Effect Of AP318 On Tobacco Smoke-induced Oxidative Stress In HBEpCs And HPMVECs

Aim:Investigate the role of AP318 in oxidative stress induced by tobacco in human bronchial epithelial cells (HBEpC) and human pulmonary microvascular endothelial cells (HPMVECs). Ap318 is a cyclic peptide derived from the lectin-like domain of tumor necrosis factor (TNF). The investigation will contribute more evidence to the field of animal model of COPD. The long term goal is to practice potential treatment of AP318 in human COPD.Methods:Incubate the AP318 with primary HBEpC and HPMVECs for 30 minutes at a final concentration of 50ug/mI. Then introduce 5%TSE for 5 hours and 24 hours treatment. Control groups are a) only cell culture medium b) only Ap318 solution and cell culture medium c) only TSE and cell culture medium. The readout for oxidative stress is to stain 8-OHdG and quantify the positive cell numbers by Confocal microscopy. The readout for apoptosis is TUNEL assay by confocal microscopy and quantify the positive cell numbers; and Caspase-3 activity measurement by Elisa.Results: 1) Under both 5 and 24 hours treatment of TSE alone, in both cell lines, there was significant increase of oxidative stress, which is qualified by 8-OHdG staining, comparing with controls; p value was less than 0.05. In 24 hours treatment, AP318 with TSE treatment in both cell lines showed less oxidative stress than TSE treatment alone, and it was statistically significant. In 5 hours treatment, there was no significant difference between TSE treatment alone and TSE treatment with AP318 in HBEpC but not in HPMVECs.2) TUNEL assay results showed that in both 5 and 24 hours treatment, TSE treatment caused apoptosis in both cell lines. With AP318, TSE group showed less apoptosis than in TSE alone group. It was statistically significant.3) Caspase-3 activity measurement exploded that only 24 hours treatment showed significant apoptosis in TSE group while not in 5 hours treatment. AP318 with TSE group showed less Caspase-3 activity than TSE only group, it is statistically significant.Conclusions:1) TSE can cause the higher ROS production, which intrigued the oxidative stress and apoptosis in HBEpC and HPMVEC cells.2) AP318 is an effective drug to prevent or decrease the ROS production and cell apoptosis in HBEpC and HPMVEC cells.