FIZZ1 Promotes Airway Remodeling Through The PI3K/AKT Signaling Pathway In Asthma

ObjectivesAsthma is a serious public health problem throughout the world, and the disease incidence rate assumes the rise tendency in recent years.The three main pathological change of asthma is airway inflammation, smooth muscle dysfunction and airway remodeling. Airway remodeling is the pathological basis of impaired lung function, resulting in loss of labor, increasing the social burden of medical care. To reveal the occurrence and development mechanism of asthma airway remodeling, and to prevent or even reverse the pathological process, is the key to improve prognosis of asthma, which is also the hotspot and difficult point of asthma research. Our previous study demonstrated that FIZZ1 was vital in airway remodeling in asthma and was capable of increasing the expression levels of α-SMA and type I collagen in the early stages of airway remodeling.In the present study, the hypothesis that FIZZ1 may activate the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway through promoting Akt phosphorylation in vitro was investigated. However, the mechanism by which FIZZ1 functions in the process of airway remodeling remains unclear.We hypothesize that FIZZ1 may promotes airway remodeling via the PI3K/Akt signaling pathway.The main purpose of this experiment was conducted as follows:(1)FIZZ1 plays a role in airway remodeling in asthma;(2) FIZZ1 can promote epithelial-mesenchymal transition by regulating PI3K/AKT signaling pathway;(3)Blocking the PI3K/Akt signaling pathway may attenuate the early stages of airway remodeling induced by OVA by regulating the abnormal process of epithelial-mesenchymal transition.Methods(1)Animal experements:Specific-pathogen-free female BALB/c mice (age,8-10 weeks;weight,20±2g) were randomly divided into control group,OVA group, LY294002 group and AKT-inhibitor-IV group.Mice were sensitized on days 1 and 14 by intraperitoneal injection of ovalbumin and A1(OH)3 (Sigma-Aldrich) suspension. On days 21-23 following the initial sensitization, the mice were challenged for 30 min with an aerosol of OVA using an ultrasonic nebulizer,while saline alone was used to challenge the control group. LY294002, Akt inhibitor IV or saline were administered intranasal 2h prior to each OVA aerosol challenge. After 24h of the last challenge,measurements of airway hyperresponsiveness were conducted using an animal pulmonary instrument.Lungs tissues were stained with hematoxylin and eosin for examination of inflammatory cell infiltration,and the expression of FIZZ1, type I collagen, E-cadherin and fibronectin-1 protein was measured by immunohistochemical technique.The expression of FIZZ1,p-Akt,a-SMA,type I collagen and β-actin protein was measured by Western blot analysis.The expression of a-SMA mRNA and type I collagen mRNA was analyzed by quantitative real-time PCR.(2) Cell culture in vitro:The MEL-12 cell line was cultured with the FIZZ1 recombinant protein or FIZZl-shRNA plasmid.The protein was extracted from the cell line for analysis of the Akt phosphorylation levels and the expression levels of a-SMA and type I collagen.Statistical analysis:Data was presented as the mean ± SEM. Continuous variables were analyzed usingthe studentps t-test between the groups studied.P<0.05 was considered to indicate a statistically significant difference.Results(1) Performance of asthma model in mise:Mice in the OVA group showed frequent activity, shortness of breath, abdominal muscle twitching, incontinence and hair up in the modeling process.Some mice appeared unresponsive and slow performance. OVA-treated mice developed airway hyperresponsiveness to inhaled methacholine compared to the saline-changed group. And OVA aerosol challenge induced the infiltration of inflammatory cells around the airway.(2) FIZZ1 and p-Akt expression in mice:The expression of FIZZ1 in mice airway epithelium was up-regulated in OVA-induced asthma mice compared to the saline control group. FIZZ1 expression and Akt phosphorylation were enhanced in OVA group by western blot.(3) Expression of a-SMA,type I collagen and Akt phosphorylation in MLE-12 cells:To verify whether FIZZ1 can promote Akt phosphorylation, we cultured MLE-12 cell line with FIZZ1 recombination protein and FIZZ1 sh-RNA transfection and then detected the expression of a-SMA,type I collagen and Akt phosphorylation. After FIZZ1 recombination protein co-culture, Akt phosphorylation,a-SMA and type I collagen expression were up-regulated. The level of FIZZ1 expression in MLE-12 cells after FIZZ1 sh-RNA transfection was down-regulated, and so as the level of Akt phosphorylation, a-SMA and type I collagen expression.(4) Effects of LY294002 and AKT inhibitor IV on OVA-induced inflammatory cells infiltration and airway hyperresponsiveness:The histological analysis showed that LY294002 and AKT inhibitor IV can attenuate the inflammatory cells infiltration compared to the OVA-induced asthma group.(5) LY294002 and AKT inhibitor IV can overcome airway remodeling:Fibronetin-1 and type I collagen were highly expressed in the epithelial cells of the airway in asthma mice. LY294002 and AKT inhibitor IV reduced their expression compared with the OVA group. In the contrary, the expression of E-cadherin was decreased in OVA-induced mice and LY294002 and AKT inhibitor IV up-regulated its expression. At the same time, we also found the higher expression of a-SMA and type I collagen both in mRNA levels and protein levels in OVA-induced mice, and blocking the PI3K/Akt pathway could inhibit the production of a-SMA and type I collagen.Conclusions(1) The expression of FIZZ1 in the OVA group was significantly increased compared with control group;(2) FIZZ1 can promote epithelial-mesenchymal transition by regulating PI3K/AKT signaling pathway;(3) Blocking the PI3K/AKT signaling pathway may attenuate the early stages of airway remodeling induced by OVA by regulating the abnormal process of epithelial-mesenchymal transition.