P akt

Granulation tissues were homogenized and lysed in ice-cold lysis buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, and 1% Triton X-100) containing protease inhibitors (2 mmol/L sodium orthovanadate, 0.2 mmol/L PMSF, 2 μg/mL leupeptin, and 2 μg/mL aprotinin). After centrifuged at 13 000 g for 15 minutes at 4°C, the supernatant was collected. Equal amounts of denatured proteins in the supernatants were separated by 12% SDS-PAGE, and transferred to nitrocellulose membranes. Then the membranes were blocked in TBS-T (10 mmol/L Tris, 150 mmol/L NaCl, and 0.1% Tween20, pH 7.5) containing 5% skim milk for 2 hours. The membranes were incubated with rabbit polyclonal antibodies β-actin, COX-2, VEGF, FGF-2, ERK1/2, p-ERK1/2, Akt, and p-Akt (1: 500) (Bio Basic Inc., Canada) in TBS-T with 5% nonfat milk overnight at 4°C. After the membranes were incubated with anti-rabbit HRP-conjugated antibody (1: 10 000) in TBS-T, target proteins were determined via visualization with DAB (Bio Basic Inc., Canada).

Pentobarbital sodium, STZ, and a horseradish peroxidase-conjugated secondary antibody were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA). Rabbit polyclonal antibodies β-actin, COX-2, vascular endothelial growth factor (VEGF), FGF-2, CD34, ERK1/2, p-ERK1/2, Akt, and p-Akt were purchased from Bio Basic Inc. (Canada). Lysozyme-antimicrobial peptide fusion protein was purchased from HebeiChuangyue Biotech. Co., Ltd. (egg-white lysozyme and active fragment of Drosophila melanogaster antimicrobial peptide genes were used to construct fusion genes and subclones) (Langfang, China).
Healthy Sprague-Dawley (SD) rats weighing 220 g to 260 g were purchased from Nanjing Qinglongshan animal breeding ground (Nanjing, China) and raised in our standard animal facility with 22±2°C room temperature and 12-hour day/night alternate. All experimental procedures were handled according to Chinese Community guidelines for the use of experimental animals.