J Dent Anesth Pain Med.  2017 Mar;17(1):21-28. 10.17245/jdapm.2017.17.1.21.

Propofol protects human keratinocytes from oxidative stress via autophagy expression

• Yoon JY
• Jeon HO
• Kim EJ
• Kim CH
• Yoon JU
• Park BS
• Yu SB
• Kwak JW

• KMID: 2374720
• DOI: http://doi.org/10.17245/jdapm.2017.17.1.21

Abstract

BACKGROUND
The skin consists of tightly connected keratinocytes, and prevents extensive water loss while simultaneously protecting against the entry of microbial pathogens. Excessive cellular levels of reactive oxygen species can induce cell apoptosis and also damage skin integrity. Propofol (2,6-diisopropylphenol) has antioxidant properties. In this study, we investigated how propofol influences intracellular autophagy and apoptotic cell death induced by oxidative stress in human keratinocytes. METHOD: The following groups were used for experimentation: control, cells were incubated under normoxia (5% COâ‚‚, 21% Oâ‚‚, and 74% Nâ‚‚) without propofol; hydrogen peroxide (Hâ‚‚Oâ‚‚), cells were exposed to Hâ‚‚Oâ‚‚ (300 µM) for 2 h; propofol preconditioning (PPC)/Hâ‚‚Oâ‚‚, cells pretreated with propofol (100 µM) for 2 h were exposed to Hâ‚‚Oâ‚‚; and 3-methyladenine (3-MA)/PPC/Hâ‚‚Oâ‚‚, cells pretreated with 3-MA (1 mM) for 1 h and propofol were exposed to Hâ‚‚Oâ‚‚. Cell viability, apoptosis, and migration capability were evaluated. Relation to autophagy was detected by western blot analysis.
RESULTS
Cell viability decreased significantly in the Hâ‚‚Oâ‚‚ group compared to that in the control group and was improved by propofol preconditioning. Propofol preconditioning effectively decreased Hâ‚‚Oâ‚‚-induced cell apoptosis and increased cell migration. However, pretreatment with 3-MA inhibited the protective effect of propofol on cell apoptosis. Autophagy was activated in the PPC/Hâ‚‚Oâ‚‚ group compared to that in the Hâ‚‚Oâ‚‚ group as demonstrated by western blot analysis and autophagosome staining.
CONCLUSION
The results suggest that propofol preconditioning induces an endogenous cellular protective effect in human keratinocytes against oxidative stress through the activation of signaling pathways related to autophagy.

Keyword

Keratinocytes; Oxidative Stress; Propofol

MeSH Terms

Apoptosis
Autophagy*
Blotting, Western
Cell Death
Cell Movement
Cell Survival
Humans*
Hydrogen Peroxide
Keratinocytes*
Methods
Oxidative Stress*
Propofol*
Reactive Oxygen Species
Skin
Water
Hydrogen Peroxide
Propofol
Reactive Oxygen Species
Water

Figure

• 

  Fig. 1 The experimental protocols followed for all in vitro experiments are shown as follows. Control = normoxia group, H2O2 = H2O2 treatment group, PPC/H2O2 = propofol preconditioning + H2O2 treatment group, 3-MA/PPC/H2O2 = both 3-MA and propofol preconditioning + H2O2 treatment group.

• 

  Fig. 2 The cell viability in human keratinocytes was assessed by MTT assay. (A) Propofol preconditioning had no influence on cell viability at the normoxia state. (B) 3-MA treatment had no influence on cell viability in the normoxic state. (C) More than a 300 µM concentration of H2O2 decreased cell viability.

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  Fig. 3 Cell viability comparison. The H2O2 and 3-MA/PPC/H2O2 groups had lower cell viabilities than the control group.

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  Fig. 4 Hoechst staining: morphological changes in human keratinocytes treated with propofol and 3-MA under oxidative stress. The majority of cells in the control and PPC/H2O2 groups showed round regular nuclei considered as normal morphology. Apoptotic bodies were seen in cells of the H2O2 and 3-MA/PPC/H2O2 groups.

• 

  Fig. 5 Flow cytometric assay was used to detect the percentage of apoptotic cells. Detection of apoptosis and propidium iodide staining. Compared to that in the control and PPC/H2O2 groups, the number of apoptotic cells increased in the H2O2 and 3-MA/PPC/H2O2 groups.

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  Fig. 6 Effects of propofol on the expression of Bak, caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP) in human keratinocytes. Western blot analysis. In the PPC/H2O2 group, cellular expression of caspase-9, caspase-3, and PARP was elevated while that of Bak was down-regulated.

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  Fig. 7 Monodansylcadaverine (MDC) staining of cytoplasmic vacuoles after propofol treatment in human keratinocytes. Significant accumulation of autophagy-specific staining of MDC was observed around the nuclei in PPC/H2O2 group cells.

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  Fig. 8 AO staining of cytoplasmic vacuoles after propofol treatment in human keratinocytes. AO staining, indicated by red fluorescent spots, is shown in PPC/H2O2 group cells, while the control, H2O2, and 3-MA/PPC/H2O2 groups showed mainly green cytoplasmic fluorescence.

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  Fig. 9 In vitro wound healing. Confluent monolayers of human keratinocytes were wounded by scratching the surface as uniformly as possible with a 1-ml pipette tip. The PPC/H2O2 group treated with propofol showed increased wound healing capability.

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