| Objective:To study the expression of leptin and its receptor in asthma rat under intermittent hypoxia.Through establishing intermittent hypoxia asthma rat model,to observe its changes in the serum leptin concentration,the expression of leptin and its receptor mRNA,inflammatory cell in bronchoalveolar lavage fluid,airway wall and airway smooth muscle thickness.To investigate the effects of intermittent hypoxia on leptin and its receptors,and the roles in airway inflammation and remodeling,and to study the mechanism of intermittent hypoxia on airway inflammation and remodeling of asthma.Method:Forty cleaning Sprague-Dawley rats(female,6weeks old,between 120 g and 150g)were randomly divided into normal control group,intermittent hypoxia group,asthma group and intermittent hypoxia asthma group.Each group has 10 rats.The rats of asthma group were intraperitoneally injected with 100 mg of ovalbumin(OVA)and 100 mg of aluminum hydroxide(mixed with 2mL of normal saline)on day 1 and day 8 to sensitize,from day15 The rats inhaled aerosolized OVA solution 30 minutes in the self-made and,not completely enclosed box,once in every other day,adjust the drug concentration every four inhalations(from high to low in order 1%,1.5%,2%,2.5%,3%),totaling 20 times.The rats of intermittent hypoxia group were placed in the hypoxic equipment from the first day of the experiment,the equipment was circularly filled with pure nitrogen 45 minutes to decrease the oxygen concentration from 21% to 5%,and then filled with compressed air for75 minutes to restore the oxygen concentration,this was a cycle,4cycles/day(from 9am to 5pm),totaling four weeks,the rest of the time regular feeding.The rats of intermittent hypoxia asthma group were placed in the hypoxic equipment after each excitation on the day of challenge,only gave intermittent hypoxia at the time of non-excitation day.The rats of normalcontrol group sensitized and stimulated use the same amount of saline instead.Twenty-four hours after the last challenge,rats were sacrificed with 1%pentobarbital.Collected the bronchoalveolar lavage fluid,serum and lung tissue from the rats of each group.The cell classification and counting in BALF;The lung tissues were stained with hematoxylin-eosin(HE)and observed the histopathological changes.Using computer image analysis software to measure the thickness of bronchial wall(total wall area/bronchial basement membrane circumference),bronchial smooth muscle thickness(total wall smooth muscle area/bronchial wall circumference)and vascular density.The serum leptin concentration was measured by radioimmunoassay.The expression of leptin and its receptor mRNA in lung tissue were examined by RT-PCR.Results:1 The establishment of rats modelsThe model of asthma was successfully established by intraperitoneal injection of ovalbumin to sensitive and repeatedly aerosol inhalation to excite,during the progress.The rats appear shortness of breath,rhythm irregular,apathetic,unresponsive,etc.By placing the rats in hypoxic chambers,circulating hypoxia-reoxygenation processes,we Successfully established intermittent hypoxia models.and the rats appear apathetic,unresponsive,etc.2 The cells counting and classification in BALF(×10~6/L):The total number of cells:The difference among the four groups had statistically significant(F=2375.37,P<0.01),differences between any two had statistically significant(P<0.01).From high to low were intermittent hypoxia asthma group(35.16±1.08),asthma group(15.34±0.83),intermittent hypoxia group(10.26±0.55),the normal control group(9.48±0.55).The neutrophils:The difference among the four groups had statistically significant(F=424.57,P<0.01),differences between any two had statistically significant(P<0.01).From high to low were intermittent hypoxia asthma group(0.98±0.07),asthma group(0.51±0.05),intermittent hypoxia group(0.36±0.03),the normal control group(0.25±0.04).The lymphocytes:The difference among the four groups had statistically significant(F=1896.37,P<0.01),differences between any two had statistically significant(P<0.01).From high to low were intermittent hypoxia asthma group(2.91±0.36),asthma group(2.22±0.39),intermittent hypoxia group(1.65±0.74),the normal control group(1.25±0.51).The eosinophils:The difference among the four groups had statistically significant(F=5234.02 P<0.01),differences between any two had statistically significant(P<0.01).From high to low were intermittent hypoxia asthma group(3.52±0.07),asthma group(1.75±0.04),intermittent hypoxia group(0.57±0.05),the