In a COPD model of LPS induced airway inflammation and remodeling in guinea pigs, tiotropium abrogated the selleck chemicals Imatinib LPS induced in crease in neutrophils, goblet cells, collagen deposition and muscularised microvessels, but had no effect on em physema. These results suggested that endogenous acetylcholine plays a major role in the pathogenesis of this disease. EMT takes center stage as the convergence point between inflammation and airway diseases. Inflammatory mediators are known to induce downregulation of epithe lial cell cell adhesion and promote mesenchymal gene ex pression both in vitro and in vivo, and consequently inflammatory mediators have emerged as decisive factors in EMT induction. Although a number of molecules in volved in ACh mediated airway inflammation and remo deling have been identified, little is known regarding the role of ACh in EMT.

In the current study, we explored whether TGF B1 induced EMT can be influenced by non neuronal choli nergic system in lung epithelial cells, and if so, whether mAChR activation has similar effects to TGF B1 in EMT induction. Moreover, which of the two main pathways, the Smad pathway or the ERK pathway, both of which can be activated by mAChR agonists, was involved in EMT in lung epithelial cells. Methods Cell culture and treatment The human alveolar epithelial cell line A549 and human tracheobronchial epithelial cell line 16HBE were obtained from the American Type Culture Collection, and were maintained in Hams F 12 medium or Dulbeccos modi fied Eagles medium /HIGH Glucose supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator with 5% CO2 at 37 C.

Cells were grown in 96 or 6 well plastic tissue culture plates until confluence and then transferred into serum free medium containing 0. 1% FBS for 24 h. After that, cells were treated as designed. Western blot analysis Cells were lysed in RIPA lysis buffer and a protease inhibitor cocktail on ice for 5 min and scraped into a centrifuge tube. The lysates were centri fuged at 13,000 g for 5 min at 4 C. Total protein was mixed with sodium dodecyl sulfate sample buffer and heated at 100 C for 5 min. Equal amounts of samples were separated by 10% SDS polyacrylamide gel electro phoresis and transferred to polyvinylidene fluoride mem branes, which were then blocked with 5% non fat milk in TBS T for 1 h at room temperature and then incubated with primary antibodies overnight at 4 C.

After washing the membranes three times for 5 min each with TBS T, they were incubated with horseradish peroxidase conjugated secondary antibodies for 1 h at RT, followed by an additional three washes for 5 min each with TBS T. Bands were subsequently visualized on film using enhanced chemiluminescence reagents. Results were expressed relative to glyceraldehyde 3 phosphate dehydrogenase Drug_discovery band density used as a loading control.