All of these factors observations indicate that Nogo B plays a pivotal role in vascular remodeling and tissue repair. Airway smooth muscle remodeling in asthma is basically GW786034 a SMC repair response to inflammatory mediates and cytokines, the role of Nogo B in the process of airway smooth mus cle remodeling has not yet been reported. We evaluated the role of Nogo B in ASM in a mouse model of chronic asthma and then determined the effects of Nogo B on PDGF induced proliferation, migration and contraction of HBSMCs in vitro using a siRNA strategy. Proteomic analysis was then performed to unveil the underlying mechanisms. Our results demonstrate a novel mechanism through which Nogo B regulates airway smooth muscle cells. Materials and methods Animal models Four to six week old male BALB c mice were used in our experiments.
The mice were sensitized intra peritoneally with Ovalbumin in alum. Control mice received the same volume of PBS in alum, as previously described. Chronic allergic airway remodeling was induced when mice were subsequently exposed to aerosolized OVA challenges three times a week from Days 21 to 72. Mice were sacrificed at the indicated times and the lungs were harvested, either into 4% formalin for histological evalua tion or snap frozen into liquid nitrogen for protein preparations. Animals were treated humanely according to Institutional Animal Care procedures. Cell culture Primary human bronchial smooth muscle cells and smooth muscle growth medium were pur chased from ScienCell. HBSMCs were cultured in SmGM containing 5% FBS.
The cells were incubated at 37 C in a 5% CO2 humidified atmosphere. Cells from pas sages 4 to 10 were used for the experiment. PDGF BB was purchased from R D and dissolved in PBS to yield a stock solution of 1 ug ml. Histological examination Mouse lung tissues were collected and embedded in paraffin for histological analysis. Lung sections were stained with hemotoxylin and eosin for examina tion of airway remodeling. For the immunohistochemis try, 5 um thick sections were cut, and the Envision method was performed according to the instructions. Anti SM 22 antibody, anti Nogo B antibody were applied. 3, 3 Diaminobenzi dine was used as a chromogen with a subsequent hema toxylin counter stain. All of the above siRNAs were designed and Entinostat synthesized by Qiagen. For 6 well plate trans fection, human bronchial smooth muscle cells were transfected with 300 ng siRNA using 12 ul Hiperfect according to the manufacturers instructions. Efficacy of siRNA interference of Nogo B was assayed at 24 to 60 h post transfection by Western blotting. Western blotting analysis The protein concentration was determined using the Bio Rad protein assay system. HBSMCs were dissolved and boiled in Laemmli buffer for 5 min.