Protective Effect of Propofol against Hypoxia-reoxygenation Injury in HaCaT Human Keratinocytes
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The aim of this study was to determine the beneficial effectof propofol on human keratinocytes that have undergonehypoxia reoxygenation (H/R) injury and to investigate whetherautophagy is associated with the protective mechanism. Thus,we evaluated how propofol influences the intracellularautophagy and apoptosis during the H/R process in the HaCaTcells. The cultured human keratinocyte cells were exposed to24 h of hypoxia (5% CO2, 1% O2, 94% N2) followed by 12 hof reoxygenation (5% CO2, 21% O2, 74% N2). The experimentwas divided into 4 groups: (1) Control=Normoxia ; (2)H/R=Hypoxia Reoxygenation ; (3) PPC+H/R=PropofolPreconditioning+Hypoxia Reoxygenation; (4) 3-MA+PPC+H/R=3-MA-Methyladenine+Propofol Preconditioning+Hypoxia Reoxygenation. In addition, Western blot analysiswas performed to identify the expression of apoptotic pathwayparameters, including Bcl-2, Bax, and caspase 3 involved inmitochondrial-dependent pathway. Autophagy was determinedby fluorescence microscopy, MDC staining, AO staining, andwestern blot. The H/R produced dramatic injuries inkeratinocyte cells. In our study, the viability of Propofol in H/R induced HaCaT cells was first studied by MTT assay. Thetreatment with 25, 50, and 100 μM Propofol in H/R inducedHaCaT cells enhanced cell viability in a dose-dependentmanner and 100 μM 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 HaCaTcells. The Atg5 were increased when autophagy was inducedby Propofol, and they were decreased when autophagy wassuppressed by 3-MA. These data provided evidence thatpropofol preconditioning induced autophagy and reducedapoptotic cell death in an H/R model of HaCaT cells, which wasin agreement with autophagy playing a very important role incell protection.
Abstract
The aim of this study was to determine the beneficial effectof propofol on human keratinocytes that have undergonehypoxia reoxygenation (H/R) injury and to investigate whetherautophagy is associated with the protective mechanism. Thus,we evaluated how propofol influences the intracellularautophagy and apoptosis during the H/R process in the HaCaTcells. The cultured human keratinocyte cells were exposed to24 h of hypoxia (5% CO2, 1% O2, 94% N2) followed by 12 hof reoxygenation (5% CO2, 21% O2, 74% N2). The experimentwas divided into 4 groups: (1) Control=Normoxia ; (2)H/R=Hypoxia Reoxygenation ; (3) PPC+H/R=PropofolPreconditioning+Hypoxia Reoxygenation; (4) 3-MA+PPC+H/R=3-MA-Methyladenine+Propofol Preconditioning+Hypoxia Reoxygenation. In addition, Western blot analysiswas performed to identify the expression of apoptotic pathwayparameters, including Bcl-2, Bax, and caspase 3 involved inmitochondrial-dependent pathway. Autophagy was determinedby fluorescence microscopy, MDC staining, AO staining, andwestern blot. The H/R produced dramatic injuries inkeratinocyte cells. In our study, the viability of Propofol in H/R induced HaCaT cells was first studied by MTT assay. Thetreatment with 25, 50, and 100 μM Propofol in H/R inducedHaCaT cells enhanced cell viability in a dose-dependentmanner and 100 μM 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 HaCaTcells. The Atg5 were increased when autophagy was inducedby Propofol, and they were decreased when autophagy wassuppressed by 3-MA. These data provided evidence thatpropofol preconditioning induced autophagy and reducedapoptotic cell death in an H/R model of HaCaT cells, which wasin agreement with autophagy playing a very important role incell protection.
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