The remifentanilduring H/R dramatically increased formation of autophagosomes and the autophagy pathway inhibitor 3-MA blocked formation of autophagosomes by remifentanil

The remifentanilduring H/R dramatically increased formation of autophagosomes and the autophagy pathway inhibitor 3-MA blocked formation of autophagosomes by remifentanil

Cells handled with remifentanil exhibited more rapidly migration into the wounded 5-Carboxy-X-rhodamine biological activity region in contrast to the management group and 5,7-Dihydroxy-4′-methoxyflavone three-MA team cells (p < 0.05).Figure 4. Hoechst staining: Morphological changes in H/R-induced HaCaT cells treated with remifentanil (1ng/ml), 3-MA and naloxone. H/R-induced HaCaT cells treated with remifentanil, 3-MA and naloxone as observed by fluorescence microscopy. Apoptotic bodies were seen in control and 3-MA group cells. In contrast they were markedly reduced in RPT and NLX group cells. Normoxia = normoxia group, Control = no remifentanil treatment group, RPT = remifentanil post treatment group, 3-MA = both 3-MA and remifentanil treatment group, NLX = both naloxone and remifentanil treatment group.Figure 5. Effects of remifentanil on the expression of caspase-3, caspase-9, Bcl-xl, and Bax in human keratinocytes. Western blot analysis and densitometry. In RPT and NLX groups, cellular expression of cleaved caspase-3, 9, and BAX were down-regulated while that of Bcl-xl was elevated. P < 0.05 as compared with the control group.Figure 6. MDC staining of cytoplasmic vacuoles after remifentanil treatment in human keratinocytes. Fluorescence microscopic (00) analysis of autophagosome in the H/R injured HaCaT cells. H/R caused accumulation of autophagosomes containing partially digested cytoplasmic contents compared to the control group. The remifentanilduring H/R dramatically increased formation of autophagosomes and the autophagy pathway inhibitor 3-MA blocked formation of autophagosomes by remifentanil. But naloxone did not block that.Figure 7. AO staining of autophagosomes after remifentanil treatment in human keratinocytes. Fluorescence microscopic (00) analysis of autophagosome in the H/R injured HaCaT cells. Stained with acridine orange the green shows where the dye has stained the nucleus and the red is where the cell is starting to’digest’ parts of itself in small capsules called autophagasomes. H/R caused accumulation of autophagosomes containing partially digested cytoplasmic contents compared to the control group. The remifentanil during H/R dramatically increased formation of autophagosomes and the autophagy pathway inhibitor 3-MA blocked formation of autophagosomes by remifentanil. But naloxone did not block that.Figure 8. Effects of remifentanil on autophagy markers in human keratinocytes. Western blot analysis and densitometry. ATG5, Becline-1, LC-3 II, and P62 were elevated in RPT and naloxone group cells. P < 0.05 as compared with the control group.The object of the current study is to determine the beneficial effect of remifentanil on human keratinocytes in hypoxia-reoxygenation injury and to investigate whether autophagy is associated with the protective mechanism. This study provides four principal findings. First, we showed that remifentanil treatment increased the proliferation of human keratinocytes in hypoxia-reoxygenation injury (Fig. 2A). Second, remifentanil treatment protected human keratinocytes against hypoxia-reoxygenation induced apoptosis. Using western blot analysis, we showed that remifentanil treatment decreased cleaved caspase-3,-9 levels, related to caspase-dependent pathway, and reduced Bax/Bcl-xl ratio, which is associated with mitochondrial related pathway. Activation of the upstream regulators cleaved-caspase-3 and-9 is a key to the initiation and induction of apoptosis [12,13]. It is known that the mitochondria-dependent apoptotic pathway is regulated by Bcl-xl protein family, such as the anti-apoptotic protein Bcl-xl and proapoptotic protein Bax, which are critical downstream regulators in caspase activation [14,15].Figure 9. In vitro wound healing. Remifentanil restored cell proliferation and migration, which had been decreased by hypoxia. Investigation of cell migration capability after H/R was performed.

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