Protective Effect and Mechanism of Alprostadil in Acute Respiratory Distress Syndrome Induced by Oleic Acid in Rats

Yan, Xiujuan; Li, Yingxiu; Choi, Yun Ho; Wang, Chongyang; Piao, Yihua; Ye, Jing; Jiang, Jingzhi; Li, Liangchang; Xu, Huixian; Cui, Qingsong; Yan, Guanghai; Jin, Minggen

Total proteins were isolated from the rest of the right lung tissue homogenate. Protein concentration was determined using the BCA Protein Assay Kit (Beyotime, China). Equal quantities of protein samples were separated by SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk, and incubated with anti-ACE (1: 3000), anti-TNF-a (1: 3000), anti-IL-1b (1: 1500), anti-Bcl-2 (1: 1500), anti-Bax (1: 1500), anti-NF-kB p65 (1: 1500), anti-p-p38MAPK (1: 1500), anti-p38MAPK (1: 1500), anti-p-ERK1/2 (1: 1500),anti-ERK1/2 (1: 1500), anti-p-JNK(1: 1500), anti-JNK (1: 1500), and anti-b-actin (1: 1500) antibodies at 4°C overnight. The goat anti-rabbit (or mouse) secondary antibody conjugated with horseradish peroxidase (1: 5000) was added as secondary antibody. All the antibodies used were purchased from Elabscience Biotechnology Co., Ltd. (China). Immunodetection was carried out with ECL detection reagent (Beyotime, China). b-actin was used as a loading control. Results of all protein expression by Western blot are expressed as ratios of the optical density value of the target bands to those of internal controls by using Quantity One software (Bio-Rad Technical Service Department).