山莨菪碱对血管内皮细胞损伤保护机制的实验研究

An experimental study on the mechanisms of anisodamine in protection against vascular endothelial cellular damages

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 investigate the mechanisms of anisodamine （654-2）in protection against the vascular endothelial cellular（VEC） damages. Methods：An in vitro culture system of human umbilical venous endothelial cells （HUVEC） was established. After intervention by the combination of TNF-α stimulation and 654-2 treatment, the content of GSH in HUVEC was measured by fluorescence speetrophotometry, and the activity of NF-κB by electrophoretie mobility shift assay （EMSA） and expression of ICAM-1 in HUVEC by immunohistochemistry. Results：The GSH level in HUVEC obviously decreased after TNF-α stimulation（ P 〈0.01 ）, and reached the lowest level at 3 h. The activity of NF-κB and the expression of ICAM-1 increased significantly（P 〈 0.01 ）;their peak value was reached at 6 h and 12 h, respectively. The GSH level was negatively correlated with NF-κB activity （ r = - 0.315, P = 0.012 ） and ICAM -1 expression （ r = - 0.389, P = 0.001 ）, while NF-κB activity was positively correlated with ICAM-1 expression （r = 0. 261, P = 0. 026）. After TNF-α stimulation and 654-2 intervention,the GSH level was obviously reduced but was much higher than after TNF-αstimulation alone（P 〈 0.05） ,while NF-κB activity and ICAM-1 expression （ P 〈 0.05 ） were lower than after TNF-α stimulation alone （ P 〈 0.05）. Conclnsions：The activation of NF-κB and excessive expression of ICAM-1 could be seen in TNF-α-stimulated VEC. 654-2 plays a protective role in prevention of MODS induced by SIRS/sepsis byinhibiting GSH level reduction by TNF-α stimulation and downregulating the expression of ICAM-1 triggered by activation of NF-κB.