The Preventive Effects Of Bacillus Calmette-guerin Vaccinated On Th1/Th2 Balance And Airway Extracellular Matrix Remodeling In A Mouse Model Of Asthma

Objective: The rat asthmatic model is created successfully .Two groups of rats are vaccination with BCG ,the aims to investigate: (1)The immunologic mechanism of BCG vaccination effecting on Th1/Th2 balance in asthmatic rats. (2)Whether BCG vaccination can decrease the airways inflammatory and airway extracellular Matrix remodeling. (3)Whether the inflammatory cells infiltration and airway remodeling of multiple BCG vaccination rats are more significantly ameliorated compared than those of rats with BCG single vaccination.Method: Thirty-two male Sprague-Dawley rats were randomly divided into four groups:control group, asthmatic group,BCG single vaccination group(BCG1) and multiple BCG vaccination group(BCG3). The control group rats were sensitized and challenged with normal saline. The asthmatic group rats were sensitized and challenged with ovalbumin(OVA). The BCG vaccination group rats were respectively intradermally single or multiple injected BCG. Twenty-four hours after last challenge rats were measured by pulmonary function. Bronchoalveolar lavage fluid (BALF) cells was collected and counted.The lung tissues were sliced and stained with H.E,then the bronchial basement membrane perimeter(Pbm),total bronchial wall area(WAt),and smooth muscle area(WAm) were measured by image analysis system. The level of interferon-γ(IFN-γ), interleukin-4(IL-4), matrix metalloproteinase-9 (MMP-9) and transforming growth factor-β1(TGF-β1) in serum was measured by enzyme linked immunosorbent assay(ELISA). The expression of TGF-β1 and MMP-9 in airway tissues were observed by immunohistochemistry,and Masculine gray value of MMP-9 and TGF-β1 were measured by image analysis software. Results:(1) Bronchoalveolar lavage fluid (BALF)cells are collected and counted(×106/L):The numbers of total cells and eosinophils have a significant increase in asthma group (17.44±5.89,2.21±0.98)rats compared with those of control group(5.40±1.52,0.47±0.29),BCG single vaccination group (14.61±2.15,2.15±0.34) and multiple BCG vaccination group (8.78±1.26, 1.19±0.27). The number of total cells and eosinophils are significiantly different among the four groups(p<0.05).The number of eosinophils and the serum levels of MMP-9 are positive correlation(r= 0.551,P<0.01).(2) The lung tissue pathology of rats in each groups :Obvious infiltration of inflammatory cells,damage of airway mucosa,proliferation of goblet cells and increasing of the mucus secretion as well as enhancement of basement membrane and vascular smooth muscle hyperplasia can be seen in the lung tissue of asthmatic rats. The pathology characteristics of lung tissue in asthmatic rats are decreased after BCG vaccination. The lung tissue change of rats with multiple BCG vaccination are decrease compared with those of rats with BCG single vaccination .(3) The concentration of IFN-γin serum of control group (30.36±3.65) rats and multiple BCG vaccination group(22.66±2.05) rats are significantly higher than that of asthmatic group rats(10.92±1.93)(all P<0.01). The concentration of IFN-γin serum of BCG single vaccination group rats(18.20±1.73) are higher than that of asthmatic group rats and lower than that of multiple BCG vaccination group rats.(F 87.58, all P<0.01). The serum levels of IFN-γand MMP-9 are significantly negative correlation(r= -0.919,P<0.01).The concentration of IL-4 in serum of asthmatic group rats (10.92±1.93)are significantly higher than that of control group (11.67±1.48) rats and multiple BCG vaccination group rats(15.82±0.88 )(all P<0.01). The concentration of IL-4 in serum of BCG single vaccination group rats (18.83±1.11)are higher than multiple BCG vaccination group rats and lower than asthmatic group rats(F=297.21, all P<0.01).The serum levels of IL-4 and MMP-9 are significantly positive correlation(r= 0.964,P<0.01). (4) Results of pulmonary function observation: The basic airway pressure peaks of asthmatic group(10.71±2.47) rats and BCG single vaccination group(10.81±1.05)rats are lower than control group (13.02±2.44)rats and multiple BCG vaccination group rats (13.15±1.471)(F 16.64,P<0.05). The basic airway pressure peaks of multiple BCG vaccination group rats and control group rats have not a statistics significance(P>0.05).The peaks of airway pressure are lower in rats of asthma group and BCG single vaccination group than the rats of control group after inject Ach (F23.909,52.465,P<0.05).