there was no significant difference at the high doses of histamine stimulation(P>0.05) 。 The α-Galcer can reduced the AHR in 1% OVA+α-Galcer group and 5%OVA+α-Galcer group at the concentration of 4mg/ml, 8mg/ml of histamine.Conclusion:1. 1% OVA and 5%OVA can induce different degrees of bronchial asthma in mice. 2. NKT cells can improve the changes of behavior,inhibit airway inflammation and reduce AHR in 1%OVA-stimulate slight bronchial asthma model, buteffects on 5%OVA-stimulate mild bronchial asthma model is weak.