Methods Materials Male Sprague Dawley rats weighing 200 250 g were housed under specific pathogen free con ditions in a temperature and humidity controlled envir onment and given Bortezomib order free access to food and water with the Guide for the Care and Use of Laboratory Animals. Reagents for cell culture were provided by the Institute of Life Science, Chongqing Medical University. Lipopolysac charide, LY294002, wortmannin, amiloride, sodium pentobarbital and Evans blue were purchased from Sigma. Akt inhibitor Inhibitors,Modulators,Libraries 2 Omethyl 3 O octadecylcarbonate was purchased from Enzo Life Sciences. Serum and glucocorticoid regulated protein kinase1 inhibitor was purchased from Tocris bioscience. Rabbit anti a ENaC,b ENaC and g ENaC antibodies were purchased from Santa Cruz Biotechnology Rabbit anti Phospho Akt and total Akt monoclonal antibodies were obtained from Cell Signaling Technol ogy.

Rabbit anti Nedd4 2 polyclonal antibody was purchased from ABcam. The study was approved Inhibitors,Modulators,Libraries by the Ethics Committee Inhibitors,Modulators,Libraries of the Second Affiliated Hospital of Chongqing Medical University. Animal model and intervention Rats were anesthetized by intraperitoneal administration of sodium pentobarbital. ALI model was established by LPS with intraperitoneal injection followed by insertion of an internal jugular Inhibitors,Modulators,Libraries vein catheter for drug administration. Human Insulin was administered at a dose of 0. 1 U/kg/h and at a rate of 2. 5 mU/h/rat via micro osmotic pumps 16 hours before LPS exposure. Wortmannin were injected retro orbitally three times at 90, 90, and 360 minutes relative to the LPS injection.

Rats in control group were received an equivalent volume of saline. Rats were killed 8 hours after LPS or saline treat ment. Blood samples, bronchoalveolar lavage fluid and lung tissue were obtained for analysis. Cell isolation, culture and treatment Inhibitors,Modulators,Libraries Alveolar epithelial type II cells were isolated from male Sprague Dawley rats by elastase digestion of lung tissue and then differentially adhered on IgG coated plates as previously described. Purity of the ATII cells were determined by microscopic analysis, indicative of epithelial cell lineage and by immunohis tochemistry for surfactant protein C, indicative of ATII cell. ATII cells were seeded onto plastic culture dishes and cultured in a 5% CO2, 95% air atmosphere in DMEM containing 10% fetal bovine serum, 100 U/ml penicillin and 0. 1 mg/ml streptomycin after isolation.

On day 3 after isolation, the cells were pre incubated with LY294002, Akt inhibitor and SGK1 inhibitor for 30 minutes before insulin treatment for 2 hours and the experiments were performed. Measurement of glucose and insulin levels Blood samples were withdrawn from the catheter by cen trifuging at 3000 rpm at 4 C for 15 minutes. Glucose levels in the plasma were analyzed by Glucometer Ceritinib cancer OneTouch. Human insulin levels in the plasma were analyzed by a ELISA kit for only human insulin. Total insulin levels in the plasma were analyzed by a ELISA kit for rat plus human insulin.