Expression Of FIZZ1 And NOTCH1 And The Effect Of Rosiglitazone On Their Expression In Mices With Asthma

Airway is 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, mucus gland and goblet cell hyperplasia, subepithelial fibrosis, increased airway smooth muscle (ASM) mass, decreased distance between epithelium and ASM cells, airway edema proliferation and remodeling of blood vessels. Airway remodeling not only play a significant role in the pathogenesy of bronchial asthma, but also is considerable pathology characteristic of bronchial asthma. The pulmonary myofibroblast differentiate in the process of early stage airway remodeling which is know to be a critical modulator of a-smooth muscle actin(a-SMA). In asthma airway remodeling A-SMA is regards as the main parameters. The pulmonary interstitial fibrosis has similar pathology with early stage airway remodeling in asthma, so we hypothesis that the inflammatory factors which play significant role in the pulmonary interstitial fibrosis also play considerable effect in early stage airway remodeling in asthma. FIZZ1(found in inflammatory zone 1), a novel factor which was found in 2000.NOTCH1 is a transmembrane receptor proteinum and evolutionarily conserved receptor that regulatesed cell fate, including such events as differentiation, proliferation, and apoptosis. Recently studies showed that FIZZ1 and NOTCH1 induced myofibroblast differentiation in the process of pulmonary interstitial fibrosis, at the same time, NOTCH 1 participated the process of FIZZ1 inducing of myofibroblast differentiation. However, if FIZZ1 and NOTCH1 produce a marked effect in the occurrences of early stage airway remodeling in asthma,its mechanism of action and correlation between FIZZ1 and NOTCH 1 remain unknown. Many researchs showed that TZDs, such as rosiglitazone, could restrain the expression of FIZZ1. In this experiment we set up the mice asthma model, study the expression of FIZZland NOTCH in the airway and the effect of rosiglitazone on their expression, aim at offering effective channel and wide thinking of the treatment and earlier intervention of asthma.ObjectiveThe objective of this study was to investigate the expression of a-SMA,FIZZ1 and NOTCH 1 in the lung tissue from asthmatic rat and thus explore the potential role of FIZZ1 and NOTCH1 in airway remodeling in asthma and the depressant effect of rosiglitazone in airway remodeling.Methods1 animal group and asthma modelForty-five SDF male mices, aged 6-8 weeks, were randomly separate into asthmatic group, rosiglitazone group and control group,15 mices every group. The asthma group were induced by hypodermic injection in each fold inguen of 10% ovalbumin (OVA)suspension 0.2ml and intraperitoneal injection of 10% ovalbumin (OVA)suspension 0.6ml on days 1,8 and 15, and then from days 22 to 35, chanllenged by inhalation of 2% OVA aerosol every day. The rosiglitazone group received same treatment, but was intragastric administration with rosiglitazone[10 mg/(kg-d)] from days 22 to 35. The control group receive the normal saline instead both on the intraperitioneal injection,hypodermic injection and aerosol inhalation stage.2 Gathering the sampleAll the mice were anesthetized by pentobarbital sodium (40mg per kg weight), then opened the chest, taken the left lung into liquid nitrogen. Then through the right ventricle, intubated the washpipe into the pulmonary artery and lavaged quickly with normal sodium. After that, dislodged the middle lobe of right lung and put into 4% methanal, then fix them by imbeding in paraffin more than 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.On HE staining slice, observed the pathology. Using the same analysis system to analyze the immunohistochemistry staining slice. Under high power lens(×400), on each slice, five representative filed of view were choosed randomly. Then selected positive area and measured 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 FIZZ1,NOTCH1 and GAPDH. 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 seal of FIZZ1,NOTCH1 and GAPDH. The ratio of FIZZ1to GAPDH and the ratio of NOTCH 1 to GAPDH represent the expression quantity of the FIZZ1 and NOTCH1 in the lung.5 Statistics analysisThe results use SPSS 17.0 statistical package to analyze, measurement data were demonstrated by mean±tandard deviation, using one-way ANOVA to compare the mean of each group, linear correlation analysis was used to assess the relation of variables,α=0.05 is the significance level.Results1 Pato-manifestation by HE in Lung TissuesStudies in the OVA-induced model had demonstrated that a subset of inflmmatory reaction as a major histopathological features of asthma. When compared with rats in control group, the lung tissue from asthmatic group showed the airway wall thickening, lumina narrowing, epithelial detachment, increased airway smooth muscle (ASM) mass, inflammatory cell infiltration. In the rosiglitazone group, these inflammatory reactions were not as obvious as in asthma group. In control group, inflammatory reactions were not observed.2 Immunohistochemical Staining of a-SMA in Lung TissuesPositive a-SMA staining was highly expressed in peribronchial lung sections isolated from the asthma group. In peribronchial lung sections isolated from the rosiglitazone group, the expression of the a-SMA was lower than that in asthma group. In control group, the expression of the a-SMA was negative.3 RT-PCRThe expression of the FIZZ1mRNA and NOTCH1mRNA was higher in the asthma group than that in control group and rosiglitazone group of the same period.4 linear Correlation Analysis Between FIZZ1mRNA and A-SMAThe FIZZ1mRNA expression has positive correlation with the expression of a-SMAin lung.5 linear Correlation Analysis Between a-SMA and NOTCH1mRNAThe NOTCH1mRNA expression has positive correlation with the expression of a-SMAin lung.6 linear Correlation Analysis Between FIZZ1mRNA and NOTCH1mRNA The NOTCH1mRNA expression has positive correlation with the expression of FIZZ1mRNA in lung.Conclusions1 In the pathogenesis process of airway remodeling,FIZZ1 and NOTCH1 play a critical function, and they had synergistic effect.2 Rosiglitazone can delay the process of airway remodeling.