(5) Results of the concentration of MMP-9 and TGF-β1 in serum :The serum levels of MMP-9 and TGF-β1 in asthmatic group rats are 207.13±5.30, 268.20±2.00 respectively ,they are significantly higher than those of control group(107.59±4.56, 147.96±3.01)rats respectively, and in multiple BCG vaccination group(130.75±7.00,183.12±4.21)rats respectively, and in BCG single vaccination group(157.31±7.42,199.12±4.50)rats (F 382.70,1031.25,all P<0.01).The line related analysis result: The serum levels of MMP-9 and airway wall thickness are positive correlation (r=0.720,P<0.01).The serum levels of TGF-β1 and airway wall thickness are positive correlation (r=0.796,P<0.01).(6) The expression of MMP-9 and TGF-β1 in airway tissues are observed by immunohistochemistry and the system of micro-image analysis,the Masculine gray value was used as statistic:MMP-9: The lung tissues expression of MMP-9 in asthmatic group (128.86±3.80) rats are higher than that of control group(161.58±3.33)rats (P<0.05); The lung tissues expression of MMP-9 in multiple BCG vaccination group (155.41±4.35) rats are higher than that of control group rats but lower than that of asthmatic group rats (P<0.05); The difference between BCG single vaccination group (128.39±4.69)and control group are no statistics significance (F=146.104,P>0.05).The Masculine gray value of MMP-9 and airway wall thickness are significantly negative correlation(r= -0.919,P<0.01). It means that the increase of the expression of MMP-9 have a positive correlation with the increase of airway wall thickness.TGF-β1: The lung tissues expression of TGF-β1 in asthmatic group (128.86±3.80)rats are higher than that of control group(165.56±6.37)rats (P<0.05). The lung tissues expression of TGF-β1 in multiple BCG vaccination group (159.43±5.77)rats and BCG single vaccination group(123.94±2.43)rats are higher than that of control group rats but lower than that of asthmatic group (P<0.05)rats.There are significiantly statistics difference among the four groups rats.(F=177.316,P<0.05)The Masculine gray value of TGF-β1 and airway wall thickness are negative correlation(r= -0.535,P<0.05). It means that the increase of the expression of TGF-β1 have a positive correlation with the increase of airway wall thickness.(7) Results of the thickness of airway wall and smooth muscle in the four groups by optical microscope and image analysis system:the airway wall thickness and smooth muscle thickness of asthmatic group(98.17±9.38, 26.77±6.79)rats respectively were thicker than those of control group (61.71±8.78,12.22±2.63)rats respectively, and in multiple BCG vaccination group(72.11±8.70,16.98±1.66) rats respectively, and in BCG single vaccination group (81.21±8.71,22.53±4.03)rats, and the thickness of airway wall and smooth muscle in multiple BCG vaccination group rats and BCG single vaccination group rats are thinner than those in asthmatic group(P<0.05)rats, the thickness of airway wall and smooth muscle in multiple BCG vaccination group rats thinner than those in BCG single vaccination group rats(P<0.05).Conclusions:1 We Succesfully to created the asthmatic model of rats by sensitized with ovalbumin and aluminum hydroxide and challenged with repeated exposure to aerosolized ovalbumin.2 The numbers of total cells and eosinophils in bronchoalveolar lavage fluid (BALF)have a significant increase in asthma group rats, and following with the concentration of IL-4 in serum have a significantly higher than control group rats , as well as following with the concentration of IFN-γin serum have a significantly lower than those of control group rats. The number of eosinophils have a significant increase ,and comparing with obviously infiltration around the airways in the pulmonary pathology.3 The concentration of TGF-β1 and MMP-9 in serum of asthmatic group rats is significantly higher than that of control group rats,as well as the expression of TGF-β1 and MMP-9 in asthmatic group rats have a significantly increased. The lung tissue expression of TGF-β1 ,MMP-9 and airway wall thickness are positive correlation . Both of them were correlated with the airway remodeling.4 BCG vaccination were significantly decreased eosinophils infiltration and the airway hyperresponsiveness. It is the effect of BCG vaccination to increase the concentration of IFN-γin rats serum ,and to decrease the concentration of IL-4 in rats serum. the BCG vaccination can improve the balance of Th1/Th2 partially.5 BCG vaccination are significantly decreased the concentration of TGF-β1 and MMP-9 in serum of asthmatic group rats,as well as decrease the lung tissue expression of TGF-β1 and MMP-9 in airway tissues of asthmatic rats, and then to descreat the proliferation of airway smooth muscle and collagen deposition,to improve the pulmonary function of asthmatic rats. So it may be delay the development of airway remodeling.6 The preventive effect of multiple BCG vaccination on airway inflammatory cells infiltration and airway remodeling are better than single vaccination with BCG.