| Objective: To investigate the effect of doxycycline on airway inflamma-tion and remodeling in asthmatic rats, and to explore a new way for clinicalprevention and treatment of asthma. In order to observe the changes ofinflammatory markers of IL-5, IL-13, TNF-α and the airway smooth muscleremodeling marker α-SMA of asthma, this experiment established the asthmarat model and the changes of MMP-9, IL-5, IL-13, TNF-α, α-SMA by theintervention of doxycycline were studied.Method: The33cleaning male SD rats, which were4weeks old, andweighted from50to100grams, were divided randomly into three groups (11in each group): control group, asthma group, doxycycline intervention group.The asthma group rats were sensitized on the first day and the eighth day byintraperitoneal injection OVA (100mg) and Al(OH)3(100mg). And then thatrats were challenged with aerosol inhalation OVA in a not completely closedbox from the fifteenth day to the end of experiment. Rats were challenged30minutes each time, every other day, and the challenge had20times in total.Doxycycline intervention group was given intragastric administration withdoxycycline (30mg/Kg)30minutes prior aerosol inhalation challenged, otherprocedures were the same as the asthma group. The control group rats weresensitized and challenged replaced the OVA with normal saline; otherprocedures were the same as the asthma group. After24hours of the lastchallenge, rats were anesthetized by1%pentobarbital sodium and then werekilled. Then we collected bronchoalveolar lavage fluid (BALF), the serum andlung tissues of rats in each group. The total and different cell’s numbers inBALF were counted. The concentrations of IL-5、IL-13in serum were assayedby ELISA, and the concentration of TNF-α in serum was determined byradioimmunoassay, and the expression of MMP-9, α-SMA in lung tissues were observed by immunohistochemistry. Lung tissues were sliced and stained withHE. These parameters such as bronchial basement membrane perimeter (Pbm),total bronchial wall area (WAt) and smooth muscle area (WAm), whichreflected the thickness of airway wall, were measured by image analysissystem.Results：1The count of total cells and eosinophils in BALF (×106/L) were asfollow: control group were9.42±3.67,1.24±0.72; asthma group were35.53±7.06,4.84±0.65; doxycycline intervention group were14.66±5.81,2.14±0.58. The count of total cells and eosinophils of asthma group anddoxycycline intervention group were increased significantly compared withcontrol group（F=64.840P＜0.05，F=90.598P＜0.05). And the count of totalcells and eosinophils of doxycycline intervention group were decreased thanthat of asthma group (P＜0.05).2Pathological findings: The obvious infiltration of inflammatory cells,thickness of airway wall, basement membrane and vessel wall, hyperplasia ofvascular and smooth muscle can be found in the lung tissues of asthma group.The changes of the above mentioned in the lung tissues of doxycyclineintervention group were less than asthma group.3The thickness (μm2/μm) of bronchial wall and smooth muscle layerwere measured by the image analysis: control group were65.47±7.22,20.09±6.09; asthma group were109.95±13.85,56.94±8.23; doxycyclineintervention group were86.37±5.85,40.64±4.53. The thickness of bronchialwall and smooth muscle layer of asthma group and doxycycline interventiongroup were thicker significantly compared with normal group (F=58.737P＜0.05，F=89.782P＜0.05). And the thickness of bronchial wall and smoothmuscle layer of doxycycline intervention group were thinner than that ofasthma group (P＜0.05).4Results of the concentrations (pg/ml) of IL-5, IL-13in serum wereassayed by ELISA:The concentration of IL-5in serum of asthma group (38.63±6.64) and doxycycline intervention group (20.42±4.50) were higher compared withcontrol group (15.92±3.66)(F=61.348, P＜0.05). And that was lower indoxycycline intervention group than that in asthma group (P＜0.05).The concentration of IL-13in serum of asthma group (57.58±14.09) anddoxycycline intervention group (35.74±8.91) were higher significantlycompared with normal control group (16.98±7.53)(F=40.713, P＜0.05). Andthat was lower in doxycycline intervention group than that in asthma group(P＜0.05).5Result of the concentration (ng/ml) of TNF-α in