Altavilla D, Saitta A, Guarini S, Galeano M, Squadrito G, Santamaria L B, Venuti F S, Bazzani C, Bertolini A, Squadrito F
Department of Experimental and Clinical Medicine and Pharmacology, Section of Pharmacology, School of Medicine, University of Messina, Messina, Italy.
Cardiovasc Res. 2001 Oct;52(1):143-52. doi: 10.1016/s0008-6363(01)00362-5.
Cyclosporin is an immunosuppressive drug that blocks Nuclear Factor kappaB (NF-kappaB) activation. We investigated the role of NF-kappaB in acute hypovolemic hemorrhagic (Hem) shock and the effects of cyclosporin in this model of experimental shock.
Hem shock was induced in male anesthetized rats by intermittently withdrawing blood from an iliac catheter over a period of 20 min (bleeding period) until mean arterial blood pressure (MAP) fell and stabilized within the range of 20-30 mmHg. Two minutes after bleeding cessation, animals received intravenously cyclosporin (1 mg kg(-1)) or its vehicle. Survival rate and survival time were evaluated for 120 min after bleeding was discontinued. Plasma TNF-alpha levels were investigated at different time points after bleeding cessation. Moreover we investigated levels of TNF-alpha mRNA in the liver, vascular reactivity, liver NF-kappaB binding activity and levels of the inhibitory protein IkappaBalpha in the cytoplasm.
Hemorrhagic shocked rats died in 27+/-6 min following the cessation of bleeding, experienced a marked hypotension (mean arterial blood pressure=20-30 mmHg) and had enhanced plasma levels of Tumor Necrosis Factor-alpha (208+/-22 pg ml(-1), 20 min after the end of bleeding). Furthermore, aortas taken 20 min after bleeding from hemorrhagic shocked rats showed a marked hypo-reactivity to phenylephrine (PE: 1 nM-10 microM) compared with aortas harvested from sham shocked rats. Hem shocked rats also had increased levels of TNF-alpha mRNA in the liver (15-20 min after the end of bleeding). Electrophoretic mobility shift assay showed that liver NF-kappaB binding activity increased in the nucleus 10 min after the end of hemorrhage and remained elevated until the death of animals. Western blot analysis suggested that the levels of inhibitory protein IkappaBalpha in the cytoplasm decreased at 5 min after the end of bleeding. Cyclosporin inhibited the loss of IkappaBalpha protein from the cytoplasm and prevented NF-kappaB binding activity in the nucleus. Furthermore, cyclosporin increased survival time (118+/-7 min; P<0.01) and survival rate (vehicle=0% and cyclosporin=80%, at 120 min after the end of bleeding), reverted the marked hypotension, decreased liver mRNA for TNF-alpha, reduced plasma TNF-alpha (28+/-7 pg ml(-1)), and restored to control values the hypo-reactivity to PE.
Our results suggest that acute blood loss (50% of the estimated total blood volume over a period of 20 min) causes early activation of NF-kappaB which triggers an inflammatory cascade leading to a fatal outcome. Cyclosporin blocks NF-kappaB activation and protects against hypovolemic hemorrhagic shock.
环孢素是一种免疫抑制药物，可阻断核因子κB（NF-κB）的激活。我们研究了NF-κB在急性低血容量性出血性（Hem）休克中的作用以及环孢素在该实验性休克模型中的效果。
在雄性麻醉大鼠中诱导Hem休克，通过在20分钟（出血期）内从髂静脉导管间歇性抽血，直至平均动脉血压（MAP）下降并稳定在20 - 30 mmHg范围内。出血停止两分钟后，动物静脉注射环孢素（1 mg kg⁻¹）或其溶媒。在出血停止后120分钟评估存活率和存活时间。在出血停止后的不同时间点研究血浆肿瘤坏死因子-α（TNF-α）水平。此外，我们研究了肝脏中TNF-α mRNA水平、血管反应性、肝脏NF-κB结合活性以及细胞质中抑制蛋白IκBα的水平。
出血性休克大鼠在出血停止后27±6分钟死亡，经历明显低血压（平均动脉血压 = 20 - 30 mmHg），并且出血结束后20分钟血浆肿瘤坏死因子-α水平升高（208±22 pg ml⁻¹）。此外，与假手术休克大鼠获取的主动脉相比，出血性休克大鼠出血20分钟后获取的主动脉对去氧肾上腺素（PE：1 nM - 10 μM）表现出明显的低反应性。Hem休克大鼠肝脏中TNF-α mRNA水平也升高（出血结束后15 - 20分钟）。电泳迁移率变动分析显示，出血结束后10分钟肝脏细胞核中NF-κB结合活性增加，并一直升高直至动物死亡。蛋白质印迹分析表明，出血结束后5分钟细胞质中抑制蛋白IκBα水平降低。环孢素抑制IκBα蛋白从细胞质中丢失，并阻止细胞核中NF-κB结合活性。此外，环孢素增加了存活时间（118±7分钟；P<0.01）和存活率（出血结束后120分钟，溶媒组 = 0%，环孢素组 = 80%），逆转了明显的低血压，降低了肝脏中TNF-α的mRNA水平，降低了血浆TNF-α（28±7 pg ml⁻¹），并将对PE的低反应性恢复到对照值。
我们的结果表明，急性失血（20分钟内估计总血容量的50%）导致NF-κB早期激活，触发炎症级联反应，导致致命结局。环孢素阻断NF-κB激活并预防低血容量性出血性休克。
