The Effects Of Doxycycline On MMP-9/TIMP-1Balance And TGF-β₁in Asthmatic Rats

Objective: To investigate the effects of doxycycline on MMP-9/TIMP-1and TGF-β₁expression in asthmatic rats model. We established the rat asthmamodel and observed the change of MMP-9, TIMP-1in lung tissue and theTGF-β₁in serum to explore the effects and mechanisms of doxycycline onairway remodeling and airway inflammation in asthmatic rats. Thedoxycycline intervention may provide a novel way for clinical prevention ofasthma remodeling and airway inflammation.Methods: Thirty-three SD rats （male）, six weeks old, weights from50-100grams were randomly divided into three groups （11in each group）:normal control group, asthma group, doxycycline intervention group. Both theasthma group and doxycycline intervention group were sensitized byintraperitoneal injection of a mixed solution2ml mixing by OVA （100mg）mixed and AL（OH3）（100mg）（resolved in0.9%sodium chloride solution） onthe first and the8thday. Subsequently, the animals were located in a self-madenon-closed box inhaled aerosolized OVA repeatedly since the15thday, theconcentration ranged as1%,1.5%,2%,2.5%,3%. The challenge was carriedout every other day lasting30minutes each time and20times in total. Theconcentration was changed after every four times.The control group replacedOVA and AL（OH3） with equivalent sodium chloride solution and the challengemeasure is similar to the asthma group and doxycycline intervention group.Doxycycline intervention group was gavaged with doxycycline （30mg/kg）mixed with sodium chloride solution half an hour before OVA challenge. Allrats were anaesthetized by1%sodium pentobarbital24hours after the lastintervention. Then the rats were sacrificed and the serum, bronchoalveolarlavage fluids （BALF） and lung tissue of each group rats were obtained. Thennumbers of total cells, neutrophils, eosinophils and lymphocytes were measured. The expressions of TGF-β₁in serum were also detected by ELISA.HE staining was used to observe the morphology lung tissue. The parameterssuch as bronchial basement membrane perimeter, total bronchial wall area,which reflect the thickness of airway wall, were measured by computer imageanalysis system. The expression levels of MMP-9and TIMP-1in lung tissuewere observed by immunohistochemistry. The mean optical densities ofMMP-9and TIMP-1were measured by image analysis software.Results:1Rats asthma modelThrough the sensitization and repeated challenge of OVA, wesuccessfully established rat asthma model. The asthma group rats showedshortness of breath and irritability during the challenges; In the period of thewhole experiment, rats showed less activity, slow response, weight loss, etc.2The total cells in BALF （106/L） and classificationThe total cells in BALF asthma group （35.53±7.06） was significantlyhigher control group （9.42±3.67）（P<0.05） and doxycycline intervention group（14.65±5.81）（P <0.01）; the total cells of doxycycline intervention group washigher than that of control group （P <0.01）; there are significant difference inthe three groups （F=64.840, P <0.01）.The eosinophils in BALF asthma group （4.83±0.65） was significantlyhigher control group （1.24±0.72）（P<0.05） and doxycycline intervention group（2.14±0.58）（P<0.01）; the eosinophils of doxycycline intervention group washigher than that of control group （P<0.01）; there are significant differencebetween the three groups （F=96.598, P<0.01）.The NEUT in BALF asthma group （1.12±0.30） was significantly highercontrol group (0.23±0.14（P<0.05） and doxycycline intervention group（0.59±0.19）（P<0.01）; the neutrophils of doxycycline intervention group washigher than that of control group （P<0.01）; there are significant differencebetween the three groups （F=96.598, P<0.01）.The LYM in BALF asthma group (2.63±0.33was significantly highercontrol group (1.18±0.38（P<0.05） and doxycycline intervention group （2.14±0.58）（P<0.01）; the lymphocytes of doxycycline intervention group washigher than that of control group （P<0.01）; there are significant differencebetween the three groups （F=96.598, P<0.01）.3measurement of the airway wall thickness and the smooth muscle thicknessby optical microscope and computer image analysis systemThe airway wall thickness and the smooth muscle thickness in asthmagroup （109.95±13.85,56.94±8.23） were significantly thicker than doxycyclineintervention group （86.37±5.85,40.64±4.53）（P＜0.01, P＜0.01） and that incontrol group （P＜0.01）; doxycycline group were thicker than control group（P＜0.01）; there are significant difference between the three groups （F=58.737P<0.01, F=89.782, P<0.01）.4MMP-9, TIMP-1and MMP-9/TIMP-1MMP-9: The expression in asthma group （0.52±0.06） was significantlyhigher than that of control group（0.24±0.06）（P＜0.01） and doxycycline group（0.39±0.06）（P＜0.01）; doxycycline group was higher than control group （P＜0.01）; there are significant difference between the three groups （F=57.996,P<0.01）.TIMP-1: The expression in asthma group （0.62±0.06）was significantlyhigher than that in control group（0.24±0.63）（P＜0.01） and doxycycline group（0.41±0.07）（P＜0.01）; doxycycline group was higher than control group （P＜0.01）; there are significant difference between the three groups （F=102.496,P<0.01）.MMP-9/TIMP-1: The ratio in asthma group （0.83±0.04） was significantlylower than that in control group （1.02±0.04）（P＜0.01） and doxycycline group（0.94±0.06）（P＜0.01）; doxycycline group was lower than control group （P＜0.01）; there are significant difference between the three groups （F=41.839,P<0.01）.5The comparison of TGF-β₁among groupsThe concentration of TGF-β₁in asthma group （27.66±4.01） wassignificantly higher than that in control group （13.15±2.01）（P＜0.01） anddoxycycline group （18.39±2.88）（P＜0.01）; doxycycline group was higher than control group （P＜0.01）; there are significant difference between thethree groups （F=62.632, P<0.01）.6Results of related analysisThe related analysis showed that the expression of MMP-9was positivecorrelation with TIMP-1（r=0.82, P<0.01）, TGF-β₁（r=0.81, P<0.01） and theairway wall thickness （r=0.93, P<0.01）; the expression of TGF-β₁is positivecorrelation with the smooth muscle thickness （r=0.79, P<0.01）. The ratio ofMMP-9/TIMP-1is negative correlation with the airway wall thickness（r=-0.77, P<0.01）.Conclusion: Doxycycline can reduce the number of inflammatorycells in BALF, thereby reduce airway inflammation; doxycycline candown-regulate the expression of MMP-9, TIMP-1and the concentration ofTGF-β₁, thereby decrease airway remodeling. Doxycycline can ameliorateairway inflammation and airway remodeling by decreasing the ratio ofMMP-9/TIMP-1and reducing the concentration of TGF-β₁, which is one ofcrucial mechanisms for the asthma therapy. Therefore, doxycycline may be auseful for clinical treatment of asthma.