serum was measuredby radioimmunoassay:The concentration of TNF-α in serum of asthma group (1.57±0.20) anddoxycycline intervention group (1.20±0.24) were higher significantlycompared with normal control group (0.43±0.25)(F=68.254, P＜0.05). Andthat was lower in doxycycline intervention group than that in asthma group(P＜0.05).6The expression of MMP-9、α-SMA in lung tissues were observed byimmunohistochemistry:The average optical density of MMP-9in asthma group (0.52±0.06) anddoxycycline intervention group (0.39±0.06) were higher than control group(0.24±0.06)(F=57.966, P＜0.05). And that was lower in doxycyclineintervention group than that in asthma group (P＜0.05).The average optical density of α-SMA in asthma group (0.55±0.06) anddoxycycline intervention group (0.35±0.06) were higher than control group(0.20±0.04)(F=112.663, P＜0.05). And that was lower in doxycyclineintervention group than that in asthma group (P＜0.05).7Results of correlation analysis: There were significant positivecorrelation between the concentration of IL-5, IL-13and the count ofeosinophils (r=0.817, P＜0.05；r=0.739, P＜0.05), there was a significantpositive correlation between the concentration of TNF-α and the count oftotal cells (r=0.807, P＜0.05), there was a significant positive correlationbetween the expression of MMP-9and the count of total cells (r=0.738, P＜ 0.05), there was a significant positive correlation between the expression ofMMP-9and the concentration of TNF-α (r=0.793, P＜0.05), there was asignificant positive correlation between the expression of α-SMA and thethickness of smooth muscle layer (r=0.841, P＜0.05). There were alsosignificant positive correlation between the expression of MMP-9and thethickness of bronchial wall and smooth muscle layer, the expression ofα-SMA(r=0.819, P＜0.05；r=0.883, P＜0.05; r=0.841, P＜0.05).Conclusion:1We have successfully established the airway inflammation and airwayremodeling in asthmatic model of rats by sensitizing with ovalbumin andaluminum hydroxide, and challenging with repeated exposure to aerosolizedovalbumin.2In asthma group of rats, lung tissues pathology showed a large numberof inflammatory cells’ infiltration, and the count of total cells and eosinophilsin BALF were significantly increased than control group. The serumconcentrations of IL-5, IL-13and TNF-α were obviously higher than those ofcontrol group. Inflammatory cells’ infiltration in the lung tissues pathology,the count of total cells and eosinophils in BALF and the serum concentrationsof IL-5, IL-13, TNF-α in doxycycline intervention group were lower than thatin asthma group. There were significant positive correlation between theconcentration of IL-5, IL-13and the count of eosinophils in BALF, and therewas also a significant positive correlation between the concentration of TNF-αand the count of total cells in BALF. It is suggested that doxycycline candecrease asthmatic airway inflammation by influencing the production ofinflammatory mediators.3The expression of MMP-9in lung tissues of asthma group was higherthan control group, and that was lower in doxycycline intervention group thanthat in asthma group. There were significant positive correlation between theexpression of MMP-9and the count of total cells in BALF, the concentrationof TNF-α. It is suggested that MMP-9can influence asthmatic airwayinflammation, it is also suggested that doxycycline can reduce airway inflammation of asthma through inhibiting the activity of MMP-9.4The thickness of bronchial wall and smooth muscle layer weresignificantly thicker, and the expression of α-SMA in lung tissues wassignificantly higher in asthma group than in control group. In the doxycyclineintervention group, the thickness of bronchial wall and smooth muscle layerwere thinner than asthma group, and the expression of α-SMA was lower thanthat in asthma group. There were significant positive correlation between theexpression of MMP-9and the thickness of bronchial wall, the thickness ofsmooth muscle layer, the expression of α-SMA. It is suggested that MMP-9involved asthmatic airway remodeling, and it is also suggested thatdoxycycline can reduce airway remodeling of asthma through inhibiting theactivity of MMP-9. |
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