| Objective:1 To study the effect of Chronic intermittent hypoxia on OVA induced animal models of asthma inflammation and airway remodeling.2Through the monitoring of MMP9, TIMP1,VEGF and VEGFR,We study hypoxia effects on airway remodeling.3. We explored the role of NF-?B and HIF-1 in the airway inflammation and remodeling and study the role of the hypoxia on that chang.through observing the change of NF-?B and HIF-1 in the lung tissue.Methods: Thirty cleaning Sprague-Dawley(SD) rats(female, 6 weeks, weight 120-150 grams)were randomly divided into 3 groups, normal control group, asthma model group, chronic intermittent hypoxia asthma group(intermittent hypoxia asthma group,hypoxia asthma group), 10 rats in each group. Asthma group rats were respectively in experiment on the first day and the 8th day by intraperitoneal injection with OVA(100mg) and aluminum(100mg)that mixed in normal saline 2ml.From the 15 th day, these rats were inhaled aerosolized OVA(OVA concentration increases gradually, from 1% every inspire 4 times increase, respectively, 1%, 1.5%, 2%, 2.5%, 3%) in a not completely closed atomization chamber. The rats were inhaled, 30 min every time, every other day at a time, a total of 20 times. Appears to be agitated, choking cough, shortness of breath, activity decline in typical asthma symptoms, to stimulate the success. Normal control group:sensitization and inspire all use normal saline instead of OVA,method as above. Chronic intermittent hypoxia asthma group: put rats in low oxygen cabin that is filling with pure nitrogen to 45 minutes, in order to oxygen concentration to 5%,and than is filling with pure oxygen to 45 minutes, in order to oxygen concentration to 21%, before OVA challenge. At other times, hypoxia method as above. The rat in hypoxic box to 8 hours every day(morning 9:00 to afternoon17:00),4 cycles/d, 7 d/week, and the rest of the time of conventional breeding, a total of four weeks. The last excitation after 24 hours, rats were anesthetized by 1% pentobarbital and sacrificed to determine the corresponding indicators.The blood serum, lung tissue and BALF of the rats were collected.The number of eosinophils,lymphocytes, neutrophils and total cells in the BALF were detected. Line hematoxylin- eosin hematoxylin- eosin(HE) staining, observed the morphological changes, the application of computer image analysis software testing bronchial wall thickness(the total wall area/week of bronchial basement membrane diameter), bronchial smooth muscle thickness,vascular density;.the expression of NF-?Bp65 in lung tissue is measured by immunohistochemistry and RT-PCR respectively; We measured the expression of HIF-1?,VEGFR1,VEGFR2 in lung tissue by immunohistochemistry; We measured the expression of VEGF,MMP9,TIMP1 in serum by ELISA. Application of image analysis software to determine the average optical density of NF-?Bp65, VEGFR1, HIF- 1?, VEGFR2.Results:1 Rats bronchial asthma modelBy intraperitoneal injection of ovalbumin and repeatedly atomization inhalation way successfully established rat model of asthma is observed in the experimental process of asthma rats unstable, abdominal cramps, shortness of breath, unresponsive, depression, irritability, etc.2 The cell count and classification in bronchoalveolar lavage fluid(×106/L)The total number of cells in BALF of intermittent hypoxia asthma group( 35.76±1.96) was higher than that of asthma group(15.24±1.37).The difference is statistically significant(P<0.01).And the normal control group(9.57±0.55) were lower than the two groups; among the three groups,there were significant difference(F=944.535,P<0.01).The neutrophils in BALF of intermittent hypoxia asthma group(0.98±0.09) was higher than that of asthma group(0.51±0.05).The difference is statistically significant(P < 0.01). And that of normal control group(0.25±0.03)were lower than the two groups. The difference is statistically significant among the three groups(F=335.811,P<0.01).The lymphocyte in BALF of intermittent hypoxia asthma group(2.93±0.06) was higher than that of asthma group(2.22±0.04).The difference is statistically significant(P < 0.01).And that of normal control group(1.24±0.05)were lower than the two groups The difference is statistically significant among the three groups(F=2014.357,P<0.01).The eosinophils in BALF of intermittent hypoxia asthma group(3.51±0.09) was higher than that of asthma group(1.75±0.04).The difference is statistically significant(P < 0.01).And that of normal control group(0.57±0.05) were lower than the two groups.The difference is statistically significant among the three groups(F=5576.645,P<0.01).3 the pathological image analysis:We can discover Significantly increased inflammatory cells around the blood vesselsand windpipe, mucus secretion increased,The falling off of epithelial cells, the damaged bronchial mucosa, obvious thickening of airway smooth muscle in the lung tissue of asthmatic rats. The intermittent