EGb761对LPS诱导THP-1细胞释放HMGB1蛋白表达的调节

The effect of EGb761 on the Expression of HMGB1 in LPS-induced THP-1

目的:探讨EGb761对LPS诱导THP-1细胞释放HMGB1蛋白表达的调节,为EGb761的临床运用提供可行的依据。方法:LPS(1μg/m L)诱导不同时间后,western blotting检测THP-1细胞上清液中HMGB1蛋白含量变化及不同浓度EGb761对LPS诱导THP-1细胞释放HMGB1蛋白的表达和NF-κB的活性;酶联免疫吸附法(ELISA)检测细胞中IL-1β、IL-6、TNF-α的含量。共聚焦显微镜观察EGb761对LPS诱导THP-1细胞释放HMGB1蛋白核转位变化。结果:(1)LPS组IL-1β、IL-6、TNF-α的含量在刺激6-12 h后明显高于空白对照组,而EGb761+LPS组IL-1β、IL-6、TNF-α的含量均显著低于LPS组(P<0.05)。(2)EGb761处理LPS诱导THP-1细胞6 h后细胞上清液NF-κB活性表达量较空白对照组低,随着处理时间延长至12 h,NF-κB的活性表达量呈明显下降趋势(P<0.05)。(3)LPS诱导THP-1细胞18 h后,细胞上清液中HMGB1蛋白含量呈明显升高趋势(P<0.05)。(4)不同浓度EGb761对LPS诱导THP-1细胞18 h后,HMGB1蛋白含量较空白对照组有下降趋势,HMGB1蛋白含量随着EGB761浓度增加至100μg/m L呈下降趋势并呈浓度依赖效应(P<0.05)。(5)LPS诱导THP-1细胞后,在共聚焦显微镜下可见胞浆中大量HMGB1蛋白标记分布,而EGb761+LPS共同诱导THP-1细胞后胞浆中可见少量HMGB1蛋白分布。结论:LPS可诱导THP-1细胞IL-1β、IL-6、TNF-α表达增多及NF-κB活化,导致HMGB1蛋白表达增多及核转位,而EGB761能抑制THP-1细胞IL-1β、IL-6、TNF-α表达及NF-κB活化,调节HMGB1蛋白的表达及核转位。

Objective： To investigate the effect of EGb761 on the expression of HMGB1 in LPS-induced THP-1 cells. We expect this study could provide a viable basis for the clinical application of EGb761. Methods： THP-1 cells were induced by LPS（1 μg/mL） for a certain period of time, then the amount of HMGB1 in the THP-1 cell supernatant was measured by a Western blot. EGB761 with different concentrations was added to the LPS-induced THP-1 and then the amount of HMGB 1 and the level of phosphorylated NF-KB were tested. ELISA was used to test the content of IL-1β, IL-6 and TNF-α. A confocal microscope was used to observe the nuclear translocation of HMGB1 in the LPS induced THP-1 cells to evaluate the effect of EGb761 in this progress. Results：（1） The amount of IL-113, IL-6 and TNF-α in the LPS-induced group was significantly increased when compared with the control group after induced for 6-12 h, while the amount of those makers in the EGb761＋LPS group was slightly increased compared to the control group but significantly less than that in the control group （P〈0. 05）. （2） The amount of NF-KB in the EGb761＋LPS group was less than that in the control group at 6 h, and showed a significantly downward trend at 12 h （P〈0.05）. （3） The amount of HMGB1 in the THP-1 cell supernatant increased significantly after the procedure of LPS induction for 18 hours （P〈0.05）. （4） The amount of HMGB1 in the EGb761＋LPS group decreased significantly when compared with the control group after induced for 18 h, and showed a concentration- dependent effect when the concentration of EGb761 was increased to 100 μg/mL （P〈0.05）.（5） We found a large amount of marked HMGB1 under the confocal microscope in the LPS group, while a relatively small amount of that in the EGb761＋LPS group. Conclusions： LPS may induce the expression of IL-113, IL-6, TNF-α and the activation of NF-KB in the LPS-induced THP-1 cells to promote the expression and nuclear transloeation of HMGB1, while EGb761 may inhibit the progress of expression and activation mentioned above, thus affect the expression and the progress of nuclear tmnslocation of HMGB 1.