The Effects Of Bacillus Calmette-Guérin Immunization On Airway Inflammation And Remodeling In Asthma Rats

Objective: To investigate whether the bacillus Calmette-Guérin (BCG) immunization on asthma rats can prevent the airway inflammation and remodeling and evaluate the underlying immunological mechanisms.Through observe the level of IL-4 and IFN-γin the blood serum and the expression ofα-SMA and cyclinD1 in lung tisse,to explore whether BCG can adjust the balance of Th1/Th2 and lessen airway remodeling.Method: Forty Cleaning male SD rats,4 weeks old ,weights from 50 to 100 grams,were randomly divided into four groups(10 in each group):normal control group, asthmatic group , multiple BCG vaccination group and BCG intervention group.The rats of the multiple BCG vaccination group was injected hypodermatically BCG(0.025mg) on the 7th,3th,first days before sensitized.The asthmatic group ,multiple BCG vaccination group and BCG intervention group rats were sensitized on the first day and the 8th day with ovalbumin(OVA) (100mg) combinated with AL(OH)3(100mg) by intraperitoneal injection. And then those rats were challenged repeatedly with inhaled aerosolized OVA in a 20cm×30cm×40cm close tightly box from fifteen day,rats were challenged 30minutes each time,every other day ,20 times in all , The rat of the BCG intervention group was injected hypodermatically BCG(0.025mg) on the 7th day before challenged .From the 15th day on,The rat of the BCG intervention group was injected hypodermatically BCG(0.025mg)besides be challenged. The control group rats were sensitized and challenged with normal saline. After 24 hours of last challenge, rats were anesthesiaed by 1% Pentobarbitalin and measured Pulmonary function(the airway basic expiratory resistance and airway expiratory resistance after injected Ach) . Lung tissues were sliced and stained with H.E. The parameters such as bronchial basement membrane perimeter(Pbm),total bronchial wall area(WAt),and smooth muscle area(WAm) , which reflect the thickness of airway wall, were measured by image analysis system. The level of IFN-γ,IL-4in serum was measured by ELISA. The expression of CyclinD1,α-SMAin airway tissues were observed by immunohistochemistry combined with the micro-image analysis,and mean optical density of CyclinD1,α-SMAwere measured by image analysis software.Results:1 The pulmonary function among study groups:The airway basic expiratory resistance was higher in asthmatic .it is effected by respiratory peak.the respiratory peak of the asthma group and multiple BCG vaccination group,BCG intervention group were significantly lower than that in normal control group (P<0.01),and that was lower in multiple BCG vaccination group and BCG intervention group than in asthmatic group(S-N-K analysis ,P<0.05).2 The level of IL-4,IFN-γin blood serum among study groups:The concentration of IL-4 in serum of asthmatic group(33.98±2.35)ng/L , multiple BCG vaccination group (16.46±1.92)ng/Land BCG intervention group(20.89±2.29)ng/Lwas significantly higher than that of control group(11.43±2.72 )ng/L(F:136.414 ,all P<0.01), the concentration of IL-4 in serum of multiple BCG vaccination group(16.46±1.92)and BCG intervention group(20.89±2.29)ng/L were significantly lower than asthmatic group(33.98±2.35)ng/L(S-N-K analysis ,P<0.01),while the concentration of IL-4 between multiple BCG vaccination group (16.46±1.92)ng/Land BCG intervention group(20.89±2.29)ng/L were statistical significance ( S-N-K analysis ,P<0.01).The concentration of IFN-γin serum of asthmatic group(10.9±1.9) ng/L, multiple BCG vaccination group (22.7±2.1)ng/Land BCG intervention group(18.5±1.5)ng/Lwas significantly lower than that of control group(30.4±3.6)ng/L(F:90.030,all P<0.01), the concentration of IFN-γin serum of multiple BCG vaccination group(22.7±2.1)ng/Land BCG intervention group(18.5±1.5)ng/L were significantly higher than asthmatic group(10.9±1.9)ng/L(S-N-K analysis ,P<0.01),the concentration of IFN-γof multiple BCG vaccination group(22.7±2.1)ng/Lwas better than BCG intervention group(18.5±1.5)ng/L (S-N-K analysis ,P<0.05).3 The sum of cells in Broncho-alveolar Lavage Fluid ( BALF ) and classification:The sum cells in BALFof asthmatic group(17.15±3.02)×10~6 , multiple BCG vaccination group (8.78±2.00)×10~6and BCG intervention group(11.87±2.31)×10~6was significantly higher than that of control group(5.40±1.52 )×10~6(F=47.930:P<0.01), multiple BCG vaccination group and BCG intervention group were significantly lower than that of asthmatic group(.S-N-K analysis ,P<0.01).multiple BCG vaccination group were better than BCG intervention group.