Role of Bcl-2 family members in caspase-3/9-dependent apoptosis during Pseudomonas aeruginosa infection in U937 cells
Role of Bcl-2 family members in caspase-3/9-dependent apoptosis during Pseudomonas aeruginosa infection in U937 cells
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DOI：
10.1007/s10495-008-0197-6
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发表时间：
2008-06-01
期刊：
影响因子：
7.2
通讯作者：
Chen, B. Y.
中科院分区：
文献类型：
--
作者：
Chai, W. S.;Zhu, X. M.;Chen, B. Y.
铜绿假单胞菌是一种革兰氏阴性条件致病菌，对多种真核细胞具有细胞毒性。为了研究该细菌对单核细胞的影响，我们在体外用铜绿假单胞菌菌株感染人U937细胞。为探讨铜绿假单胞菌诱导U937细胞凋亡过程中Bcl-2、Bax的表达及caspase-3/9的活化，采用Hoechst 33258染色、Giemsa染色及流式细胞术检测细胞凋亡率，RT-PCR及Western blotting检测Bcl-2、Bax的表达。免疫沉淀法检测Bax蛋白构象变化。细胞色素c释放通过蛋白质印迹法测量。此外，将U937细胞暴露于铜绿假单胞菌测量半胱天冬酶-3/9活性。结果表明，铜绿假单胞菌能诱导人U937细胞凋亡，且呈剂量和时间依赖性。Bax蛋白表达水平升高，Bcl-2蛋白表达水平降低，细胞色素c释放水平升高，caspase-3/9活性升高，Bax蛋白构象改变。为了评价半胱天冬酶的作用，分别使用半胱天冬酶-3/9抑制剂Z-DEVD-FMK和Z-LEHD-FMK。caspase抑制剂Z-DEVD-FMK和Z-LEHD-FMK阻断铜绿假单胞菌诱导的U937细胞凋亡进一步证实了上述结果。结论：铜绿假单胞菌可诱导U937细胞凋亡，上调Bax蛋白表达，下调Bcl-2蛋白表达，导致细胞色素c释放增加，caspase-3和caspase-9表达增加。
Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that is cytotoxic towards a variety of eukaryotic cells. To investigate the effect of this bacterium on monocyte, we infected human U937 cells with the P. aeruginosa strain in vitro. To explore the expression of Bcl-2 and Bax as well as caspase-3/9 activation in the apoptosis of human U937 cells induced by P. aeruginosa, Hoechst 33258 staining and Giemsa staining as well as Flow cytometry analysis were used to determine the rate of apoptosis, and the expressions of Bcl-2 and Bax were assayed by RT-PCR and Western blotting respectively. Bax protein conformation change was assayed by immunoprecipitation. Cytochrome c release was measured by Western blotting. Moreover, exposure of U937 cells to P. aeruginosa measured caspase-3/9 activity. It was found that the apoptosis of human U937 cells could be induced by Pseudomonas aeruginosa in a dose- and time-dependent manner. Also, there were a tendency of alterations with an increased expression level of Bax and a reduced expression level of Bcl-2, increased levels of cytochrome c release, and also with an increased activation of caspase-3/9 and Bax protein conformation change. For the evaluation of the role of caspases, caspase-3/9 inhibitors Z-DEVD-FMK and Z-LEHD-FMK respectively were used. The results were further confirmed by the observation that the caspase inhibitors Z-DEVD-FMK and Z-LEHD-FMK blocked P. aeruginosa-induced U937 apoptosis. It is concluded that P. aeruginosa can induce apoptosis with an up-regulated expression of Bax and a down-regulated expression of Bcl-2, which resulted in increased levels of cytochrome c release and increased caspase-3 and -9 in human U937 cells.