Anti-inflammatory Effects Of Sevoflurane Pretreatment On TNF-α Induced Vascular Endothelial Cells In Vitro And Its Mechanism

Objective (1) To establish culturing technique of HUVECs in vitro. To investigate the effects of TNF-a on the expression of ICAM-1, VCAM-1 of HUVECs and then to decide the optimal action time course and concentration. To investigate the inhibitory effects of sevoflurane pretreatment with different concentration on the expression of ICAM-1, VCAM-1 in TNF-a-induced HUVECs and neutrophils adhesion to HUVECs induced by TNF-a. (2) To investigate the effects of sevoflurane pretreatment on the activity of NF-κB signal transduction pathway in TNF-a-induced HUVECs, so as to provide theoretical and experimental evidence for the mechanism of anti-inflammatory effects of sevoflurane pretreatment. (3) To explore the effects of eNOS-NO signal system on anti-inflammatory effects of sevoflurane pretreatment on vascular endothelial cells in vitro.Methods The study is divided into three parts:(1) HUVECs were cultured according to references with minor modifications. Endothelial cells were identified by both morphology and immunocytochemical staining response to factorⅧrelated antigen. We measured the expression of ICAM-1, VCAM-1 of HUVECs induced by TNF-a at different concentrations (0, 1ng/ml,2.5ng/ml, lOng/ml,40ng/ml) by different time course (0, 1h,2h,4h,12h) by methods of Western blot and realtime quantitative chain reaction polymerase (qRT-PCR). Cultured HUVECs in vitro were randomly divided into six groups:vehicle, 1.5MAC sevoflurane, lOng/ml TNF-a,0.5MAC sevoflurane+lOng/ml TNF-a,1.5MAC sevoflurane+10ng/ml TNF-a,2.5MAC sevoflurane +10ng/ml TNF-a, the expression of ICAM-1 and VCAM-1 of HUVECs were detected by methods of Western blot and qRT-PCR and the adhesion ratio of neutrophils to HUVECs was measured by methods of myeloperoxidase. (2) In vitro study was performed on cultured HUVECs subjected to lOng/ml TNF-a, the expression of IκBa, p-IκBa and NF-KB/p65 subunit in nucleus or endochylema at different time course were detected by Western blot; the cultured HUVECs were randomly exposed to one of the following treatments:vehicle,1.5MAC sevoflurane, lOng/ml TNF-a,1.5MAC sevoflurane+10ng/ml TNF-a, the protein levels of IκBα, p-IκBa and NF-κB/P65 subunit in nucleus or endochylema were measured at respective alterative peak time points. (3) HUVECs were randomly exposed to one of the following treatments:vehicle,0.5MAC sevoflurane,1.5MAC sevoflurane,2.5MAC sevoflurane,30 min aftter sevoflurane pretreatment, the protein levels of p-eNOS and eNOS were detected by Western blot, the total NO content in cell medium by methods of Griess reagent and the NO content in vascular endothelial cells by methods of DAF-FM DA fluorescent probe; HUVECs were randomly assigned to receive lOng/ml TNF-a for 4 hours (control),1.5MAC sevoflurane sequently combined with TNF-a in the presence or absence the eNOS inhibitor N-nitro-L-arginine methyl ester (L-NAME; 1mM) pretreatment, the NO content in vascular endothelial cells and cultured medium, the protein levels of p-eNOS\eNOS, IKBa\p-IκBa, NF-κB/p65 subunit in nucleus\endochylema, the protein and mRNA levels of ICAM-1\VCAM-1 in vascular endothelial cells and the adhesion ratio of neutrophils to HUVECs were determined at corresponding time points.Results (1) HUVECs presented cobblestone-shaped and a positive immunochemistry reaction (97.5%). The increased expression of ICAM-1, VCAM-1 protein and mRNA levels in TNF-a-induced HUVECs was in a dose-dependent and time-dependent manner, HUVECs subjected to 10ng/ml TNF-a for 4 hours had a stable expression of ICAM-1, VCAM-1 protein and mRNA levels.sevoflurane pretreatment with 1.5MAC and 2.5MAC concentration both siginificantly inhibited the the expression of ICAM-1/VCAM-1 protein and mRNA levels in HUVECs, and the adhesion ratio of PMNs to HUVECs was also down-regulated (P<0.05). (2) Phosphorylation and degradation of IκBa, subsequent nucleus-bound translocation of p65 subunit of NF-κB were activated by lOng/ml TNF-a, the alterative peak time was respectively 30 minutes,60 minutes after TNF-a stimulti (P＜0.01).1.5 MAC sevoflurane pretreatment could siginificantly inhibit phosphorylation and degradation of IκBαand nucleus-bound translocation of p65 subunit compared with 10ng/ml TNF-a group (P<0.05). (3) sevoflurane exposure evoked an increased NO content in vascular endothelial cells and cell medium, followed by phosphorylation of eNOS in HUVECs (P<0.05). Compared with sevoflurane pretreatment group, L-NAME decreased p-eNOS/eNOS and NO content both in cells and medium, L-NAME aslo increased phosphorylation and degradation of IκBα, subsequent nucleus-bound translocation of NF-αB p65, mRNA and protein expression of ICAM-1 and VCAM-1 in HUVECs and the adhesion ratio of neutrophils to HUVECs (P＜0.05).Conclusion (1) Sevoflurane pretreatment inhibited the expression of ICAM-1, VCAM-1 induced by TNF-a and neutrophils adhesion to HUVECs. (2) Sevoflurane pretreatment down-regulated the activation of NF-κB signal pathway induced by TNF-a, which may play a major role in anti-inflammatory effects of sevoflurane pretreatment on vascular endothelial cells in vitro. (3) The anti-inflammatory effects of sevoflurane pretreatment could be mediated by eNOS phosphorylation and subsequent NO release, which inhibited NF-κB signal pathway activation and inflammatory response on TNF-a-induced vascular endothelial cell.