乙肝病毒X蛋白抑制高迁移率族蛋白B1的表达和活性氧的产生

HBX inhibits the expression of high - mobility group protein box 1 and the generation of reactive oxygen species via nuclear factor - kB signaling pathway

目的观察乙肝病毒X蛋白（HBX）表达对宿主细胞高迁移率族蛋白B1（HMGB1）和活性氧（ROS）的影响。方法在正常肝细胞系LO2中过表达HBX蛋白；利用Western blot和ROS检测剂双氯荧光素（DCFH-DA）检测HMGB1的表达、ROS的产生和核因子（NF）-κB信号通路的变化；然后利用NF-κB和ROS的激活剂分别刺激细胞，验证NF-κB对HMGB1和ROS的调节作用。结果Western blot结果显示HBX的过表达显著抑制HMGB1的表达，下调2.4倍（P＝0.025）。通过对绿色荧光的观察发现HBX对ROS的产生具有抑制作用。Western blot结果证明HBX过表达可以抑制NF-κB（p65）及其抑制子IκB的磷酸化；鱼藤酮和LPS分别刺激HBX过表达细胞后发现，NF-κB（p65）及其抑制子IκB的磷酸化增强、HMGB1、ROS和Toll样受体4（TLR4）的表达量增加，提示HMGB1与NF-κB信号通路间是相互正调控关系。结论HBX通过抑制NF-κB信号通路，进而抑制HMGB1的表达和ROS的产生，同时抑制宿主细胞对炎性细胞因子的释放。

ObjectiveThis study focus on the effect of HBX on the expression of HMGB1 in vitro.Methodswe overexpress the HBX protein in LO2 cell （a normal hepatocyte cell line）. Then the activity of nuclear factor （NF）-κB, the expression of HMGB1 and the generation of ROS were detected by Western blotting and 2’, 7’-dichlorodihydrofluorescin diacetate （DCFH-DA） （ROS detector） staining. Afterwards, retenone and lipopolysaccharide （LPS）, which were the activor of ROS and NF-κB separately, were used to stimulate the HBX-overexpressed cells and the expressed differentiation of, HMGB1 and ROS or the activity alternation of NF-κB were detected again.ResultsThe results of Western blotting indicated that HBX significantly inhibited expression of HMGB1 （more than 2.4 fold, P=0.025）, ROS and the phosphorylation levels of p65 and IκB. For further confirm these results, we stimulated the LO2-HBX cell, which are the stimulators of ROS and NF-κB, respectively. And the results suggested that the phosphorylation levels of p65 and IκB were enhanced after stimulation. All of these results revealed that the NF-κB and HMGB1 could directly regulate each other.ConclusionHBX inhibits the expression of HMGB1 and the generation of ROS via NF-κB signaling pathway.