Xue Hui, Chen Boting, Fan Yaomin, Palikhe Muna, Li Yingchao
Department of Gastroenterology, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, China.
Scand J Gastroenterol. 2011 Jul;46(7-8):931-40. doi: 10.3109/00365521.2011.568516. Epub 2011 Apr 20.
To study whether alcohol and endotoxin induce inflammation, apoptosis, and expression of downstream genes TNF-α and caspase-3 through the nuclear transcription factor NF-κB, i.e., the IκB-NF-κB-importin α pathway.
Flow cytometry was used to observe the apoptosis rate in human hepatoma cells (HepG(2)) and murine macrophages (RAW264.7) after being stimulated by alcohol and endotoxin at different concentrations. The caspase-3 activity was determined by spectrophotometry and TNF-α level in cell culture supernatant by ELISA. Western blot was used to examine the expression level of P50, importin α3 and IκBα, and indirect immunofluorescence to determine the activation level of P50, importin α3 and IκBα.
In human hepatoma cells, a transmembrane polypeptide, cSN50, inhibited the elevation in the level of TNF-α and caspase-3 and the expression of nuclear transport receptor importin α3 stimulated by alcohol and endotoxin. Immunofluorescence showed the nuclear translocation of NF-κB and importin α3 and cytosolic degradation of IκBα. In murine mononuclear macrophages, addition of alcohol or endotoxin or both resulted in a significant elevation of NF-κB and its downstream factors TNF-α and caspase-3 and apoptosis of RAW264.7 cells. These effects were remarkably suppressed by cSN50. However, the expression of importin α3 was not detected by Western blotting or immunofluorescence in this experiment, indicating the existence of other pathways.
The nuclear translocation of NF-κB plays an important role in acute alcoholic hepatic injuries and the induced nuclear NF-κB activity, and its downstream gene expression can be partially suppressed by cSN50 in HepG(2) and RAW264.7 cells.
研究酒精和内毒素是否通过核转录因子NF-κB即IκB-NF-κB-importinα途径诱导炎症、细胞凋亡以及下游基因TNF-α和半胱天冬酶-3的表达。
采用流式细胞术观察不同浓度酒精和内毒素刺激后人肝癌细胞（HepG(2)）和小鼠巨噬细胞（RAW264.7）的凋亡率。用分光光度法测定半胱天冬酶-3活性，用酶联免疫吸附测定法检测细胞培养上清液中的TNF-α水平。采用蛋白质印迹法检测P50、importinα3和IκBα的表达水平，用间接免疫荧光法测定P50、importinα3和IκBα的激活水平。
在人肝癌细胞中，一种跨膜多肽cSN50抑制了酒精和内毒素刺激引起的TNF-α水平、半胱天冬酶-3水平升高以及核转运受体importinα3的表达。免疫荧光显示NF-κB和importinα3的核转位以及IκBα的胞质降解。在小鼠单核巨噬细胞中，添加酒精或内毒素或两者均导致NF-κB及其下游因子TNF-α和半胱天冬酶-3显著升高以及RAW264.7细胞凋亡。这些作用被cSN50显著抑制。然而，在本实验中通过蛋白质印迹法或免疫荧光未检测到importinα3的表达，表明存在其他途径。
NF-κB的核转位在急性酒精性肝损伤及诱导的核NF-κB活性中起重要作用，其下游基因表达在HepG(2)和RAW264.7细胞中可被cSN50部分抑制。