normal control group(0.57±0.05).3 Pathological results of lung tissue analysisThe sections of lung tissue of HE staining results show:Asthma group:lung tissue structure disorder,a large number of inflammatory cell infiltration,shedding of epithelial cells,airway smooth muscle and vascular proliferation.Intermittent hypoxia group:bronchial alveolar structure disorder,inflammatory cells infiltration,alveolar epithelial cells,airway smooth muscle and vascular proliferation.Intermittent hypoxic asthma group:the pathological changes were significantly increased.Pathological image analysis results show:The airway wall and smooth muscle thickness(?m2/?m)and vascular density(A/mm2)were significantly higher in intermittent hypoxia asthma(139.47±3.34,60.19±4.34,82.80±3.11)than in asthma group(98.49±5.36,42.75±3.83,59.35±2.80),intermittent hypoxia group(80.72 ± 6.27,28.04 ± 2.95,41.40 ± 2.62),and the asthma group was higher than the intermittent hypoxia group,The difference had statis-tically significant(P<0.01);The three groups were all higher than the normal control group(67.06±4.58,15.08±2.50,19.18±1.55),the difference among the four groups had statistically significant(F=394.298,P<0.01;F=311.23,P<0.01;F=1089.44,P<0.01).4 The concentration of serum leptin by radioimmunoassay(ng/ml)The concentration of leptin in intermittent hypoxic asthma group(2.74±0.24)was higher than that in asthma group(2.06±0.15),intermittent hypoxiagroup(1.72±0.16),and asthma group was higher than hypoxia group,The differences all had statistically significant(P<0.01).The three groups were all higher than the normal control group(0.93±0.17),the difference among the four groups had statistically significant(F=172.33,P<0.01).5 The expression of leptin and its receptor mRNA in lung tissue by RT-PCRThe expression of leptin mRNA in the intermittent hypoxic asthma group(0.90±0.07)was higher than that in the asthma group(0.73±0.01)and the intermittent hypoxia group(0.46±0.02),and the asthma group was higher than the hypoxia group,The differences all had statistically significant(P<0.01).The three groups were all higher than the normal control group(0.36 ± 0.01),the difference among the four groups had statistically significant(F=479.901,P<0.01).The expression of leptin receptor mRNA in the asthma group(0.61±0.04)was lower than that in intermittent hypoxia asthma group(0.83±0.04),intermittent hypoxia group(0.88±0.02),and the intermittent hypoxia asthma group was lower than the intermittent hypoxia group,The differences all had statistically significant(P<0.01).The three groups were all lower than the normal control group(0.93±0.01),the difference among the four groups had statistically significant(F=188.629,P<0.01).6 The result of correlation analysisThere was a significant positive correlation between serum leptin concentration and neutrophil count,and the total cells in BALF(r=0.886,P<0.01;r=0.834,P<0.01).The serum leptin concentration was positively correlated with airway smooth muscle thickness,airway wall thickness(r=0.943,P<0.01;r=0.916,P<0.01).There was a significant positive correlation between leptin mRNA expression and serum leptin concentration(r=0.893,P<0.01).Conclusion:1 Through antigen sensitization and repeated atomization exciting and cyclic hypoxia intervention.We successfully established intermittent hypoxiaasthma model.The results showed that there was more obvious inflammatory cell infiltration and airway wall and airway smooth muscle thickening than in the asthma group.The presence of intermittent hypoxia may aggravate asthma through airway inflammation and remodeling.2 There was a significant positive correlation between leptin concentration and inflammatory cell numbers,airway wall and airway smooth muscle thickness,that suggested leptin can aggravate asthma both in airway inflammation and airway remodeling.When simultaneously exist intermittent hypoxia,the expression of leptin at the level of protein and gene was significantly higher than that in the other groups(P<0.05),Indicating that intermittent hypoxia may aggravate asthma by increasing leptin concentration.3 The leptin receptor mRNA expression of asthma group rats was reduced,when merge with intermittent hypoxia would be relatively elevated.Suggesting that asthma may exist leptin resistance,and with intermittent hypoxia would reduce this situation,so the leptin can bind to more leptin receptor to play a biological activity that exacerbates asthma. |
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