J Dent Anesth Pain Med.  2016 Sep;16(3):175-184. 10.17245/jdapm.2016.16.3.175.

Remifentanil induces autophagy and prevents hydrogen peroxide-induced apoptosis in Cos-7 cells

• Yoon JY
• Baek CW
• Woo MN
• Kim EJ
• Yoon JU
• Park CH

• KMID: 2354652
• DOI: http://doi.org/10.17245/jdapm.2016.16.3.175

Abstract

BACKGROUND
This study investigated the effect of remifentanil pretreatment on Cos-7 cells exposed to oxidative stress, and the influence of remifentanil on intracellular autophagy and apoptotic cell death.
METHODS
Cells were divided into 4 groups: (1) Control: non-pretreated cells were incubated in normoxia (5% COâ‚‚, 21% Oâ‚‚, and 74% Nâ‚‚). (2) Hâ‚‚Oâ‚‚: non-pretreated cells were exposed to Hâ‚‚Oâ‚‚ for 24 h. (3) RPC+Hâ‚‚Oâ‚‚: cells pretreated with remifentanil were exposed to Hâ‚‚Oâ‚‚ for 24 h. (4) 3-MA+RPC+Hâ‚‚Oâ‚‚: cells pretreated with 3-Methyladenine (3-MA) and remifentanil were exposed to Hâ‚‚Oâ‚‚ for 24 h. We determined the cell viability of each group using an MTT assay. Hoechst staining and FACS analysis of Cos-7 cells were performed to observe the effect of remifentanil on apoptosis. Autophagy activation was determined by fluorescence microscopy, MDC staining, and AO staining. The expression of autophagy-related proteins was observed using western blotting.
RESULTS
Remifentanil pretreatment increased the viability of Cos-7 cells exposed to oxidative stress. Hoechst staining and FACS analysis revealed that oxidative stress-dependent apoptosis was suppressed by the pretreatment. Additionally, fluorescence microscopy showed that remifentanil pretreatment led to autophagy-induction in Cos-7 cells, and the expression of autophagy-related proteins was increased in the RPC+Hâ‚‚Oâ‚‚ group.
CONCLUSIONS
The study showed that remifentanil pretreatment stimulated autophagy and increased viability in an oxidative stress model of Cos-7 cells. Therefore, we suggest that apoptosis was activated upon oxidative stress, and remifentanil preconditioning increased the survival rate of the cells by activating autophagy.

Keyword

Apoptosis; Autophagy; Cos-7 cells; Oxidative stress; Remifentanil

MeSH Terms

Animals
Apoptosis*
Autophagy*
Blotting, Western
Cell Death
Cell Survival
COS Cells*
Hydrogen*
Microscopy, Fluorescence
Oxidative Stress
Survival Rate
Hydrogen

Figure

• 

  Fig. 1 Cell viability of Cos-7 cells assessed by MTT assay. (A) The effect of remifentanil and 3-MA + remifentanil preconditioning on Cos-7 cell viability at various concentrations (0, 0.1, 0.5, 1, 2 ng/ml) without exposure to H2O2, (B) Cell viability of Cos-7 cells upon exposure to various concentrations (0, 50, 100, 200, 400) of H2O2.

• 

  Fig. 2 The effect of remifentanil pretreatment on Cos-7 cell viability with or without autophagy inhibition, as examined by MTT assay. *P < 0.05 as compared with control group. Control: no H2O2, remifentanil treatment group, H2O2: hydrogen peroxide exposure only, RPC + H2O2: remifentanil pretreatment group plus hydrogen peroxide exposure, 3-MA + RPC + H2O2: pretreatment with 3-MA and remifentanil plus hydrogen peroxide exposure.

• 

  Fig. 3 Hoechst staining: Morphological changes in Cos-7 cells upon pretreatment with remifentanil alone or in combination with 3-MA before exposure to oxidative stress. Cos-7 cells were pretreated with remifentanil or remifentanil and 3-MA, then exposed to oxidative stress. Cells were stained with Hoechst stain and observed by fluorescence microscopy. Apoptotic bodies were seen in H2O2 and 3-MA + RPC + H2O2 group cells. In contrast, they were remarkably reduced in RPC + H2O2 group cells. Control: no H2O2, remifentanil treatment group, H2O2: hydrogen peroxide exposure only, RPC + H2O2: remifentanil pretreatment group plus hydrogen peroxide exposure, 3-MA + RPC + H2O2: pretreatment with 3-MA and remifentanil plus hydrogen peroxide exposure.

• 

  Fig. 4 Fluorescence activated cell sorting (FACS) analysis of Cos-7 cells to determine apoptotic cell populations of each experimental group. Control: no H2O2, remifentanil treatment group, H2O2: hydrogen peroxide exposure only, RPC + H2O2: remifentanil pretreatment group plus hydrogen peroxide exposure, 3-MA + RPC + H2O2: pretreatment with 3-MA and remifentanil plus hydrogen peroxide exposure.

• 

  Fig. 5 Fluorescence microscopic analysis of autophagosomes in Cos-7 cells under oxidative stress to observe autophagy activation upon different pretreatment conditions. (A) MDC staining of cytoplasmic vacuoles after preconditioning with remifentanil in Cos-7 cells followed by exposure to oxidative stress. Cellular oxidative stress resulted in greater accumulation of autophagosomes containing partially digested cytoplasmic contents compared to the control group. The remifentanil pretreatment group showed increased formation of autophagosomes. Compared with the RPC + H2O2 group, the autophagy pathway inhibitor, 3-MA, blocked formation of autophagosomes. Magnification: 400 ×, (B) AO staining of autophagosomes after remifentanil pretreatment in Cos-7 cells followed by exposure to oxidative stress. AO staining indicated by red fluorescent spots appeared in RPC + H2O2 group cells, while the control, H2O2, and 3-MA + RPC + H2O2 groups showed mainly green cytoplasmic fluorescence. The green shows where the dye has stained the nucleus and the red indicates autophagosome formation. Magnification: 400 ×. Control: no H2O2, remifentanil treatment group, H2O2: hydrogen peroxide exposure only, RPC + H2O2: remifentanil pretreatment group plus hydrogen peroxide exposure, 3-MA + RPC + H2O2: pretreatment with 3-MA and remifentanil plus hydrogen peroxide exposure.

• 

  Fig. 6 Western blot analysis and densitometry to determine the effects of remifentanil treatment ATG5, Beclin-1, LC3-II, and p62 protein levels in each experimental group of Cos-7 cells. In RPC + H2O2 groups, cellular expression of ATG5, Beclin-1, LC3-II and p62 were upregulated, while these proteins were downregulated in 3-MA+RPC+H2O2 groups. Control: no H2O2, remifentanil treatment group, H2O2: hydrogen peroxide exposure only, RPC+H2O2: remifentanil pretreatment group plus hydrogen peroxide exposure, 3-MA+RPC+H2O2: pretreatment with 3-MA and remifentanil plus hydrogen peroxide exposure. *P < 0.05 as compared with the control group.

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