Remifentanil, an ultra-short-acting mu-opioid receptor agonist, is unique from other opioids because of its esterasebased metabolism, minimal accumulation, and very rapid onset and offset of clinical action. Remifentanil can prevent the inflammatory response and can suppress inducible nitric oxide synthase expression in a septic mouse model. However, the effects of remifentanil on human keratinocyte and autophagy have yet to be fully elucidated during hypoxia-reoxygenation. Here we investigated whether remifentanil confers protective effect against hypoxia-reoxygenation in human keratinocyte and, if so, whether autophagy mediates this effect.

The human keratinocytes were cultured under 1% oxygen tension. The cells were gassed with 94% N2, and 5% CO2 and incubated for 24 h at 37℃. To determine whether the administration of affects human keratinocytes hypoxia-reoxygenation injury, cells were then exposed to various concentrations of remifentanil (0.01, 0.1, 0.5 and 1 ng/ml) for 2 h. After remifentanil treatment, to simulate reoxygenation and recovery, the cells were reoxygenated for 12 h at 37℃. Control group did not receive remifentanil treatment. Normoxia group did not receive hypoxia and remifentanil treatment for 36 h. 3-MA group was treated 3-methyladenine (3-MA) for 1h before remifentanil treatment. Cell viability was measured using a quantitative colorimetric assay with MTT, showing the mitochondrial activity of living cells. Cells were stained with fluorescence and analyzed with Western blot analysis to find out any relations with activation of autophagy.

Prominent accumulation of autophagic specific staining MDC was observed around the nuclei in RPT group HaCaT cells. Similarly, AO staining, red fluorescent spots appeared in RPT group HaCaT cells, while the Normoxia, control and 3-MA groups showed mainly green cytoplasmic fluorescence. We here examined activation of autophagy related protein under H/R-induced cells by Western blotting analysis. Atg5, Beclin-1, LC3-II (microtubule-associated protein 1 light chain 3 form II) and p62 was elevated in RPT group cells. But they were decreased when autophagy was suppressed by 3-MA (Fig. 5).

Although the findings of this study are limited to an in vitro interpretation, we suggest that remifentanil may have a beneficial effect in the recovery of wound from hypoxia-reoxygenation injury.