The Protective Effect Of Desflurane Preconditioning On The Injury Of Endothelial Cells Induced By Anoxia/Reoxygenation And Its Molecular Mechanism

PartⅠ: The Protective Effect of Desflurane Preconditioning on the Injury ofEndothelial Cells Induced by Anoxia/ReoxygenationObjective To elucidate the damage on human endothelial cells induced byAnoxia/Reoxygenation(A/R) and the protective effect through desfluranepreconditioning.Methods This study is based on human umbilical vein endothelial cell line (ECV304) . The ECV304 cells were divided into 5 groups: Control group (C group), A/R Group, A/R + TNF-α10ng/ml Group, Des + A/R Group, and Des + A/R + TNF-α10ng/ml Group. The apoptosis or necrosis of cells in each group was detected by the methods of fluorescence flow cytometry, terminal deoxynucleotide mediated nick end labeling (TUNEL) and electron microscope (EM) observation.Results Cells of A/R group has a higher percentage of apoptosis and necrosis as compared to cells of control group. Cell apoptosis and necrosis rates were further elevated in A/R+TNF-αgroup. Des+A/R Group and Des+A/R+TNF-αhave significantly lower percentage of apoptosis and necrosis as compared to A/R or A/R+TNF-αGroup, respectively. The EM observed that there were more necrotic cells in A/R Group, A/R+TNF-αGroup, as cell swelling, damage of ultra-micro structure, massive vacuole, melting down of nuclear chromatin, nuclear fragmentation and damage of cell membrane could be seen more often. However, through desflurane preconditioning, the cell structure in those two groups remains normal. There were abundant endoplasmic reticulum and mitochondria in the cytoplasm, and much more nucleoli in the nuclei, which indicates that the cells were in the status of duplicating and self-repairing.Conclusions Desflurane preconditioning can reduce endothelial cell death and injury caused by A/R, and enhance the capability of cell repairing and proliferation. PartⅡ: Effect of Desflurane Preconditioning on NF-κB Signal Transduction Pathway of Endothelial Cells Induced by Anoxia/Reoxygenation InjuryObjective To explore the effect of desflurane preconditioning on NF-κB signal transduction pathway of endothelial cells induced by anoxia/reoxygenation injury, and its molecular mechanism.Methods This study is based on human umbilical vein endothelial cell line (ECV304) . The ECV304 cells were divided into 5 groups: Control group (C group), A/R Group, A/R + TNF-α10ng/ml Group, Des + A/R Group, and Des + A/R + TNF-α10ng/ml Group. The intracellular localization of NF-κB/p65 was analyzed by indirect immumofluorescence assay. The protein levels of NF-κB/ p65 and the key signaling molecules in NF-kappaB pathway, such as IκB-α(the inhibitory factor of the NF-κB), its phosphorylated form, p-IκB-α, and the phosphorylated form of IκB kinase, p-IKKα/IKKβ, were detected by Western blot. The protein (tumor necrosis factor receptor-associated factor 2, TRAF2 and IκB kinase, IKKα) expression in receptor signal transduction compounds of cell membrane were determined by Co-Immunoprecipitation-Western blot. To examine the protein expression of tumor necrosis factor receptor 1(TNFR1) in cell membrane, the fluorescence flow cytometry assay (FCA) was used.Results In TNF-αstimulated cells, NF-κB/ p65 nucleus translocation, which is the indication of NF-κB activation, was significantly inhibited by 1．0MAC desflurane preconditioning. The results of subcellular fractionation and Western blot confirmed the observed immumofluorescence result. The IκBαexpression level by Western blot was lower in A/R group than that in control group and significantly decreased in A/R+TNF-αgroup. In Des+A/R group or Des+A/R+TNF-αgroup, the level was significantly higher than that in A/R group or A/R+TNF-αgroup, respectively. There was no significant difference between the levels in Des+A/R and Des+A/R+TNF-αgroup. Furthermore, the phosphorylation levels of IκBα,IKKα/IKKβin A/R group elevated and significantly increased in A/R+TNF-αgroup as compared to that in control group. While in Des+A/R group and Des+A/R+TNF-αgroup, the protein levels were lower than those in A/R group and A/R+TNF-αgroup, respectively. The formation of signal transduction complex was detected by Co-Immunoprecipitation-Western blot in order to evaluate whether desflurane preconditioning can influence the formation of receptor-adaptor complex. The recruitment of TRAF2 and activation of IKKαwere slightly induced in A/R group and significantly increased in A/R+TNF-αgroup as compared to control group. Whereas, recruitment of TRAF2 and activation of IKKαsignificantly decreased in Des+A/R group or Des+A/R+TNF-αgroup. The protein expression of TNFR1 in the membrane of endothelial cells in A/R group was comparable to that in control group. The internalization of the receptors could significantly increase as stimulated by TNF-αalone to activate NF-κB signal transduction pathway. The desflurane preconditioning made no significant difference of the TNFR1 internalization as compared to the non-preconditioning groups, respectively.Conclusions Desflurane preconditioning can inhibit the activation of the NF-κB signal transduction pathway induced by A/R injury, which could be one of the protective mechanisms against A/R injury. In the initiation of activation of NF-κB signal transduction pathway, the inhibition of recruitment of TRAF2 and activation of IKKαother than TNFR1 internalization may play the key role in the protective effect of desflurane preconditioning. PartⅢ: Effect of Desflurane Preconditioning on the Effective Factors of Activated NF-κB Signal Transduction Pathway in Endothelial Cells Induced byAnoxia/Reoxygenation InjuryObjective To evaluate the effect of desflurane preconditioning on the effective factors(inflammatory mediators and adhesion molecules) of activated NF-κB signaltransduction pathway induced by anoxia/reoxygenation injury.Methods This study is based on human umbilical vein endothelial cell line (ECV304) .The ECV304 cells were divided into 5 groups: Control group (C group), A/R Group,A/R + TNF-α10ng/ml Group, Des + A/R Group, and Des + A/R + TNF-α10ng/mlGroup. The gene expression of intercellular adhesion molecule-1 (ICAM-1) , vascularcell adhesion molecule-1 (VCAM-1) and interleukin-8 (IL-8) was detected by reversetranscription-polymerase chain reaction (RT-PCR).Results There was few mRNA expression of ICAM-1, VCAM-1 and IL-8 in controlgroup. In A/R group, the expression was higher than that in control group,significantly increased in A/R+TNF-αgroup, but significantly decreased in Des+A/Rgroup and Des+A/R+TNF-αgroup. There was no significantly expression differencebetween Des+A/R group and Des+A/R+TNF-αgroup.Conclusions Desflurane preconditioning could inhibit the expression levels ofinflammatory mediators and adhesion molecules induced by anoxia/reoxygenationinjury.