Expression Of Stromal Cell Derived Factor-1 And CXC Chemokin Receptor 4 And The Effect Of Budesonide On Their Expression In Mices With Asthma

Bronchial asthma is a chronic airway disease that involved many cells, include inflammatory cells and airway structural cells and celelular component, characterized by airway inflammation, airway remodeling and airway hyperresponsiveness.Airway was injured by the repeated airway inflammation and repaired once and again, then the airway remodeling occurred gradually. Main changes of the airway remodeling include epithelial detachment, increased airway smooth muscle (ASM) mass, decreased distance between epithelium and ASM cells, subepithelial fibrosis, mucus gland hyperplasia, goblet cell hyperplasia, proliferation and remodeling of blood vessels and airway edema. Airway remodeling is closely related to the airway hyperresponsiveness and irreversible airway obstruction, but the specific mechanisms how airway remodeling developed was not well understood. Stromal cell derived factor-1(SDF-1) and CXC chemokine4(CXCR4), which belong to CXC chemokine family, can attract inflammatory cells, promote angiogenesis and regulator other cytokine, maybe play a role in the process of airway remodeling. In this experiment we set up the mice asthma model,study the expression of SDF-1 and CXCR4 in the airway and the effect of budesonide on their expression, aim at offering effective channel and wide thinking of the treatment and earlier intervention of asthma.ObjectiveThrough building the asthma airway remodeling model in mices, to study the expression of stromal cell derived factor-1(SDF-1)and CXC chemokine receptor 4(CXCR4) in the airway and the effect of budesonide on their expression in mices with asthma. Methods1 animal group and asthma modelThirty BALB/c male mices, aged 6-8 weeks, were randomly divided into three groups:placebo control,untreated asthma, and budesonide-treated asthma,10 mices every group. The untreated asthma group were induced by intraperitoneal injection of 10% ovalbumin (OVA)suspension(containing 0.2ml normal saline, lmg aluminum hydroxide,20μg OVA)on days 1,8 and 15,and then from days 22 to 34, chanllenged by inhalation of 2% OVA aerosol every other day. The budesonide-treated asthma group received same treatment, but was additionally given an inhalation of budesonide(1 mg)before OVA challenge.The placebo control group receive the normal saline instead both on the intraperitioneal injection and aerosol inhalation stage.2 Collecting the sampleAll the mice were anesthetized by pentobarbital sodium (45mg per kg weight), then opened the chest, intubated into the pulmonary artery through the right ventricle, using normal sodium to lavage quickly, taken the middle lobe of right lung into 4% methanal to fix them, then imbed in paraffin within 24 hours. Then made 5μm thickness slice, and did HE staining and immunohistochemistry staining.3 Analysis of the HE staining and immunohistochemistry stainingUsing computer image analysis system Image Pro Plus 6.0,chosing completed cross section of brochhi, each section was quantified under a×400 objective microscope to measure the perimeter of basement membrane (μm) and area of the airway wall (μm2), area of the tracheal epithelium(μm2), area of the airway smooth muscle(μm2), then use the ratio of area to perimeter to represent the relative thickness.On immunohistochemistry staining slice, using the same analysis system, chosing five representative filed of view randomly on each slice under high power lens(×400), selecting positive area and measuring the optical density value, then calculate the mean optical density value as the last result. 4 RT-PCRUsing the trizol method to get the total RNA from the frozen left lung, then reversed transcript into cDNA.Add a specific primer and do the polymerase chain reaction to amplify the objective fragment CXCR4 andβ-actin. The agarose gel electrophoresis is conducted to display the DNA. The gel analysis system Band Scan 5.0 can be used to detect the gray scal of CXCR4 andβ-actin. The ratio of CXCR4 to P-actin represent the expression quantity of the CXCR4 in the lung.5 Statistics analysisUsing SPSS13.0 statistical package to analyze, measurement data were demonstrated by mean±standard deviation, using one-way ANOVA to compare the mean of each group, linear correlation analysis was used to assess the relation of variables, a=0.05 is the significance level.Results1 Pathologic changesIn the lung sample of mice in the untreated asthma group, we can see the airway wall thickening, lumina narrowing, epithelial detachment, increased airway smooth muscle (ASM) mass, inflammatory cell infiltration. In the budesonide-treated asthma group, thickening of airway wall and infitration of inflammatory cells were both less than those in asthma group.But there were not such changes in control group.2 Immunohistochemistry stainingThe expression of the SDF-1 was higher in the model group than that in control group and the therapeutic group of the same period,P<0.05.3 RT-PCRThe expression of the CXCR4 was higher in the model group than that in control group and the therapeutic group of the same period,P<0.05.4 linear correlation analysis The expression of SDF-1 and CXCR4 was positive correlation with the thickness of airway wall (r=0.744, r=0.553,respectively, P<0.05).ConclusionsSDF-1 and CXCR4 are tightly correlated with airway remodeling,and glucocorticoids can reduce the expression of SDF-1 and CXCR4 in the asthma airway remodeling process.