Qin Wenjuan, Guan Dongfang, Ma Rongji, Yang Rentan, Xing Guoqiang, Shi Hongjuan, Tang Guangyao, Li Jiajie, Lv Hailong, Jiang Yufeng
Department of Histology and Embryology, School of Medicine, Shihezi University, Shihezi 832000, China.
Department of Ultrasound Diagnosis, The First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832000, China.
Acta Biochim Biophys Sin (Shanghai). 2017 Aug 1;49(8):696-705. doi: 10.1093/abbs/gmx067.
The aim of this study was to investigate the impact of trigonelline (TRG) on Echinococcus granulosus, and to explore the inhibition impact of nuclear factor erythroid-2-related factor 2 (Nrf2) signaling pathway on E. granulosus protoscoleces. Echinococcus granulosus protoscoleces were incubated with various concentrations of TRG, and then Nrf2 protein expression and its localization in protoscoleces were detected by western blot analysis and immunofluorescence assay, respectively. Reactive oxygen species (ROS) level in protoscoleces was measured using ROS detection kit. Caspase-3 activity was measured using a caspase-3 activity assay kit, and NAD(P)H quinone oxidoreductase (NQO)-1 and heme oxygenase (HO)-1 activities in protoscoleces were measured by ELISA. The effect of TRG on protoscoleces viability was investigated using 0.1% eosin staining, and ultrastructural alterations in protoscoleces were examined by scanning electron microscopy (SEM). Immunolocalization experiment clearly showed that Nrf2 protein was predominantly present in cells of protoscoleces. TRG treatment reduced NQO-1 and HO-1 activities in protoscoleces, but could increase ROS level at early time. Protoscoleces could not survive when treated with 250 μM TRG for 12 days. SEM results showed that TRG-treated protoscoleces presented damage in the protoscoleces region, including hook deformation, lesions, and digitiform protuberance. Nrf2 protein expression was significantly decreased and caspase-3 activity was clearly increased in protoscoleces treated with TRG for 24 and 48 h, respectively, when compared with that in controls (P < 0.05). Our results demonstrated that TRG had scolicidal activity against E. granulosus protoscoleces. Nrf2 protein was mainly expressed in the cells and TRG could efficiently inhibit the Nrf2 signaling pathway in E. granulosus.
本研究旨在探讨胡芦巴碱（TRG）对细粒棘球绦虫的影响，并探究核因子红细胞2相关因子2（Nrf2）信号通路对细粒棘球绦虫原头节的抑制作用。将细粒棘球绦虫原头节与不同浓度的TRG孵育，然后分别通过蛋白质免疫印迹分析和免疫荧光测定法检测原头节中Nrf2蛋白的表达及其定位。使用活性氧（ROS）检测试剂盒测量原头节中的ROS水平。使用caspase-3活性检测试剂盒测量caspase-3活性，并通过酶联免疫吸附测定法测量原头节中NAD（P）H醌氧化还原酶（NQO）-1和血红素加氧酶（HO）-1的活性。使用0.1%伊红染色研究TRG对原头节活力的影响，并通过扫描电子显微镜（SEM）检查原头节的超微结构改变。免疫定位实验清楚地表明，Nrf2蛋白主要存在于原头节细胞中。TRG处理降低了原头节中NQO-1和HO-1的活性，但在早期可增加ROS水平。用250μM TRG处理12天时，原头节无法存活。SEM结果表明，经TRG处理的原头节在原头节区域出现损伤，包括钩变形、病变和指状突起。与对照组相比，用TRG处理24小时和48小时的原头节中，Nrf2蛋白表达显著降低，caspase-3活性明显增加（P < 0.05）。我们的结果表明，TRG对细粒棘球绦虫原头节具有杀头节活性。Nrf2蛋白主要在细胞中表达，TRG可有效抑制细粒棘球绦虫中的Nrf2信号通路。