hypoxia asthma group group can markedly attenuate those pathological change. Image analysis results that the airway wall thickness(?m2/?m), airway smooth muscle thickness(?m2/?m) vascular density(mean/mm2)hypoxia asthma group(139.47±3.34;59.79±5.01;82.80±3.11) were significantly thicker than the asthma group(98.49±5.36;42.75±3.83;59.35±2.80).The difference is statistically significant between the groups(P < 0.01); And that of normal control group(67.06±4.58;15.08±2.50;19.18±1.55)were thinner than the two groups.There were significant difference among the three groups(F=649.435,P<0.01;F=339.951,P<0.01;F=1561.07,P<0.01).4 The expression of NF-?Bp65mRNA by RT-PCR:In lung tissue, the expression of NF-?Bp65mRNA of intermittent hypoxia asthma group(0.73±0.042) was higher than that of asthma group(0.479±0.059).The difference is statistically significant(P < 0.01).And that of normal control group(0.297±0.020)were thinner than the two groups.There were significant difference among the three groups(F=1267.128,P<0.01).5 The expression of NF-?Bp65,HIF-1?,VEGFR1,VEGFR2 in lung tissue by immunohistochemistry:In lung tissue, the average optical density of NF-?Bp65 of intermittent hypoxia asthma group(0.539±0.065) was higher than that of asthma group(0.400±0.02).The difference is statistically significant(P < 0.01).And that of normal control group(0.247±0.035) were lower than the two groups.The difference is statistically significant among the three groups(F=91.862,P<0.01).In lung tissue, the average optical density of HIF-1? of intermittent hypoxia asthma group(0.66±0.12) was higher than that of asthma group(0.50±0.09).The difference is statistically significant(P < 0.01).And that of normal control group(0.31±0.07) were lower than the two groups.The difference is statistically significant among the three groups(F=36.107,P<0.01).In lung tissue, the average optical density of VEGFR1 of intermittent hypoxia asthma group(0.529±0.074) was higher than that of asthma group(0.406±0.045).The difference is statistically significant(P < 0.01).And that of normal control group(0.251±0.028) were lower than the two groups.The difference is statistically significant among the three groups(F=70.788,P<0.01).In lung tissue, the average optical density of VEGFR2 of intermittent hypoxia asthma group(0.537±0.075) was higher than that of asthma group(0.402±0.045).The difference is statistically significant(P < 0.01).And that of normal control group(0.248±0.025) were lower than the two groups.The difference is statistically significant among the three groups(F=75.549,P<0.01).6 The concentration of VEGF,MMP9,TIMP1 in serum by ELISA :In serum,the concentration of VEGF,MMP9,TIMP1 of asthma group(24.93±1.93,29.72±2.77,30.49±2.85)was lowwer than that of intermittent hypoxia asthma group(32.982±0.04,39.66±2.84,40.44±2.99).The difference is statistically significant between the groups(P < 0.01).And that of normal control group was lowwer than others( 18.57±2.01, 14.92±2.59,14.921±2.276).There were significant difference( F=70.52,P < 0.01 F=198.98,P<0.01 F=251.02,P<0.01).7 The result of correlation analysis :The results showed that NF-?Bp65and NF-?Bp65mRNA in lung tissue were significant positive correlation with the concentration of the total cells of BALF(r=0.979,P<0.01;r=0.777,P<0.01),were significant positive correlation with smooth muscle(r=0.949,P<0.01;r=0.920,P<0.01).HIF- 1?in lung tissue and vascular density was significantly positive correlation(r=0.922,P<0.01).Conclusion:1 By the antigen sensitization and repeated nebulization, OVA-inducded asthma model of rats was successfully established.In asthma group of pathologic results, we can find that airway clear airway wall thickening and inflammatory cells infiltration existed in the rats with, smooth muscle thickness. After hypoxia intervention, that increased obviously.2 The expression of NF-?Bp65 and HIF-1? of control normal group were apparently lower than that of asthma group. The average optical density results the activation of NF-?B signal transduction pathways may be related to asthma. The expression of NF-?Bp65 and HIF-1? in chronic intermittent hypoxia asthma group were higher than that of asthma group,and normal control group.It means that intermittent hypoxia may increased the activation of NF-?B and HIF-1 signal transduction pathways.3Intermittent hypoxia can aggravate the levels of MMP9?TIMP1 ?VEGF?VEGFR1?VEGFR2 and airway remodeling.4. According to the results of correlation analysis, the related literature and combined the experimental data.It demonstrated that Intermittent hypoxia may aggravate the remodeling of asthma and airway inflammation by increased the activation of signal transduction pathways that HIF-1 that influence the expression of VEGF, VEGFR of vascular remodeling indicators and NF-?B that influence the expression of MMP- 9, TIMP1 of matrix remodeling indicators. |
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