Desflurane Preconditioning Induces Oscillation Of NF-κB In Endothelial Cells

Part I Desflurane preconditioning proctects HUVECs against A/RObjective:To isolate and culture primary human umbilical vein endothelial cells (HUEVCs). To elucidate the protective effect against anoxia and reoxygenation induced by deflurane preconditioning.Method:HUVECs were isolated from human umbilical veins by the digestion of collagen, and identificated by Ⅷ factor immunofluorescence assay. The HUVECs were divided into3groups:control group (CON group), anoxia and reoxygenation group (A/R group), desflurane preconditioning group (DesPC group).30min desflurane (1MAC) treatment was initiated before anoxia in the DesPC group. Cells apoptosis was analyzed by flow cytometry using Annexin V-fluorescein isothiocyanate staining and cell viability was evaluated by modified tertrozalium salt (MTT) assay. The cellular SOD activitiy were tested by water-soluble tetrazolium salt (WST-1) assay.Results:Primary HUVECs achieved confluency in5-7days and presented a cobblestone appearance. Cells expressed factor Ⅷ antigen in the cytoplasm. Cells in A/R group revealed a higher apoptosis rate (35.73±5.43%=mean±D) compared with CON group (23.62±4.56%). Desflurane preconditioning significantly decreased the apoptosis rate in DesPC group to (27.39±3.22%). Desflurane preconditioning also increased the cell viability in DesPC group to (96.58±6.52%) compared with A/R group (86.25±3.61%). The SOD activity of A/R group (23.55±7.84U/mgprotein) was significantly lower than CON group (38.86±6.40U/mgprotein). Cells in DesPC group had much higher SOD activity (35.49±9.36U/mgprotein) than A/R group.Conclusion:Primary HUVECs were successfully isolated and the vitro model of A/R was established. Desflurane preconditioning decreased cell apoptosis and reserved the activity of SOD, and protected HUVECs against A/R. Part II Desflurane preconditioning induces oscillation of NF-κB and increases anti-apoptotic proteins in HUVECsObjective:NF-κB has been implicated in anesthetic preconditioning (APC) induced protection against anoxia and reoxygenation (A/R). To investigate the influence of desflurane preconditioning on the expression of NF-κB and anti-apoptotic proteins in HUVECs and to elucidate the mechanism of desflurane preconditioning.Method:HUVECs were divided into6groups:control group (CON group), deflurane group (DES group), desflurane preconditioning group (DesPC group), anoxia and reoxygenation group (A/R group), BAY+DesPC group and BAY+A/R group.30min desflurane (1MAC) treatment was initiated before ischemia in the DesPC group, cells in DES group exposed to desflurane without following A/R. BAY11-7082were used in BAY+DesPC and BAY+A/R group before desflurane preconditioning. Cells apoptosis was analyzed by flow cytometry using annexin V-fluorescein isothiocyanate staining and cell viability was evaluated by modified tertrozalium salt (MTT) assay. The nuclear tanslocation of NF-κB p65was analyzed by immunofluorescence assay. Expressions of c-IAP1, Bcl-2, caspases-3, ⅠκBα and phosphorylated NF-κB p65(p-p65) were determined by western blot.Results:In the DES group, the nuclear translocation of NF-κB p65was activated by1MAC desflurane, the expression level of ⅠκBα was lower and phosphorylated NF-κB p65was higher than CON group. A/R significantly caused NF-κB p65phosphorylation. In the DesPC group, the expression level of ⅠκBα was higher and phosphorylated NF-κB p65was lower than A/R group, accompanied with higher expression of c-IAP1、Bcl-2and pro-caspase-3. Cell viability and apoptosis rate were not significant different in BAY+DesPC group and BAY+A/R group, and the expression level of ⅠκBα, phosphoryltated NF-κB p65and pro-caspase-3were also not different。Conclusion:The results demonstrated that desflurane activated NF-κB during the preconditioning period and negative-feedback inhibited excessive activation of NF-κB in reoxygenation. And the oscillation of NF-κB induced by desflurane preconditioning finally upregulated anti-apoptotic proteins expression and protected endothelial cells against A/R.