All experimental procedures conformed to international specifications of animal welfare, and were approved from the Institute Animal Care and Use Committee of Shanghai University of Classic Chinese Medication. Female BALB c mice had been obtained from Shanghai SLAC Laboratory Animal Co. Ltd. All mice were kept in wellcontrolled animal housing amenities, and had cost-free accessibility to tap water and foods pellets all through the experimental period. Female, six eight week previous BALB c mice had been divided into three groups: OVA treated group , OVA dexamethasone handled group and a saline group . Mice were challenged with Ovalbumin by intraperitoneal and intranasal routes. OVA handled and dexamethasone treated groups had been immunized by intraperitoneal injections of 100 g of OVA mixed with potassium aluminum sulfate on days 0 and 14 . Mice obtained an intranasal dose of 500 g OVA on days 14, 25, 26, 27. The management group obtained standard saline with alum i.p. on days 0 and 14 and typical saline not having alum intranasally on days 14, 25, 26, 27 .
The group of dexamethasone taken care of mice was administered with dexamethasone intraperitoneally beginning on day 28 with the protocol and continuing until day 41. Animals have been sacrificed by i.p. injection chemical catalogs of pentobarbital at day 42, as well as lungs and extrahilar tracheobronchial airways were quickly dissected out. Tissue processing and immunohistochemistry examination Immunohistochemistry detection of PTEN was done as described elsewhere . Tissue sections through the right lungs had been 1st taken care of with PTEN antibody . Just after incubation at four C overnight, tissue sections were washed with PBS, and treated with ligation enhancing buffer for 30 min at area temperature. Tissue sections have been then washed with PBS, and taken care of for thirty min with horseradish peroxidase anti rabbit IgG . The shade was created employing diaminobenzidine . The intensity of PTEN protein staining was established as an typical optical density by IPP computer software . A nonstained region was picked and set since the background.
Cell culture The lung epithelial cell line, A549, was obtained from your Institute syk inhibitor of Cell Biology , and cultured in RMPI1640 medium supplemented with 10% fetal bovine serum, penicillin and streptomycin. A549 cells were handled using the indicated concentrations of dexamethasone for 24 h. Otherwise, the cells were taken care of with one 10 five M dexamethasone. The cells have been harvested at 24 h, 48 h, 72 h, and 96 h. PTEN expression analysis by serious time quantitative PCR Complete RNA from A549 cells have been extracted by Trizol . The RNA was reverse transcribed to cDNA, using a RevertAid 1st Strand cDNA Synthesis Kit . Quantitative serious time PCR was performed by Universal Master Mixer on a 7300 Serious time PCR Method . The primers and probes implemented are listed in Table 1.