The Effects Of Doxycycline On VEGF, MMP-9and Vascular Remodeling In Asthmatic Rats

Objective: To study the effects of doxycycline on the expression ofVEGF, MMP-9and asthma remodeling in asthmatic rats. The expression levelof VEGF, MMP-9in lung tissue and serum were detected in the asthma group.The airway wall thickness and vascular density were measured in the asthmagroup. Through comparing the differences of VEGF, MMP-9, airway wallthickness and vascular density between doxycycline intervention group andasthma group, we investigate the effects of doxycycline on asthma vascularremodeling.Method: Thirty three cleaning SD rats (male, six weeks, and weightfrom50to100grams) were randomly divided into three groups: control group,asthma group and doxycycline intervention group. Each group has11rats.The asthma group rats were sensitized on the first day and the8th day withovalbumin (100mg) and AL(OH)3(100mg) by intraperitoneal injection (mixedin normal saline2ml). And then these rats were inhaled aerosolized OVA in aclose box from the15th day. We adjusted the concentration according to therats’ symptoms. The rats were inhaled30minutes each challenge. They weretotal20times and each challenge at48-hours intervals. The successfullychallenged rats were showed such as uneasy, cough, shortness of breath, andless activity. The doxycycline intervention group rats received30mg/kgdoxycycline by gavage30minutes before OVA challenge and eachdoxycycline gavage at48-hours intervals. And the doxycycline interventiongroup challenge process was the same as the asthma group. In the controlgroup, rats were challenged by the process same as the asthma group and thedoxycycline intervention group but using only normal saline replace OVA.After24hours of last challenge, rats were anesthesiaed by1%pentobarbitalinand sacrifice. The numbers of total cells, neutrophils, lymphocytes and eosinophils in the BALF were counted. Lung tissues were sliced and stainedwith HE. The expressions of VEGF and MMP-9in lung tissues were observedby immunohistochemistry combined with the micro-image analysis, and meanoptical densities of VEGF and MMP-9were measured by image analysissoftware. The contents of VEGF in serum were measured by ELISA. Themakers such as the airway wall thickness and vascular density were measuredby image analysis software.Results:1Results of total cells and eosinophils in the BALF(×106): The numbersof total cells and eosinophils in the BALF in the three groups were as follows: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. Theasthma group were significantly more than the doxycycline intervention group(P<0.01), and all the cells were significantly more than that in the controlgroup (F=64.840P＜0.05, F=90.598P＜0.01).2Pathological findings: The obvious inflammatory cells infiltration,goblet cells and smooth muscle cells proliferation, vascular hyperplasia,airway wall thickness were observed in the asthma group. The pathologicalchanges in the lung tissue of doxycycline intervention group were all betterthan the asthma group and were not observed in the control group.3Results of image analysis: The airway wall thickness (μm~2/μm) andvascular density (A/mm~2) in the asthma group (109.95±13.85,155.55±14.31)were significantly more than the doxycycline intervention group (86.37±5.85,116.64±7.33)(P<0.01). The airway wall thickness and vascular density in thetwo groups were significantly more than the control group(65.47±7.22,95.45±7.19)(F=58.737P＜0.01, F=98.885P＜0.01).4Results of immunohistochemistry: The expression levels of MMP-9and VEGF in lung of the asthma group (0.52±0.06,0.45±0.04) weresignificantly higher than the doxycycline intervention group (0.39±0.06,0.27±0.06)(P<0.01). The expression levels of MMP-9and VEGF in lung ofthe two groups were also significantly higher than the control group (0.24±0.06,0.21±0.04)(F=57.966P＜0.01, F=75.261P＜0.01).5Result of ELISA: The content of VEGF in serum of the asthma group(202.86±16.90) was significantly higher than the doxycycline interventiongroup (169.94±16.65)(P<0.01). The contents of VEGF in serum of the twogroups were significantly higher than the control group (137.75±17.02)(F=41.043P＜0.01).6Results of correlation analysis: There was a significant positivecorrelation between the airway wall thickness and vascular density (r=0.891P＜0.01). There was also a significant positive correlation between the contentof VEGF in serum and vascular density (r=0.855P＜0.01). There weresignificant positive correlations between the expression levels of MMP-9andVEGF in lung and the vascular density (r=0.793P＜0.01, r=0.889P＜0.01).There was also a significant positive correlation between the expression levelsof MMP-9and VEGF in lung (r=0.796P<0.01).Conclusion:1We observed obviously inflammatory cells infiltration in lung tissueof the asthma group. It demonstrated that bronchial asthma is a chronic airwayinflammatory disease which usually has various inflammatory cellsparticipated. Doxycycline can obviously relieve the airway inflammation.2Doxycycline reduced the extent of glandular hyperplasia, airway wallthickening and vascular hyperplasia in lung tissue of the asthma group.Doxycycline also decreased the airway wall thickness and vascular density.There was a significant positive correlation between the airway wall thicknessand vascular density. Vascular remodeling plays an important role on airwayremodeling. And doxycycline decreased vascular remodeling and airwayremodeling.3The expression levels of MMP-9and VEGF in asthmatic rats wereobviously increased. Doxycycline also significant decreased the expressionlevels of MMP-9and VEGF. There was a significant positive correlationbetween MMP-9and VEGF. It demonstrated that MMP-9and VEGF play animportant role on the pathogenesis of bronchial asthma. And it has a cooperation effect between MMP-9and VEGF. Doxycycline significantinhibits the activity and expression of MMP-9. And doxycycline also candecrease the expression of VEGF.4The results of correlation analysis showed significant positivecorrelation between the expression level of MMP-9, VEGF and the vasculardensity. It demonstrated that MMP-9and VEGF play an important role onvascular remodeling of asthma. MMP-9and VEGF can promoteneovascularization and increase vascular remodeling. Our data showed thatdoxycycline could effectively relieve the airway inflammation, vascularremodeling and airway remodeling. This effect maybe achieved bydownregulation the expression of MMP-9and VEGF.