多巴酚丁胺通过诱导细胞热休克蛋白 70 介导体外细胞保护作用。

Dobutamine mediates cytoprotection by induction of heat shock protein 70 in vitro.

机构信息

Dept. of Anaesthesiology and Critical Care Medicine, University Medical Center, Freiburg, Germany.

出版信息

Life Sci. 2014 Mar 11;98(2):88-95. doi: 10.1016/j.lfs.2014.01.005. Epub 2014 Jan 18.

DOI:10.1016/j.lfs.2014.01.005

PMID:24447628

Abstract

AIMS

Dobutamine is cytoprotective when applied before a subsequent stress. However, the underlying molecular mechanism is unknown. Dobutamine also inhibits nuclear factor (NF)-κB in human T lymphocytes. Other inhibitors of NF-κB induce a so-called heat shock response. We hypothesized that dobutamine mediates protection from apoptotic cell death by the induction of a heat shock response.

MAIN METHODS

Jurkat T lymphoma cells were preincubated with dobutamine (0.1, 0.5 mM) before the induction of apoptosis (staurosporine, 2 μM). DNA-binding of heat shock factor (HSF)-1 was analyzed by electrophoretic mobility shift assay, mRNA-expression of heat shock protein (hsp)70 and hsp90 by Northern Blot, activity of caspase-3 by fluorogenic caspase activity assay and cleavage of pro-caspase-3 by Western Blot. Apoptosis was assessed by flow cytometry after annexin V-fluorescein isothiocyanate staining. Hsp70 and hsp90 were inhibited using N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolaetam and 17-allylamino-17-demethoxygeldana-mycin, respectively. All data are given as median and 25/75% percentile.

KEY FINDINGS

Pre-incubation with dobutamine inhibited staurosporine-induced annexin V-fluorescence (28 [20-32] % vs. 12 [9-15] % for dobutamine 0.1 mM and 7 [5-12] % for dobutamine 0.5 mM, p<0.001), cleavage of pro-caspase-3 as well as caspase-3-like activity (0.46 [0.40-0.48] vs. 0.32 [0.27-0.39] for Dobutamine 0.1 mM and 0.20 [0.19-0.23] for Dobutamine 0.5 mM, p<0.01). Dobutamine induced DNA-binding of HSF-1 and mRNA-expression of hsp70 and hsp90. While inhibition of Hsp90 had no effect, inhibition of Hsp70 increased the number of annexin V-positive cells (33 [32-36] % vs. 18 [16-24] %) and caspase-3-like activity (0.21 [0.19-0.23] vs. 0.16 [0.13-0.17], p<0.05).

SIGNIFICANCE

Dobutamine protects from apoptotic cell death via the induction of Hsp70.

摘要

目的

在随后的应激前应用多巴酚丁胺可起到细胞保护作用。然而，其潜在的分子机制尚不清楚。多巴酚丁胺还可抑制人 T 淋巴细胞中的核因子（NF）-κB。其他 NF-κB 抑制剂可诱导所谓的热休克反应。我们假设多巴酚丁胺通过诱导热休克反应来介导对细胞凋亡性死亡的保护作用。

主要方法

在诱导凋亡（星形孢菌素，2 μM）之前，用多巴酚丁胺（0.1、0.5 mM）孵育 Jurkat T 淋巴瘤细胞。通过电泳迁移率变动分析检测热休克因子（HSF）-1 的 DNA 结合，通过 Northern Blot 检测热休克蛋白（hsp）70 和 hsp90 的 mRNA 表达，通过荧光酶活性测定法检测 caspase-3 的活性，通过 Western Blot 检测前胱天蛋白酶-3 的切割。用 Annexin V-异硫氰酸荧光素染色后通过流式细胞术评估细胞凋亡。使用 N-甲酰基-3,4-亚甲二氧基苯亚甲基-γ-丁内酯和 17-烯丙基-17-去甲氧基格尔达霉素分别抑制 hsp70 和 hsp90。所有数据均以中位数和 25/75%分位数表示。

主要发现

用多巴酚丁胺孵育可抑制星形孢菌素诱导的 Annexin V 荧光（28 [20-32] % vs. 12 [9-15] %，多巴酚丁胺 0.1 mM，7 [5-12] %，多巴酚丁胺 0.5 mM，p<0.001）、前胱天蛋白酶-3 的切割以及 caspase-3 样活性（0.46 [0.40-0.48] vs. 0.32 [0.27-0.39]，多巴酚丁胺 0.1 mM 和 0.20 [0.19-0.23]，多巴酚丁胺 0.5 mM，p<0.01）。多巴酚丁胺诱导 HSF-1 的 DNA 结合和 hsp70 和 hsp90 的 mRNA 表达。虽然抑制 hsp90 没有影响，但抑制 hsp70 增加了 Annexin V 阳性细胞的数量（33 [32-36] % vs. 18 [16-24] %）和 caspase-3 样活性（0.21 [0.19-0.23] vs. 0.16 [0.13-0.17]，p<0.05）。