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目的:通过观察山莨菪碱(654-2)对激活的血管内皮细胞(VEC)中谷胱甘肽(GSH)含量、核因子-κB(NF-κB)活性和细胞间黏附分子(ICAM-1)表达的影响,探讨其对VEC损伤的保护机制。方法:建立人脐静脉内皮细胞(HUVEC)体外培养体系,经TNF-α刺激和合并应用654-2干预HUVEC后,采用荧光分光光度计法测定HUVEC胞内GSH含量,凝胶电泳迁移率改变分析法(EMSA)检测NF-κB活性和应用免疫组化方法检测HUVEC的ICAM-1表达。结果:HUVEC在TNF-α刺激后GSH含量显著降低(P<0.01),于3 h达低谷;NF-κB的活性显著增高(P<0.01),峰值在刺激后6 h;ICAM-1表达显著增强(P<0.01),并于12 h达峰值。TNF-α刺激并654-2干预后,相应时相点的GSH含量显著降低,但较单纯TNF-α刺激组显著增高(P<0.05),而NF-κB的活性、ICAM-1表达强度较单纯TNF-α刺激显著降低(P<0.05)。结论:654-2通过抑制TNF-α对HUVEC刺激所导致GSH水平的降低,减少了NF-κB活化核易位所启动ICAM-1基因高效表达,这可能是其防治全身炎症反应综合征/脓毒症介导MODS发生的作用环节。
Objective: To observe the content of glutathione (GSH), nuclear factor-κB (NF-κB) activity and intercellular adhesion molecule (ICAM-1) in an activated vascular endothelial cell (VEC) by anisodamine (654-2) The effect of expression was explored on its protective mechanism against VEC injury. METHODS: Human umbilical vein endothelial cells (HUVEC) culture system was established in vitro. HUVECs were stimulated with TNF-α and combined with 654-2. The content of intracellular GSH in HUVECs was determined by fluorescence spectrophotometry, and the electrophoretic mobility of gels was analyzed. The activity of NF-κB was detected by EMSA and the expression of ICAM-1 in HUVEC was detected by immunohistochemistry. RESULTS: HUVECs significantly decreased GSH content after TNF-α stimulation (P<0.01), reached troughs at 3 h; NF-κB activity increased significantly (P<0.01), peaked at 6 h after stimulation; ICAM-1 expression was significantly increased. Increased (P<0.01) and peaked at 12 h. After TNF-α stimulation and 654-2 intervention, the GSH content of the corresponding phase was significantly decreased, but it was significantly higher than that of the TNF-α stimulation group (P<0.05), while the activity of NF-κB and the expression intensity of ICAM-1 were higher. The TNF-α stimulation alone was significantly reduced (P<0.05). CONCLUSION: 654-2 inhibits the decrease of GSH level induced by HUVEC stimulation by TNF-α, and reduces the activation of ICAM-1 gene by NF-κB-activated nuclear translocation. This may be due to its prevention of systemic inflammatory response syndrome / pus. Toxicosis mediates the role of MODS.