Li Yuan, Zhang Xiaonan, Zhu Biao, Xue Zhanggang
Department of Anesthesiology, Zhongshan Hospital, Fudan University, Fenglin Road 180, Shanghai 200032, China.
Anesth Analg. 2008 May;106(5):1473-9, table of contents. doi: 10.1213/ane.0b013e318168b3f2.
Volatile anesthetics interfere with inflammatory cytokine production and expression of adhesion molecules which are critical for ischemia reperfusion induced injury. Nuclear factor (NF)-kappaB has been reported to be suppressed in this process, but the detailed molecular mechanism is still unclear.
In this study, ECV304 (a human umbilical vein endothelial cell line) was preconditioned with 30 min desflurane (1 minimal alveolar concentration), after 15 min washout, 30 min anoxia, and 60 min reoxygenation was performed. ECV304 was finally stimulated with tumor necrosis factor (TNF)-alpha (10 ng/mL). Control groups, which were not preconditioned and/or not stimulated, were also included in the protocol. IkappaB-alpha, phospho-IkappaB-alpha, phospho-IkappaB kinase (IKKalpha)/IKKbeta, and phospho-p38 were detected by Western blotting. The nuclear NF-kappaB p65 subunit was measured by subcellular fractionation and Western blotting. The surface expression of TNF-R1 was measured by flow cytometry. Receptor-associated signaling adaptors, e.g., TNF receptor-associated factor 2 (TRAF2) and IKK-alpha, were evaluated by immunoprecipitation by TNF-R1 antibody and subsequent Western blotting.
Desflurane preconditioning inhibits IkappaB-alpha phosphorylation, degradation, and p65 nuclear localization. Desflurane also affects p38 phosphorylation, which is needed for optimal inflammatory response. The phosphorylation of IKKalpha/IKKbeta was suppressed by preconditioning while the surface abundance of TNF-R1 was not affected. The association of TRAF2 and IKK-alpha with TNF-R1 was compromised by desflurane.
Our results suggest that the molecular target of desflurane in the NF-kappaB pathway is upstream of IKK activation. The abundance of TNF-R1 on the cell membrane is not affected by anesthetic preconditioning. We suggest that desflurane preconditioning targets the proximal end of TNF-alpha signaling.
挥发性麻醉剂会干扰炎症细胞因子的产生以及对缺血再灌注诱导损伤至关重要的黏附分子的表达。据报道，核因子（NF）-κB在此过程中受到抑制，但详细的分子机制仍不清楚。
在本研究中，用30分钟地氟醚（1个最低肺泡浓度）预处理人脐静脉内皮细胞系ECV304，冲洗15分钟后，进行30分钟缺氧和60分钟复氧。最后用肿瘤坏死因子（TNF）-α（10纳克/毫升）刺激ECV304。未进行预处理和/或未受刺激的对照组也纳入该方案。通过蛋白质免疫印迹法检测IκB-α、磷酸化IκB-α、磷酸化IκB激酶（IKKα）/IKKβ和磷酸化p38。通过亚细胞分级分离和蛋白质免疫印迹法测量核NF-κB p65亚基。通过流式细胞术测量TNF-R1的表面表达。通过用TNF-R1抗体进行免疫沉淀并随后进行蛋白质免疫印迹法评估受体相关信号衔接蛋白，如TNF受体相关因子2（TRAF2）和IKK-α。
地氟醚预处理可抑制IκB-α磷酸化、降解和p65核定位。地氟醚还影响p38磷酸化，而p38磷酸化是最佳炎症反应所必需的。预处理可抑制IKKα/IKKβ的磷酸化，而TNF-R1的表面丰度不受影响。地氟醚会破坏TRAF2和IKK-α与TNF-R1的结合。
我们的结果表明，地氟醚在NF-κB途径中的分子靶点位于IKK激活的上游。细胞膜上TNF-R1的丰度不受麻醉预处理的影响。我们认为地氟醚预处理靶向TNF-α信号传导的近端。
