J Dent Anesth Pain Med.  2016 Dec;16(4):263-271. 10.17245/jdapm.2016.16.4.263.

Protective effects of remifentanil against Hâ‚‚Oâ‚‚-induced oxidative stress in human osteoblasts

• Yoon JY
• Kim DW
• Kim EJ
• Park BS
• Yoon JU
• Kim HJ
• Park JH

• KMID: 2366154
• DOI: http://doi.org/10.17245/jdapm.2016.16.4.263

Abstract

BACKGROUND
Bone injury is common in many clinical situations, such as surgery or trauma. During surgery, excessive reactive oxygen species (ROS) production decreases the quality and quantity of osteoblasts. Remifentanil decreases ROS production, reducing oxidative stress and the inflammatory response. We investigated remifentanil's protective effects against Hâ‚‚Oâ‚‚-induced oxidative stress in osteoblasts.
METHODS
To investigate the effect of remifentanil on human fetal osteoblast (hFOB) cells, the cells were incubated with 1 ng/ml of remifentanil for 2 h before exposure to H2O2. For induction of oxidative stress, hFOB cells were then treated with 200 µM Hâ‚‚Oâ‚‚ for 2 h. To evaluate the effect on autophagy, a separate group of cells were incubated with 1 mM 3-methyladenine (3-MA) before treatment with remifentanil and Hâ‚‚Oâ‚‚. Cell viability and apoptotic cell death were determined via MTT assay and Hoechst staining, respectively. Mineralized matrix formation was visualized using alizarin red S staining. Western blot analysis was used to determine the expression levels of bone-related genes.
RESULTS
Cell viability and mineralized matrix formation increased on remifentanil pretreatment before exposure to H₂O₂-induced oxidative stress. As determined via western blot analysis, remifentanil pretreatment increased the expression of bone-related genes (Col I, BMP-2, osterix, and TGF-β). However , pretreatment with 3-MA before exposure to remifentanil and H₂O₂ inhibited remifentanil's protective effects on hFOB cells during oxidative stress.
CONCLUSIONS
We showed that remifentanil prevents oxidative damage in hFOB cells via a mechanism that may be highly related to autophagy. Further clinical studies are required to investigate its potential as a therapeutic agent.

Keyword

Autophagy; Osteoblasts; Oxidative Stress; Remifentanil

MeSH Terms

Autophagy
Blotting, Western
Cell Death
Cell Survival
Humans*
Miners
Osteoblasts*
Oxidative Stress*
Reactive Oxygen Species
Reactive Oxygen Species

Figure

• 

  Fig. 1 Experimental procedures are shown in this protocol. Control = normoxia and no remifentanil (RPC) pretreatment, H2O2 = H2O2 exposure, RPC + H2O2 = RPC pretreatment before H2O2 exposure, 3-methyladenine (3-MA) + RPC + H2O2 = pretreatment with 3-MA, then pretreatment with remifentanil before H2O2 exposure.

• 

  Fig. 2 MTT reduction was examined as a measure of viability of human fetal osteoblast (hFOB) cells after exposure to H2O2. (A) There were no significant differences in cell viability in the group that underwent remifentanil pretreatment, (B) Cell viability was maintained when the cells were pretreated with 3-MA prior to remifentanil administration, (C) The application of H2O2 alone to hFOB cells at various concentrations resulted in a decrease of cell viability above a concentration of 100 µM.

• 

  Fig. 3 Viability of human fetal osteoblast cells was evaluated in four groups. In the H2O2 group, cell viability decreased significantly, while it improved in the remifentanil (RPC) + H2O2 group. In the 3-methyladenine + RPC + H2O2 group, cell viability decreased compared to that in the RPC + H2O2 group. *P < 0.05 compared with control group.

• 

  Fig. 4 Remifentanil inhibited H2O2-induced human fetal osteoblast (hFOB) cell apoptosis. (A) Morphological changes in hFOB cells were evaluated via Hoechst 33342 staining. The cellular fluorescence changes were observed using a fluorescence microscope. Apoptotic nuclei were seen in the H2O2 and 3-methyl A+remifentanil (RPC)+H2O2 groups of hFOB cells. In contrast, they markedly decreased in the RPC+H2O2 group. (B) The rate of apoptotic cell death was also assessed using a fluorescence-activated cell sorter.

• 

  Fig. 5 The results of a mineralized matrix formation assay in human fetal osteoblast cells. Representative images of mineralized matrix formation were visualized with alizarin red S dye following 14 days of incubation. Remifentanil pretreatment counteracted the negative effects of H2O2 and increased mineralized matrix formation.

• 

  Fig. 6 Expression of bone-related genes such as Col I, BMP-2, osterix, and TGF-β was evaluated using western blot analysis. Remifentanil pretreatment increased the expression of Col I, BMP-2, osterix, and TGF-β in human fetal osteoblast cells exposed to H2O2-induced oxidative stress. Pretreatment with 3-methyladenine suppressed the expression of bone-related genes. Quantification of bone-related gene expression was also assessed using densitometry. *P < 0.05 compared with control group.

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