J Dent Anesth Pain Med.  2017 Mar;17(1):37-46. 10.17245/jdapm.2017.17.1.37.

Propofol protects against oxidative-stress-induced COS-7 cell apoptosis by inducing autophagy

• Yoon JY
• Baek CW
• Kim EJ
• Park BS
• Yu SB
• Yoon JU
• Kim EN

• KMID: 2374722
• DOI: http://doi.org/10.17245/jdapm.2017.17.1.37

Abstract

BACKGROUND
In oxidative stress, reactive oxygen species (ROS) production contributes to cellular dysfunction and initiates the apoptotic cascade. Autophagy is considered the mechanism that decreases ROS concentration and oxidative damage. Propofol shows antioxidant properties, but the mechanisms underlying the effect of propofol preconditioning (PPC) on oxidative injury remain unclear. Therefore, we investigated whether PPC protects against cell damage from hydrogen peroxide (Hâ‚‚Oâ‚‚)-induced oxidative stress and influences cellular autophagy. METHOD: COS-7 cells were randomly divided into the following groups: control, cells were incubated in normoxia (5% COâ‚‚, 21% Oâ‚‚, and 74% Nâ‚‚) for 24 h without propofol; Hâ‚‚Oâ‚‚, cells were exposed to Hâ‚‚Oâ‚‚ (400 µM) for 2 h; PPC + Hâ‚‚Oâ‚‚, cells pretreated with propofol 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 was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue (MTT) reduction. Apoptosis was determined using Hoechst 33342 staining and fluorescence microscopy. The relationship between PPC and autophagy was detected using western blot analysis.
RESULTS
Cell viability decreased more significantly in the Hâ‚‚Oâ‚‚ group than in the control group, but it was improved by PPC (100 µM). Pretreatment with propofol effectively decreased Hâ‚‚Oâ‚‚-induced COS-7 cell apoptosis. However, pretreatment with 3-MA inhibited the protective effect of propofol during apoptosis. Western blot analysis showed that the level of autophagy-related proteins was higher in the PPC + Hâ‚‚Oâ‚‚ group than that in the H2O2 group.
CONCLUSION
PPC has a protective effect on Hâ‚‚Oâ‚‚-induced COS-7 cell apoptosis, which is mediated by autophagy activation.

Keyword

Autophagy; COS-7 Cells; Oxidative Stress; Propofol

MeSH Terms

Animals
Apoptosis*
Autophagy*
Blotting, Western
Cell Survival
COS Cells*
Hydrogen Peroxide
Methods
Microscopy, Fluorescence
Oxidative Stress
Propofol*
Reactive Oxygen Species
Hydrogen Peroxide
Propofol
Reactive Oxygen Species

Figure

• 

  Fig. 1 The effect of H2O2 and propofol on COS-7 cell viability is assessed using the MTT assay. (A) Propofol at various concentrations shows no significant differences in cell viability. (B) Cell viability is maintained when the cells are pretreated with 3-MA before propofol administration. (C) The application of H2O2 to COS-7 cells at various concentrations (50-µM increments) results in a decrease in cell viability.

• 

  Fig. 2 Cell viability decreases significantly in the H2O2 group and is improved by propofol preconditioning (PPC) (100 µM). In the 3-MA + PPC + H2O2 group, cell viability is significantly lower than it is in the control group. *P < 0.05 compared with the control group.

• 

  Fig. 3 (A) Hoechst staining: Morphological changes in COS-7 cells treated with H2O2, propofol (100 µM), and 3-MA. The cellular fluorescence changes are observed under a fluorescence microscope. Apoptotic bodies are observed in the H2O2 and 3-MA + PPC + H2O2 group cells. In contrast, apoptotic bodies markedly reduced in the PPC + H2O2 group cells. Control: no propofol treatment group; H2O2: H2O2 exposure group; PPC+H2O2: propofol pretreatment before exposure to H2O2 group; 3-MA + PPC + H2O2: pretreatment with 3-MA (1 mM) before 1 h and propofol treatment before exposure to H2O2 group. (B) The rate of apoptosis is assessed using a fluorescence-activated cell sorter.

• 

  Fig. 4 (A) MDC staining of cytoplasmic vacuoles after propofol treatment in COS-7 cells. Compared to the control group, the propofol-pretreated group shows the accumulation of autophagosomes containing partially digested cytoplasmic contents. Propofol pretreatment dramatically increases the formation of autophagosomes, and the autophagy-pathway inhibitor 3-MA inhibits the propofol-mediated formation of autophagosomes. (B) AO staining of autophagosomes after propofol treatment in COS-7 cells. Autophagosomes with yellow or red vesicles during AO staining are detected in the PPC+H2O2 group, and 3-MA inhibits autophagosome formation.

• 

  Fig. 5 Effects of propofol on autophagy markers in COS-7 cells are assessed using western blot analysis and densitometry. The levels of Atg5, Beclin-1, and p62 are significantly lower in the H2O2 group than in the control group as a result of the H2O2-induced apoptotic signaling; however, their levels increase in the PPC+H2O2 group. The level of LC3-II is significantly higher in the H2O2 group than in the control group, and is even higher in the PPC+H2O2 group. The levels of Atg5, Beclin-1, and LC3-II decrease when autophagy is suppressed by 3-MA. *P < 0.05 compared with the control group.

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