The Mechanism Of Fizz1 Induces Airway Epithelial To Mesenchymal Transit In Asthma Animal Model

Asthma is a serious public health problem throughout the world, and affects people of all ages, race, and ethnicities. However, despite significant advances in our understanding of the disease and availability of more effective therapies, Asthma is a significant burden, not only in terms of health care costs but also of lost productivity and reduced participation in family life.In the present study, we demonstrate asthma is a chronic lung disease characterized by airway inflammation, hyperresponsiveness, airway remodeling. Airway wall remodeling could be an important factor responsible for irreversible airflow obstruction and an accelerated decline in lung function.Asthmatic airway remodeling is characterized by goblet cell hyperplasia, angiogenesis, smooth muscle hypertrophy, and subepithelial fibrosis. The airway epithelium in asthma is fundamentally abnormal with increased susceptibility to environmental injury and impaired repair associated with activation of the epithelial-mesenchymal trophic unit. Chiara Folli considered the interaction between the epithelium and mesenchymal elements such as fibroblasts is essential for normal airway repair. An abnormal response of this epithelial-mesenchymal trophic unit has been proposed to be central to the airway pathology and physiology characteristic of asthma. EMT is a new insights into airway remodeling.Our previous studies have demonstrated that FIZZ1 mRNA expression was increased in AECⅡisolated from the lungs of OVA sensitized and challenged rats. In addition, FIZZ1 is associated with myofibroblast differentiation, resulting in augmented expression ofα-SMA and collagen typeⅠin early stage airway remodeling. Therefore, it indicates that FIZZ1 may play an important role in the process of airway remodeling in asthma.We hypothesize that FIZZ1 participates in the process of Epithelial to Mesenchymal transit. So, we proceeded to validate this hypothesis. We investigated the mechanisms of Epithelial to Mesenchymal transit by FIZZ1 in mouse lungs, cultured lung epithelial cells, and dissected the possible signaling pathways. Our results show FIZZ1 can up-regulation mesenchymal markers (typeⅠcollagen and Fibronectin1), with concomitant reduction in expression of epithelial markers (E-Cadherin).This reveal the critical role of FIZZ1 in EMT during asthma.Methods:Twenty female BALB/c mice (8-to 10-week-old,20±2g) purchased from the Animal Experiment Center of Shandong University were randomly divided into an ovalbumin (OVA) group and control group. They were housed in a pathogen-free facility. Briefly, mice in the OVA group were sensitized by intraperitoneal injection of 10μg OVA, which was emulsified in 1 mg aluminum hydroxide (Sigma), in a total volume of 0.25mL per mouse on days 1 and 8. From day 15 to 21, mice were challenged through the airways daily with 1% (w/v) OVA solution for 30 minutes in a 30-cm×24 cm×50 cm chamber. The healthy control mice were sensitized and challenged with phosphage buffered saline (PBS). Twenty-four hours after the last challenge, the mice were sacrificed, the tracheal tissues were collected, fixed in OCT and frozen immediately in -80℃freezer for further molecular analyses. Lungs were inflated with 4% paraformaldehyde and processed in paraffin-embedded blocks. The specimens were cut at 5 um in thickness, and stained with hematoxylin & eosin to evaluate morphologic changes. The expression of FIZZ1Mrna, typeⅠcollagen, Fibronectin1 and E-Cadherin in lungs was analyzed by quantitative real-time real-time PCR. Exposure of mouse epithelial cells with FIZZ1 recombination or FIZZ1-shRNA, after 24 hours of incubation, the cells were harvested for analysis of typeⅠcollagen, Fibronectin 1 and E-Cadherin with Western blotting. Results:1. In group A, bronchus sub-mucosa edema, mucus gland hyperplasia, eosinophile granulocyte dominated inflammatory cells infiltration among all layers; In group B, no airway endepidermis hyperplasia, no inflammatory cells infiltration, intact alveolar wall.2. FIZZ1 was expressed at very low levels in bronchial epithelial cells in healthy control mice. In the models of asthma, FIZZ1 expression was significantly increased in bronchial epithelium. (p< 0.05)3. Expression of FIZZ 1 mRNA was higher in the Airway of OVA-treated mice than that of control group(p< 0.05). while the expression of type I collagen and Fibronectinl in the OVA group was significantly increased and the E-Cadherin in the OVA group was significantly decreased. The FIZZ1 had a positive correlation with type I collagen and Fibronectinl,but had a negative correlation with E-Cadherin.4. We use Cultured MLE-12, then Stimulated with FIZZ1 recombination and FIZZ1-shRNA respectively. After incubated for 24 hours, we detected the expression of type I collagen, Fibronectin 1 and E-cadherin with Western blotting. Cultured MLE-12 Stimulated with FIZZ1 recombination increases the expression of type I collagen, Fibronectin 1, while decreases the expression of E-cadherin, but incubated with FIZZ1-shRNA, there is no change in the level of type I collagen, Fibronectin 1 and E-cadherin.Conclusions:1. The expression of FIZZ1 and FIZZ1 mRNA in the OVA group was significantly increased compared with blank group.2. The expression of type I collagen and Fibronectinl in the OVA group was significantly increased but E-Cadherin in the OVA group was significantly decreased.3. FIZZ1 is a potent inducer of Epithelial to Mesenchymal transit, FIZZ1 can induce fibroblasts to express markers of myofibroblast differentiation such asα-SMA and collagen type I, which are characteristic of early stages of airway remodeling seen in asthma.