J Dent Anesth Pain Med.  2016 Dec;16(4):295-302. 10.17245/jdapm.2016.16.4.295.

Dexmedetomidine attenuates Hâ‚‚Oâ‚‚-induced cell death in human osteoblasts

• Yoon JY
• Park JH
• Kim EJ
• Park BS
• Yoon JU
• Shin SW
• Kim DW

• KMID: 2366158
• DOI: http://doi.org/10.17245/jdapm.2016.16.4.295

Abstract

BACKGROUND
Reactive oxygen species play critical roles in homeostasis and cell signaling. Dexmedetomidine, a specific agonist of the α₂-adrenoceptor, has been commonly used for sedation, and it has been reported to have a protective effect against oxidative stress. In this study, we investigated whether dexmedetomidine has a protective effect against H₂O₂-induced oxidative stress and the mechanism of H₂O₂-induced cell death in normal human fetal osteoblast (hFOB) cells.
METHODS
Cells were divided into three groups: control group"”cells were incubated in normoxia without dexmedetomidine, hydrogen peroxide (H2O2) group"”cells were exposed to Hâ‚‚Oâ‚‚ (200 µM) for 2 h, and Dex/Hâ‚‚Oâ‚‚ group"”cells were pretreated with dexmedetomidine (5 µM) for 2 h then exposed to Hâ‚‚Oâ‚‚ (200 µM) for 2 h. 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 was significantly decreased in the Hâ‚‚Oâ‚‚ group compared with the control group, and this effect was improved by dexmedetomidine. The Hoechst 33342 and Annexin-V FITC/PI staining revealed that dexmedetomidine effectively decreased Hâ‚‚Oâ‚‚-induced hFOB cell apoptosis. Dexmedetomidine enhanced the mineralization of hFOB cells when compared to the Hâ‚‚Oâ‚‚ group. In western blot analysis, bone-related protein was increased in the Dex/Hâ‚‚Oâ‚‚ group.
CONCLUSIONS
We demonstrated the potential therapeutic value of dexmedetomidine in Hâ‚‚Oâ‚‚-induced oxidative stress by inhibiting apoptosis and enhancing osteoblast activity. Additionally, the current investigation could be evidence to support the antioxidant potential of dexmedetomidine in vitro.

Keyword

Dexmedetomidine; Osteoblasts; Oxidative stress

MeSH Terms

Apoptosis
Blotting, Western
Cell Death*
Cell Survival
Dexmedetomidine*
Homeostasis
Humans*
Hydrogen Peroxide
In Vitro Techniques
Miners
Osteoblasts*
Oxidative Stress
Reactive Oxygen Species
Dexmedetomidine
Hydrogen Peroxide
Reactive Oxygen Species

Figure

• 

  Fig. 1 Measurement of cell viability in human fetal osteoblast (hFOB) cells. Cell viability was analyzed using the MTT assay. (A) Dexmedetomidine did not show any cytotoxic effect on the hFOB cells. (B) After a wide range of concentrations of H2O2 (0, 25, 50, 100, 200, and 400 µM) treatment for 2 h, the viability was significantly decreased in a dose-dependent manner.

• 

  Fig. 2 Human fetal osteoblast (hFOB) cells were pretreated with dexmedetomidine (Dex, 5 µM) for 2 h and then treated with 200 µM of H2O2 for 2 h. Cell viability was analyzed using the MTT assay. The data were calculated as a percentage of the control group and expressed as the mean of at least three experiments. Cell viability was markedly decreased in the H2O2 treatment group compared to the control, but viability was significantly improved by dexmedetomidine. *P < 0.05 compared with the control group, †P < 0.05 compared with the H2O2 group.

• 

  Fig. 3 Morphological changes of human fetal osteoblast (hFOB) cells in H2O2-induced apoptosis. The group preconditioned with dexmedetomidine (Dex, 5 µM) demonstrated weak nuclear condensation. Nuclei of normal control cells and Dex/H2O2 groups were lightly stained by the dye and morphological changes were not detected, while the nuclei of H2O2 treated cells presented with typical apoptotic features.

• 

  Fig. 4 Detection of apoptosis and necrosis with Annexin-V-FITC and propidium iodide (PI) staining. Every group of cells with Annexin-V and PI staining were measured by flow cytometry. Compared with the control group, the portion of Annexin-V(+)/PI(+) cells in the H2O2 group increased from 1.2% to 25%. However, pretreatment with dexmedetomidine 2 h prior to H2O2 treatment significantly attenuated the percentage of Annexin-V(+)/PI(+) cells to 10% (P < 0.05).

• 

  Fig. 5 Measurement of human fetal osteoblast (hFOB) cell mineralization using Alizarin red S staining. The Dex/H2O2 group showed enhanced staining compared to the H2O2 group. Dex = Dexmedetomidine.

• 

  Fig. 6 Detection of protein levels using western blot analysis. Expression of type I collagen (Col I), bone morphogenetic protein (BMP)-2, osterix, and transforming growth factor-beta (TGF-β) in human fetal osteoblast (hFOB) cells. The levels of Col I, BMP-2, osterix, and TGF-β were increased in the dexmedetomidine pretreatment group compared to the H2O2 group.

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