oftware. The following parameters were used in the search, mam malian, protein molecular mass ranged from 700 to 32, 000Da, trypsin digest with one missing cleavage, peptide tolerance of 0. 2, MS MS tolerance of 0. 6 Da and possible oxidation of methionine. Statistical analysis All values were expressed as the mean SD of n obser vations. Statistical analyses between groups were performed using one way analysis of variance or Student t tests between two groups, as appropriate. P 0. 05 was considered statistically significant. Results Down regulation of Nogo B in airway smooth muscle of chronic asthmatic mice To investigate the role of Nogo B in airway remodeling in asthma, we constructed a mouse model of chronic asthma. Evident airway inflammation and airway thick ening could be observed in mice with chronic asthma.
The asthmatic mice also had significantly increased expression of SM 22, a specific marker of differentiated ASM cells in the airway, indi cating evidence of airway smooth muscle remodeling. Immunohistochemistry revealed that Nogo B was widely expressed AV-951 in the lung, especially abundant in epithelium, alveolar epithelial cells, and airway smooth muscle cells. In chronic asthmatic mice, the distribution of Nogo B was not altered. However, there was a significant decrease in the airway smooth muscle layer. Addi tionally, Realtime analysis revealed a significant reduction of lung Nogo B mRNA expression in chronic asthmatic mice, in accordance with this, Western blot ting analysis of the total proteins collected from the lung homogenates showed that Nogo B expression was approximately 3.
08 fold lower in chronic asthmatic mice than in control mice, indicating that Nogo B may play a role in airway smooth muscle remodeling in asthma. However, incubation of cultured HBSMCs with an increasing concentration of PDGF BB for up to 48 h resulted no obvious change of Nogo B as evidenced by western blotting analysis. RNAi for Nogo B expression To determine the role of Nogo B in airway smooth mus cle cells, we used a siRNA approach to knockdown Nogo B expression in HBSMCs in vitro. Transfection of cells with two different Nogo B siRNA sequences resulted in knock down of Nogo B protein expression, as determined by Western blotting analysis. Transfection of negative control siRNA had no effect on Nogo B expression levels.
Addi tionally, NOGOi transfected cells showed a 96% reduc tion in Nogo B mRNA compared to NEGi transfected cells 60 h post transfection, as determined by quantita tive real time PCR. Effects of Nogo B on proliferation and migration of HBSMCs In the next step, we examined the effects of Nogo B on PDGF induced abnormalities of HBSMCs in vitro. HBSMCs, pretreated with either NEGi or NOGOi 2 for 48 h, were starved overnight, reseeded onto a 96 well plate at a density of 3. 5 �� 103 in 2% FBS SmGM and incubated with PDGF BB. Stimulation of HBSMCs with PDGF for 24 h and 48 h resulted in signif icant increase in cell numbers, both in untransfec
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