J Dent Anesth Pain Med.  2016 Mar;16(1):39-47. 10.17245/jdapm.2016.16.1.39.

Effects of propofol-induced autophagy against oxidative stress in human osteoblasts

• Kim EJ
• Choi IS
• Yoon JY
• Park BS
• Yoon JU
• Kim CH

• KMID: 2308764
• DOI: http://doi.org/10.17245/jdapm.2016.16.1.39

Abstract

BACKGROUND
Oxidative stress occurs during the aging process and other conditions such as bone fracture, bone diseases, and osteoporosis, but the role of oxidative stress in bone remodeling is unknown. Propofol exerts antioxidant effects, but the mechanisms of propofol preconditioning on oxidative stress have not been fully explained. Therefore, the aim of this study was to evaluate the protective effects of propofol against H2O2-induced oxidative stress on a human fetal osteoblast (hFOB) cell line via activation of autophagy.
METHODS
Cells were randomly divided into the following groups: control cells were incubated in normoxia (5% CO2, 21% O2, and 74% N2) without propofol. Hydrogen peroxide (H2O2) group cells were exposed to H2O2 (200 µM) for 2 h, propofol preconditioning (PPC)/H2O2 group cells were pretreated with propofol then exposed to H2O2, 3-methyladenine (3-MA)/PPC/H2O2 cells were pretreated with 3-MA (1 mM) and propofol, then were exposed to H2O2. Cell viability and apoptosis were evaluated. Osteoblast maturation was determined by assaying bone nodular mineralization. Expression levels of bone related proteins were determined by western blot.
RESULTS
Cell viability and bone nodular mineralization were decreased significantly by H2O2, and this effect was rescued by propofol preconditioning. Propofol preconditioning effectively decreased H2O2-induced hFOB cell apoptosis. However, pretreatment with 3-MA inhibited the protective effect of propofol. In western blot analysis, propofol preconditioning increased protein levels of collagen type I, BMP-2, osterix, and TGF-β1.
CONCLUSIONS
This study suggests that propofol preconditioning has a protective effect on H2O2-induced hFOB cell death, which is mediated by autophagy activation.

Keyword

Propofol; Oxidative Stress; Osteoblasts; Autophagy

MeSH Terms

Aging
Antioxidants
Apoptosis
Autophagy*
Blotting, Western
Bone Diseases
Bone Remodeling
Cell Death
Cell Line
Cell Survival
Collagen Type I
Fractures, Bone
Humans*
Hydrogen Peroxide
Miners
Osteoblasts*
Osteoporosis
Oxidative Stress*
Propofol
Antioxidants
Collagen Type I
Hydrogen Peroxide
Propofol

Figure

• 

  Fig. 1 The in vitro experimental protocols. Control = normal incubation, H2O2 = normal incubation after H2O2 treatment, PPC/H2O2 = propofol preconditioning (PPC) before H2O2 treatment, 3-MA/PPC/H2O2 = 3-methyladenine treatment followed by propofol treatment and H2O2 treatment.

• 

  Fig. 2 Effect of propofol and 3-MA on cell viability. (A) The viability of propofol-treated hFOB cells. Propofol did not elicit any significant toxic effect on hFOB cells. (B) 3-MA did not elicit any significant toxic effect on hFOB cells.

• 

  Fig. 3 Effect of H2O2, PPC, and 3-MA on cell viability. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT) assay. *P < 0.05 as compared with control group.

• 

  Fig. 4 Morphological changes after H2O2, PPC, and/or 3-MA treatment in hFOB cells. Fluorescence microscopy images are shown and were analyzed for presence of apoptotic bodies.

• 

  Fig. 5 Flow cytometry analysis of apoptosis and necrosis with Annexin-V-FITC and propidium iodide staining.

• 

  Fig. 6 Fluorescence microscopy (400X) analysis of autophagosomes in hFOB cells after oxidative stress. Images were taken after staining with monodansylcadaverine (MDC). Blue fluorescence indicates autophagic vacuoles and autophagosomes can be seen as punctate areas of fluorescence.

• 

  Fig. 7 Fluorescence microscopy (400X) analysis of autophagosomes in hFOB cells after oxidative stress. Images were taken after staining with acridine orange. Green indicates where the dye has stained the nucleus and red indicates where the cell is starting to 'digest' parts of itself in small capsules called autophagosomes.

• 

  Fig. 8 Photomicrographs of cultures stained with Alizarin Red after 28 days of culture. Red indicates positive staining for Ca2+ in the matrix.

• 

  Fig. 9 Western blot analysis. Expression of collagen type I, bone morphogenic protein (BMP)-2, osterix, and transforming growth factor-beta (TGF-β)1 in the experimental groups indicated. Propofol preconditioning was found to increase the expression of collagen type I, BMP-2, osterix, and TGF-β1.

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