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Journal of ZheJiang University(Medical Science)  2013, Vol. 42 Issue (5): 486-491    DOI: 10.3785/j.issn.1008-9292.2013.05.002
    
TcpC induces apoptosis of macrophages through promoting ROS production
ZHANG Da-Yong, LIN Yi-Qian, HE Fei, FANG Jie, ZHANG Chong, WANG Bao-Ming, PAN Jian-Ping
Department of Medicine,Zhejiang University City College School of Medicine,Hangzhou 310015,China
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Abstract  
Objective: To investigate the effects of Toll/interleukin 1 receptor domain-containing protein(TcpC)on macrophages and its mechanisms.
Methods: Murine macrophage J774A cells were co-cultured with TcpC producing wild type E. coli strain CFT073 (TcpCwt) or tcpc gene-deleted CFT073 mutant (TcpCmut) in Transwell system,respectively. Apoptosis of J774A cells co-cultured with TcpCwt or TcpCmut was analyzed by Annexin/PI double staining. The levels of reactive oxygen species (ROS) in J774A cells were determined by DCFH-DA staining after treatment with TcpCwt or TcpCmut at 6 h,12 h,24 h or 36 h. After the ROS was scavenged by N-acetylcysteine (NAC),the changes of J774A cell apoptosis were also examined. The expression of caspase-3 in J774A cells co-cultured with TcpCwt or TcpCmut in the presence or absence of 0.1 mmol NAC was detected by Western blot.
Results: J774A cells co-cultured with TcpCwt for 24 h or 36 h showed significantly increased apoptosis (27.39%±4.05% and 28.45%±4.55%,respectively) when compared to control group (7.96%±1.63% and 10.55%±1.44%,P<0.01) or TcpCmut group (11.45%±2.77% and 19.26%±2.89%,P<0.01). Levels of ROS in J774A cells treated with TcpCwt for 24 h (108.8±9.73) or 36 h (100.3±10.11) were significantly higher than those in control group (56.8±4.11 and 52.8±4.42,P<0.01) or TcpCmut (69.7±5.66 and 62.6±4.56,P<0.01). The pro-apoptotic effects of TcpCwt on J774A cells were reversed by 0.1 or 1 mMol NAC treatment. Expression of caspase-3 in J774A cells co-cultured with TcpCwt (0.43±0.04) decreased significantly when compared to control group (0.75±0.08,P<0.05) or TcpCmut group (0.80±0.12,P<0.05). However,total caspase-3 expression was restored in J774A cells co-cultured with TcpCwt in the presence of 0.1 mmol NAC (0.80±0.09).
Conclusion: TcpC can promote ROS production in macrophages,hereby inducing macrophage apoptosis.


Key wordsEnteropathogenic Escherichia coli      Macrophages      Reactive oxygen species      Apoptosis      Mice, Knockout      Cells, Cultured     
Received: 14 June 2013      Published: 25 September 2013
CLC:  R 742.1  
Cite this article:

ZHANG Da-Yong, LIN Yi-Qian, HE Fei, FANG Jie, ZHANG Chong, WANG Bao-Ming, PAN Jian-Ping. TcpC induces apoptosis of macrophages through promoting ROS production. Journal of ZheJiang University(Medical Science), 2013, 42(5): 486-491.

URL:

https://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2013.05.002     OR     https://www.zjujournals.com/med/Y2013/V42/I5/486


TcpC通过促进活性氧的产生诱导巨噬细胞凋亡

目的:研究含有Toll/interleukin 1 receptor结构域的蛋白(TcpC)对巨噬细胞凋亡的影响,探讨TcpC诱导巨噬细胞凋亡的分子机制。
方法:通过Transwell 将分泌TcpC的尿路致病大肠埃希菌CFT073野生株(TcpCwt)和敲除tcpc基因的CFT073突变株(TcpCmut)与小鼠巨噬细胞系J774A隔开共培养,Annexin V/PI双染法观察不同数量和不同处理时间的TcpCwt和TcpCmut对J774A细胞凋亡的影响;活性氧染色观察不同处理时间的TcpCwt和TcpCmut对J774A细胞内活性氧水平的影响,Annexin V/PI双染法观察不同浓度N-乙酰半胱氨酸清除活性氧后TcpCwt和TcpCmut对J774A细胞凋亡的影响,Western blot法检测0.1 mmol N-乙酰半胱氨酸处理前后与TcpCwt和TcpCmut共培养的J774A细胞内caspase-3表达。
结果:与对照组和TcpCmut组相比,TcpCwt在细菌数量为5×105和5×106以及处理时间为24 h和36 h时均可显著促进J774A凋亡(均P<0.01)。共培养24 h和36 h的TcpCwt组J774A细胞内活性氧水平显著高于对照组和TcpCmut组(均P<0.01)。0.1 mmol和1 mmol N-乙酰半胱氨酸处理可显著抑制TcpCwt对J774A细胞的促凋亡作用(均P<0.01)。TcpCwt处理可显著降低J774A细胞内总caspase-3表达,而N-乙酰半胱氨酸处理可抑制与TcpCwt共培养的J774A细胞内caspase-3裂解,提高总caspase-3表达水平。
结论:TcpC可促进巨噬细胞内活性氧产生,进而介导巨噬细胞凋亡。


关键词: 肠致病性大肠杆菌,  巨噬细胞,  活性氧,  细胞凋亡,  小鼠,  基因敲除,  细胞,  培养的 
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