Inhibition Of The Wnt/β-catenin Pathway Signaling By XAV939Attenuates Bleomycin-induced Pulmonary Fibrosis

Backgrounds:Idiopathic pulmonary fibrosis (IPF) is a progressive lung disorder of unknown etiology. The disease is characterized by aberrant proliferation of fibroblasts and finally by formation of scar tissues with the subsequent destruction of the lung architecture. Previous studies showed that Wnt/β-catenin signaling pathway is involved in the activation and proliferation of fibroblasts. Besides that, aberrant activation of the Wnt/β-catenin signaling cascade occurs in lungs of patients with IPF. Bone marrow-derived mesenchymal stem cells (BM-MSCs) is an excellent candidate for the repair of alveolar epithelial cells in IPF because of its capacity for multilineage differentiation potential, including epithelial cell lineages. The effects of BM-MSCs treatment in pulmonary fibrosis is debatable because BM-MSCs also have the ability to differentiate into fibroblasts. Emerging evidence suggests that Wnt/J3-catenin signaling pathway is linked to the epithelial differentiation of BM-MSCs.Considering the important roles of Wnt/β-catenin signaling pathway in pulmonary fibrosis and epithelial differentiation of BM-MSCs, we targeted this pathway for intervention in pulmonary fibrosis with XAV939, a small molecule that specifically inhibits Tankyrase1/2. Besides that, in order to observe the effect of XAV939in the epithelial differentiation of BM-MSCs, we established a co-culture system which contains BM-MSCs and alveolar type II epithelial cells(ATII cells) with the Trans-well technology. It will lay the foundation for investigating the treatment of pulmonary fibrosis by inhibiting the Wnt/β-catenin signaling pathway.Objectives:1. Proving that XAV939could attenuates bleomycin-induced pulmonary fibrosis in mice.2. Confirming that the epithelial differentiation of BM-MSCs could be promoted by XAV939in the co-culture system.Methods:1. Anesthetized mice (C57BL/6, male,10weeks) were intranasally administered with bleomycin (0.1units,50μl), control groups received saline intranasally. Take the day of bleomycin administration as day0. Mice were sacrificed on day7,14and21. Lungs were obtained for histopathology, RT-PCR, western blot and collagen assay.2. On the basis of bleomycin induced pulmonary fibrosis, XAV939was injected intraperitoneally once a day for seven times from day10to17after the intranasal instillation of bleomycin. Mice were sacrificed on day17. Lungs were obtained for histopathology, RT-PCR, western blot and collagen assay.3. Mice were intranasally instilled with bleomycin in a higher dose which found to result in lethality in the majority of mice after14days (0.3units,50μl). XAV939was intraperitoneally administered to mice once a day for seven days beginning after10days of bleomycin administration. The mortality rate of mice was counted every day after the bleomycin administration.4. To evaluate the inhibition of fibroblast proliferation of XAV939, NIH-3T3fibroblast proliferation was detected by CCK-8(cell count kit).5. To establish a co-culture system which contains BM-MSCs and alveolar type11epithelial cells (ATII cells) with the Trans-well technology for in vitro experiments. The BM-MSCs were seeded at a density of8x104cell/ml in the six-well plates and the ATII cell were seeded at a density of1x104cell/ml in a Tran-swell insert, the medium (with XAV939or not) was changed every48h. After a14days co-culture, the BM-MSCs were extracted for western blot and RT-PCR.Results:1. The pulmonary fibrosis in mice could be induced by bleomycin intranasal administration (0.3units,10μl). And the Wnt/β-catenin signaling pathway was activated in this process. 2. XAV939inhibits activation of the Wnt/β-catenin signaling in the lung and attenuates bleomycin-induced pulmonary fibrosis in mice.3. XAV939intervention improves survival of mice after lung injury which was induced by bleomycin in a higher dose (0.3units,10μl).4. XAV939inhibits the proliferation of NIH-3T3fibroblasts.5. A co-culture system which contains BM-MSCs and alveolar type II epithelial cells was established with the Trans-well technology. XAV939promotes epithelioid phenotype change and ATII cell-specific marker expression in BM-MSCs in the co-culture system.Conclusions:We report here that XAV939significantly inhibits Wnt/β-catenin signaling and attenuates bleomycin-induced lung fibrosis in mice, while concurrently improving the survival of lung injured mice. In vitro experiments demonstrates that XAV939has the ability to inhibit proliferation of NIH-3T3fibroblasts and promote differentiation of BM-MSCs into ATII cells in the co-culture system. Because no effective treatment for IPF exists, selective inhibition of Wnt/β-catenin signaling suggests a potential unique therapeutic approach for pulmonary fibrosis.