Protective Effect of Propofol against Hypoxia-reoxygenation Injury in HaCaT Human Keratinocytes

Kim, Yong Ho; Kang, Jin Mo; Kim, In Ryoung; Lee, Bo Young; Yoon, Ji Young; Kim, Cheul Hong; Park, Bong Soo

Abstract

The aim of this study was to determine the beneficial effect of propofol on human keratinocytes that have undergone hypoxia reoxygenation (H/R) injury and to investigate whether autophagy is associated with the protective mechanism. Thus, we evaluated how propofol influences the intracellular autophagy and apoptosis during the H/R process in the HaCaT cells. The cultured human keratinocyte cells were exposed to 24 h of hypoxia (5% CO2, 1% O2, 94% N2) followed by 12 h of reoxygenation (5% CO2, 21% O2, 74% N2). The experiment was divided into 4 groups: (1) Control=Normoxia ; (2) H/R=Hypoxia Reoxygenation ; (3) PPC+H/R=Propofol Preconditioning+Hypoxia Reoxygenation; (4) 3-MA+PPC+H/R=3-MA-Methyladenine+Propofol Preconditioning+Hypoxia Reoxygenation. In addition, Western blot analysis was performed to identify the expression of apoptotic pathway parameters, including Bcl-2, Bax, and caspase 3 involved in mitochondrial-dependent pathway. Autophagy was determined by fluorescence microscopy, MDC staining, AO staining, and western blot. The H/R produced dramatic injuries in keratinocyte cells. In our study, the viability of Propofol in H/R induced HaCaT cells was first studied by MTT assay. The treatment with 25, 50, and 100 microM Propofol in H/R induced HaCaT cells enhanced cell viability in a dose-dependent manner and 100 microM was the most effective dose. The Atg5, Becline-1, LC3-II, and p62 were elevated in PPC group cells, but H/R-induced group showed significant reduction in HaCaT cells. The Atg5 were increased when autophagy was induced by Propofol, and they were decreased when autophagy was suppressed by 3-MA. These data provided evidence that propofol preconditioning induced autophagy and reduced apoptotic cell death in an H/R model of HaCaT cells, which was in agreement with autophagy playing a very important role in cell protection.