(S-N-K analysis ,P<0.01).The quantity EOS of asthmatic group(2.19±0.30)×10~6 , multiple BCG vaccination group (1.19±0.31)×10~6and BCG intervention group(1.49±0.30)×10~6was significantly higher than that of control group(0.51±0.18 )×10~6(F=62.569,P<0.01), multiple BCG vaccination group and BCG intervention group were significantly lower than that of asthmatic group. ( S-N-K analysis ,P<0.01).multiple BCG vaccination group were better than BCG intervention group.(S-N-K analysis ,P<0.05).4 Airway remodeling among study groups:4.1 The pathology of lung tissue:Obvious infiltration of inflammatory cells and proliferation of goblet cells as well as enhancement of mucus and smooth muscle hyperplasia can be seen in the lung tissue of asthmatic rats.After the treatment with BCG,the decrease of the above mentioned effects were observed .Observe the airway wall area and smooth muscle area in these three groups by optical microscope and image analysis system:The airway wall area and smooth muscle area were significantly thicker in asthmatic group (98.17±9.38,26.77±6.79 )and multiple BCG vaccination group (72.10±8.70,16.98±1.66),BCG intervention group (80.34±9.23,21.33±2.91)than those in normal control group ( 61.70±8.77, 12.22±2.63 ) (F:23.308;P<0.01).The airway wall area,smooth muscle area were significantly thinner in BCG group than those in asthmatic group (S-N-K analysis,P<0.05).4.2 The expression ofα-SMA,CyclinD1 in airway tissues were observed by immunohistochemistry combined with the micro-image analysis,the masc average gray scale were used as statistic:α-SMA:the masc average gray scale ofα-SMA in asthmatic group (99.6951±5.8131),multiple BCG vaccination group(116.6681±9.4061)and BCG intervention group(109.4332±5.0131) were lower than that in control group(126.3609±6.900)(见Fig3.A) (F:15.658,P<0.01),and that was higher in multiple BCG vaccination group(116.6681±9.4061)andBCG intervention group(109.4332±5.0131)than in asthmatic group (126.3609±6.9006)(S-N-K analysis ,P<0.05).while the masc average gray scale ofα-SMA between multiple BCG vaccination group(116.6681±9.4061)and BCG intervention group(109.4332±5.0131) were not statistical significance(S-N-K analysis ,P>0.05).CyclinD1 :the masc average gray scale of CyclinD1 in asthmatic group (129.8±4.9666) ,multiple BCG vaccination group (152.2±5.0692)and BCG intervention group(148.3±2.0017) were lower than that in control group(157.7±4.5079) (F:46.997,P<0.01),and that was higher in multiple BCG vaccination group(152.2±5.0692) and BCG intervention group(148.3±2.0017) than in asthmatic group (129.8±4.9666)(S-N-K analysis ,P<0.05).while the masc average gray scaleof CyclinD1 between multiple BCG vaccination group (152.2±5.0692)and BCG intervention group(148.3±2.0017) were not statistical significance(S-N-K analysis ,P>0.05).5 Result of the related analysis: The expression of CyclinD1(immunohist- Chemical method )andα-SMA positive correlation(r=0.728,P<0.05);The related analysis between cyclinD1(1/ masc average gray scale) and IFN-γwere positive correlation (immunohistochemicalmethod)(r=-0.529,P<0.05),The related analysis between cyclinD1(1/ masc average gray scale) and IL-4were positive correlation (immunohistochemicalmethod)(r=0.767,P<0.05). Conclusion:1 Obvious infiltration of inflammatory cells and proliferation of goblet cells as well as enhancement of mucus and smooth muscle hyperplasia can be seen in the lung tissue of asthmatic rats.Compare asthma group with normal control group ,the level of IL-4 in serum was significantly increased,while the level of IFN-γwas significantly decreased, It is suggested that the balance of IL-4/IFN-γ(Th1/Th2 ) play an importment role in asthma.2 Comparemultiple BCG vaccination group , BCG intervention group with asthma goup,the level of IL-4 in serum was significantly decreased,while the level of IFN-γwas significantly increased, and turns the ratio of Th1/Th2 to normal. It is suggested that BCG can lessen airway inflammation .3 The expression ofα-SMA and CyclinD1 in asthmatic group were significantly increased compared with the control group.The related analysis between cyclinD1 andα-SMA were positive correlation (immunohistochemicalmethod) ,It is suggested that Th2 reaction cause airway remodeling.4 BCG were significantly decreased the expression ofα-SMA and CyclinD1 in airway tissues of asthmatic rats ,The related analysis between cyclinD1( 1/masc average gray scale) and IL-4 were positive correlation (immunohistochemicalmethod) ,The related analysis between cyclinD1( 1/masc average gray scale) and IFN-γwere positive correlation (immunohistochemicalmethod) ,It is suggested that IL-4 can promote the expression of cyclinD1,while IFN-γcan inhibit the expression of cyclinD1,and delay airway remodeling.5 multiple BCG vaccination group,was better than BCG intervention group .It is presumed BCG can delay airway remodeling,butcan not reverse airway remodeling.