多肽cSN50通过核质转运受体Importinα抑制HepG2细胞核因子-KB及其下游因子的表达

目的研究乙醇、内毒素是否通过核转录因子NF—kappaB即IKB—NF-κB-importina途径引起细胞炎症、凋亡和诱导下游基因TNF-α、Caspase-3的表达；cSN50作为一种穿膜多肽通过阻断NF-κB与importinct结合，从而抑制NF—KB核转位及其下游基因的表达，起到保护细胞的作用。方法应用流式细胞仪观察HepG2经不同浓度乙醇、内毒素刺激后的细胞凋亡率；分光光度计法测Caspase-3活性；ELISA法检测细胞培养上清TNF-α；Westernblot法检测NF-KBpso、importina3、IKBa，间接免疫荧光法测p50、IKBα、importinα3的活化水平。结果乙醇、内毒素刺激后TNF-α、Caspase-3的值量效上与空白对照组比差异均有统计学意义（P均〈0．05），细胞核内p50显著增加（P〈0．05），胞浆IKBa下降（P〈0．05），cSN50（100μmol／L）能部分阻断P50的核转位及其下游因子的表达（P〈0．05）。结论急性酒精肝损伤中，NF-κB的核转位在此损伤中起重要作用，cSN50能部分抑制被刺激后的HepG2细胞核中NF-κB活性及其下游基因的表达。

Objective To investigate whether polypeptide cSN50,as a transmembrane peptides,can inhibit NF-κB nuclear translocation and its downstream gene expression to play a role to protect cells by blocking the combination of NF-κB with the importinα3 during the alcohol and endotoxin-induced inflammation and apoptosis.Methods Flow cytometry method was used to observe the apoptosis rate of HepG2 by different concentration of alcohol and/or endotoxin.Spectrophotometer method was used to detect Caspase-3 activity.TNF- α in cell culture supernatant was detected by ELISA assay.p50,importinα3,and IκBαunder the stimulation of alcohol and/or endotoxin at selected optimal concentration were detected by Western blot.Indirect immunofluorescence assay was used to measure the activation of p50,IκBα,and importinα3.Results The change of TNF-α and Caspase-3 in the dose-effect course,compared with control group,was significant (P ＜0.05 ).p50 was increased in the nucleus ( P ＜ 0.05 ),and IκBα was decreased in the cytoplasm ( P ＜ 0.05 ).cSN50( 100 μmol/L) partially blocked nuclear translocation of p50 and its downstream factor( P ＜ 0.05 ).Conclusion NF-κB nuclear translocation may play an important role in acute alcoholic liver injury.cSN50 can effectively inhibit NF-κB activity and its downstream gene expression in HepG2 cells.