权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

The effect of vero toxin produced by enterohemorrhagic Escherichia coli on ion transporter

肠出血性大肠杆菌产生的维罗毒素对离子转运蛋白的影响

基本信息

批准号：
10660303
负责人：
IKEDA Masahiro
金额：
$ 1.6万
依托单位：
Miyazaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660303/
关键词：
Verotoxin enterohemorrhagic Escherichia coli intracellular signal transduction intracellular CaィイD12+ィエD1 ion channel MAPK p38 MAPK 腸管出血大腸菌パリトキシン Vero毒素 Vero細胞 WST-1 パッチクランプ fura-2

项目摘要

Background. Although shiga toxin (Stx) produced by enterohemorrhagic Escherichia coli (Stx-producing E. coli) has been shown to induce cell injury and play a central role in the pathogenesis of hemolytic uremic syndrome (HUS), the intracellular mechanism by which Stx causes damage to cells remains unclear. We examined whether the mitogen-activated protein kinase (MAPK) pathway is involved in Stx-induced Vero cell injury.Methods. We used Stx1, Stx2, and a non-toxic mutant Stx1, E167Q. The activation of MAPK pathways by Stx was assessed mainly by immunoblotting with anti-phospho-extracellular signal-regulated kinase 1/2 (ERK1/2) or -phospho-p38 MAPK antibodies. Cell viability was determined by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay. The role of MAPK and CaィイD12+ィエD1 was assessed using specific biochemical inhibitors.Results. Stx1 and Stx2 but not E167Q induced cell death in a dose-dependent and time-dependent manner. Consonant with cell injury, Stx1 and Stx2 caused a transient phosphorylation of ERK1/2 and a sustained phosphorylation of p38 MAPK. SB 203580 and PD 169316, both inhibitors of p38 MAPK, but not PD 98059, an inhibitor ERK kinase (MEK 1/2), partially inhibited the Stx1-induced cell death. BAPTA-AM, an intracellu1ar CaィイD12+ィエD1 chelator, and verapamil and nicardipine which are CaィイD12+ィエD1 channel blockers, reduced both cell injury and phosphorylation of p38 MAPK.Conclusion. These data indicate that MAPK pathways can be activated by Stx in Vero cells and that the activation of p38 MAPK, which is triggered by intracellular CaィイD12+ィエD1 elevation, plays a role in Stx-induced cell death.

背景虽然滋贺毒素（Stx）是由肠出血性大肠杆菌（Stx-producing E.大肠杆菌（Stx）可引起细胞损伤，在溶血性尿毒综合征（HUS）的发病机制中起重要作用，但Stx引起细胞损伤的细胞内机制尚不清楚。我们研究了丝裂原活化蛋白激酶（MAPK）通路是否参与Stx诱导的Vero细胞损伤。我们使用Stx 1、Stx 2和无毒突变体Stx 1 E167 Q。主要通过用抗磷酸化细胞外信号调节激酶1/2（ERK 1/2）或磷酸化p38 MAPK抗体进行免疫印迹来评估Stx对MAPK通路的激活。通过4-[3-（4-碘苯基）-2-（4-硝基苯基）-2H-5-四唑基]-1，3-苯二磺酸盐（WST-1）测定法测定细胞活力。采用特异性生化标记物检测MAPK和Ca ~（2+）D_12 + Ca ~（2+）D_1的作用。Stx 1和Stx 2以剂量依赖性和时间依赖性方式诱导细胞死亡，而E167 Q不诱导细胞死亡。与细胞损伤一致，Stx 1和Stx 2引起ERK 1/2的瞬时磷酸化和p38 MAPK的持续磷酸化。p38 MAPK抑制剂SB 203580和PD 169316可部分抑制Stx 1诱导的细胞死亡，而ERK激酶（MEK 1/2）抑制剂PD 98059则不能。细胞内Ca ~（2+）D_1通道阻断剂维拉帕米和尼卡地平均能减轻细胞损伤和p38 MAPK磷酸化。这些数据表明，丝裂原活化蛋白激酶途径可以激活的Stx在Vero细胞和p38丝裂原活化蛋白激酶，这是由细胞内的Ca ~（2+）D_12 + β_2D_1的升高，在Stx诱导的细胞死亡中发挥作用。