Effect Of KLF2 And S1PR1 On Neutrophil Migration Of Bronchial Asthma Guinea Pig

Objective: By observing the expression of KLF2 and S1PR1 in neutrophil cells of asthma guinea pig model, it is aimed to investigate KLF2 and S1PR1 affect the migration of neutrophils of bronchial asthma guinea pig, thus providing new ideas for the prevention and treatment of bronchial asthma.Methods:30 guinea pigs were divided randomly into 3 groups: normal control group, asthma group and asthma treatment group. There are 10 rats in each group. The model group and treatment group are stimulated to be the asthma model by using the sensitization of 10% OVA and FCA, and aerosol of 1%OVA. The treatment group is treated with intraperitoneal injection of dexamethasone before the aerosol excitation. The control group is treated with saline instead of OVA, as a synchronization experiment with the model group. By observing the changes of guinea pigs symptoms after the aerosol excitation and the lung tissue pathological changes, and as well, comparing the counts and differential counts of inflammatory cells in blood and Bronchoalveolarlavage fluid, detecting the changes of airway resistance by use of pulmonary function. After the separation of blood neutrophils of each groups, the migration of neutrophile granulocyte is detected by using Transwell chambers. Realtime PCR and Western blot are used to detect the expression of m RNA and protein of KLF2 and S1PR1 in neutrophil cells. ELISA is used to detect the expression of S1 P in bronchoalveolar lavage fluid. Immunofluorescence is used to detect the expression and location of KLF2 in neutrophils.Results:1.Successful construction of neutrophil cell asthma animal model(1) Observing of groups of guinea pigs the changes of physical signs after the aerosol excitation: During the period of OVA aerosol excitation, the asthma group guinea pigs shows typical asthma-like symptoms such as obviously increasing respiratory rate, forepaw nose scratching, nodding breathing, sneezing, coughing and abdominal convulsions. The treatment groups guinea pigs also shows the similar but significantly alleviated symptoms. The normal control group has no abnormal reaction.(2) Measurement of the airway resistance: The airway resistance of the asthma group increases with the increase of the concentration of methacholine, compared with the normal control group,The difference is statistically significant(P < 0.01). In comparison with the asthma group, the airway resistance levels of the asthma treatment group is decreasedunder the stimulation of different concentrations the(P < 0.01).(3) Lung tissue pathology results: The normal control group shows good integrity of the bronchus, alveolar and lung tissue structures, less inflammatory cell infiltration. The asthmatic guinea pigs shows typical pathological changes, such as bronchial remodeling, luminal stenosis, lung tissue and alveolar structure destruction, more mucous bolt and ruffle of the mucous membrane hyperplasia within the lumen, obviously thickened smooth muscle cells and inflammatory cell infiltration, among which the neutrophil infiltration is the most obvious. For the treatment group, the basement membrane, smooth muscle layer is still thickened, but the bronchial mucosa edema and inflammatory cell infiltration is significantly improved.(4) The counts and differential counts of inflammatory cell in the BALF fluid: The total cells number(124.69±7.40、45.07±2.99) and NEU%(22.05±1.57、5.04±1.07) of the asthma group is significantly higher than that of the normal group(P < 0.01), while the total number of cells(88.29±7.77、124.69±7.40)and NEU%(15.11±1.33、22.05±1.57) of the treatment group is decreased(P < 0.01).(5) Differential count of white blood cells: The NEU% in the blood of the asthmatic group is significantly higher than that of the normal group(67.04±5.56、30.74±6.02,P<0.01), while that of the treatment group is decreased(46.90±5.25、67.04±5.56,P < 0.01).2. The expression and location of neutrophil KLF2, and the expression of KLF2 m RNA and protein in the blood of each group guinea pigsImmunofluorescence, Real-time PCR and Western blot detection reveal, the average optical density of KLF2 and the expression of KLF2 m RNA and protein in the blood neutrophil of each group guinea pigs: the asthma group < the asthma treatment group < the normal group(P < 0.01).3. the expression of S1 P in the serum and bronchoalveolar lavage fluid in the each group guinea pigsELISA results show the S1 P expression in the serum and bronchoalveolar lavage fluid: the asthmatic group < the treatment group < the normal group(P < 0.01).4. Changes of the expression of neutrophil S1PR1 m RNA and protein in the blood of each group guinea pigsReal-time PCR and Western blot results show the expression of neutrophil S1PR1 m RNA and protein: the asthma group < the asthma treatment group < the normal group(P < 0.01).5. Migration experiments for detection of the migration of neutrophilsAccording to the preliminary results, the Transwell experiment is carried out by selecting the S1 P with a concentration of 0.1u M. The results show the migration rate of the blood neutrophils: the asthma group < the asthma treatment group < the normal group(P < 0.01).6. Pearson analysis on KLF2, S1PR1 m RNA and protein. The result shows that there is a negative correlation between them.7. Pearson analysis on KLF2, S1PR1 protein and asthma guinea pigs model of neutrophilic migration rate. The result shows KLF2 and neutrophil migration rate was negatively correlated, S1PR1 and neutrophil migration rate was positively correlated.Conclusions:1. For the asthma guinea pigs, the expression of neutrophil KLF2 is decreased, and the expression of S1PR1 is up-regulated. After the treatment with dexamethasone, the expression of KLF2 is increased, while the expression of S1PR1 is decreased compared with the asthma group.2. For the asthma guinea pigs, KLF2 and S1PR1 show a negative correlation.3.For the asthma guinea pigs, KLF2 may paly a role in anti-inflammatory through reduced migration of neutrophils.4.For the asthma guinea pigs model, S1 P chemokine binding to its receptor S1PR1 may through promote the migration of neutrophils, thus promoting neutrophilic inflammation in asthma.