Autophagy activation by rapamycin before hypoxia-reoxygenation reduces endoplasmic reticulum stress in alveolar epithelial cells

Abstract

Background: To determine potential effects of autophagy activation on hypoxia-reoxygenation (H/R) induced damage of a rat alveolar epithelial cell line. Methods: CCL149 cells were subjected to autophagy agonist (rapamycin, Rap), autophagy inhibitor (3-methyladenine, 3-MA) or PBS for 1 h before H/R treatment for 2 h, 4 h and 6 h. The optimal concentration of Rap (150 nM, 200 nM and 250 nM) or 3-MA (5 mM, 10 mM and 15 mM) was obtained from MTT assay. Autophagy was determined by fluorescence microscopy of eRFP-LC3 positive cells, transmission electron microscopy of autophagosome, western blot of LC3, AMPK, Beclin-1, HDAC6 and p62 proteins. Endoplasmatic reticulum stress was indicated by detecting expressions of BIP, XBP-1 and CHOP via western blot. Results: Rap at concentration of 250 nM before H/R increased the autophagy formation with more eRFP-LC3 positive cells and higher expressions of LC3-II, Beclin-1, HDAC6 and p62, but lower expressions of BIP, XBP-1 and CHOP in H/R treated CCL149. This effect seemed to be still obvious after H/R exposure for 6 h. The contrary results were obtained by treatment with 5 mM 3-MA. Conclusion: Rap might be a promising agent before mechanical ventilation or reperfusion to prevent re-damage in hypoxia related lung diseases.