Thiopental protects human T lymphocytes from apoptosis in vitro via the expression of heat shock protein 70
Thiopental protects human T lymphocytes from apoptosis in vitro via the expression of heat shock protein 70
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DOI：
10.1124/jpet.107.133108
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发表时间：
2008-04-01
影响因子：
3.5
通讯作者：
Loop, Torsten
中科院分区：
文献类型：
--
作者：
Roesslein, Martin;Schibilsky, David;Loop, Torsten
巴比妥酸盐用于治疗重型颅脑损伤后的颅内高压，一直与抗炎副作用有关。虽然所有巴比妥酸盐都抑制T细胞功能，但只有硫代巴比妥酸盐显著降低转录因子核因子-kappaB(NF-kappa B)的激活。核因子-kappaB途径的各种药物抑制物是热休克反应(HSR)的伴随非热诱导因子，热休克反应是一种与细胞和器官保护相关的细胞防御系统。我们假设硫喷妥通过诱导HSR来调节细胞保护。人CD3(+)T淋巴细胞与硫喷妥钠、戊巴比妥、依托咪酯、氯胺酮、咪达唑仑或异丙酚孵育。在硫喷妥钠孵育前，将针对热70 kDa休克蛋白(HSP70)的小干扰RNA(SiRNA)导入人Jurkat T细胞。用星形孢子素诱导细胞凋亡。凝胶迁移率改变分析HSF-1的DNA结合活性，Northern印迹分析HSP27、-32、-70、-90的mRNA表达，异硫氰酸荧光素(FITC)-HSP70-抗体染色后用Western印迹和流式细胞术分析HSP70的蛋白表达。Annexin V-FITC或Annexin V-藻红蛋白染色后用流式细胞仪检测细胞的凋亡率。用荧光半胱氨酸氨基转移酶活性测定法测定caspase-3活性。硫喷妥钠可诱导HSP27、-70和-90的表达，但不诱导HSP32的表达和HSP70蛋白的表达。硫喷妥钠呈剂量依赖性地激活HSF-1的DNA结合活性，而所研究的其他物质则没有影响。此外，硫喷妥钠可显著减弱星形孢菌素诱导的细胞凋亡和caspase样活性。在硫喷妥钠治疗前，用HSP70-siRNA转染可以减少这种衰减。硫喷妥钠特异性和差异性地诱导热休克反应，它通过表达HSP70在人T淋巴细胞上介导细胞保护。
Barbiturates, which are used for the treatment of intracranial hypertension after severe head injury, have been associated with anti-inflammatory side effects. Although all barbiturates inhibit T-cell function, only thiobarbiturates markedly reduce the activation of the transcription factor nuclear factor-kappa B (NF-kappa B). Various pharmacologic inhibitors of the NF-kappa B pathway are concomitant nonthermal inducers of the heat shock response (HSR), a cellular defense system that is associated with protection of cells and organs. We hypothesize that thiopental mediates cytoprotection by inducing the HSR. Human CD3(+) T lymphocytes were incubated with thiopental, pentobarbital, etomidate, ketamine, midazolam, or propofol. Human Jurkat T cells were transfected with small interfering RNA (siRNA) targeting heat 70-kDa shock protein (hsp 70) before thiopental incubation. Apoptosis was induced by staurosporine. DNA binding activity of HSF-1 was analyzed by electrophoretic mobility shift assay; mRNA expression of hsp27, -32, -70, and -90 was analyzed by Northern blot, and protein expression of hsp70 was analyzed by Western blot and flow cytometry after fluorescein isothiocyanate (FITC)-hsp70-antibody staining. Apoptosis was assessed by flow cytometry after annexin V-FITC or annexin V-phycoerythrin staining. Activity of caspase-3 was measured by fluorogenic caspase activity assay. Thiopental induced hsp27, -70, and -90 but not hsp32 mRNA expression as well as hsp70 protein expression. Thiopental dose-dependently activated the DNA binding activity of HSF-1, whereas other substances investigated had no effect. In addition, pretreatment with thiopental significantly attenuated staurosporine-induced apoptosis and caspase-like activity. Transfection with hsp70-siRNA before thiopental treatment reduced this attenuation. Thiopental specifically and differentially induces a heat shock response, and it mediates cytoprotection via the expression of hsp70 in human T lymphocytes.