Lu Xi, Li Chunmeng, Li Congcong, Li Pengcheng, Fu Enqing, Xie Yonghong, Jin Faguang
Department of Respiration, Tangdu Hospital, Fourth Military Medical UniversityXi'an, China.
Bacteriology Room in Department of Clinical Laboratory, Shaanxi Province Hospital of Traditional Chinese MedicineXi'an, China.
Front Cell Infect Microbiol. 2017 Jun 8;7:244. doi: 10.3389/fcimb.2017.00244. eCollection 2017.
Enterotoxigenic (ETEC) is a leading cause of diarrhea among children and travelers in developing countries, and heat-labile enterotoxin (LT) is one of the most important virulence factors. The pathogenesis of and virulence factors associated with ETEC have been well-characterized; however, the extent to which ETEC damages host cells remains unclear. In this study, we found that LT could induce decreases in intestinal epithelial cell viability and induce apoptosis in a dose- and time- dependent manner in both HCT-8 and Caco-2 cells. We analyzed the expression profiles of apoptosis-related proteins via protein array technology and found that Bax, p-p53(S46), cleaved caspase-3, and TNFRI/TNFRSF1A expression levels were significantly up-regulated in wild-type ETEC- but not in ΔLT ETEC-infected HCT-8 cells. Bax is essential for endoplasmic reticulum (ER) stress-triggered apoptosis, and our RNAi experiments showed that the PERK-eIF2-CHOP pathway and reactive oxygen species (ROS) are also main participants in this process. LT-induced ROS generation was decreased in CHOP-knockdown HCT-8 cells compared to that in control cells. Moreover, pretreatment with the ROS inhibitor NAC down-regulated GRP78, CHOP, Bim, and cleaved caspase-3 expression, resulting in a reduction in the apoptosis rate from 36.2 to 20.3% in LT-treated HCT-8 cells. Furthermore, ROS inhibition also attenuated LT-induced apoptosis in the small intestinal mucosa in the ETEC-inoculation mouse model.
产肠毒素大肠杆菌（ETEC）是发展中国家儿童和旅行者腹泻的主要病因，而热不稳定肠毒素（LT）是最重要的毒力因子之一。ETEC的发病机制和相关毒力因子已得到充分表征；然而，ETEC对宿主细胞的损伤程度仍不清楚。在本研究中，我们发现LT可诱导肠上皮细胞活力下降，并在HCT-8和Caco-2细胞中以剂量和时间依赖性方式诱导细胞凋亡。我们通过蛋白质阵列技术分析了凋亡相关蛋白的表达谱，发现野生型ETEC感染的HCT-8细胞中Bax、p-p53（S46）、裂解的caspase-3和TNFRI/TNFRSF1A表达水平显著上调，而ΔLT ETEC感染的HCT-8细胞中则未上调。Bax对内质网（ER）应激触发的细胞凋亡至关重要，我们的RNAi实验表明，PERK-eIF2-CHOP途径和活性氧（ROS）也是这一过程的主要参与者。与对照细胞相比，CHOP基因敲低的HCT-8细胞中LT诱导的ROS生成减少。此外，用ROS抑制剂NAC预处理可下调GRP78、CHOP、Bim和裂解的caspase-3表达，导致LT处理的HCT-8细胞凋亡率从36.2%降至20.3%。此外，ROS抑制还减弱了ETEC接种小鼠模型中小肠黏膜中LT诱导的细胞凋亡。
