狼毒大戟提取物通过NF-κB通路激活潜伏HIV  

Euphorbia fischeriana extract reactivates latent HIV through nuclear factor-κB pathway

作　　者：潘晓彦[1] 张明娇[2] 曾晓云[1] 林健[1] 李琳[1] 李珉珉[2] 赵伟[1] 

机构地区：[1]南方医科大学药学院,广东广州510515 [2]暨南大学附属第一医院临床医学检验中心,广东广州510630

出　　处：《南方医科大学学报》2015年第11期1614-1618,共5页Journal of Southern Medical University

基　　金：中国第54批博士后基金面上项目(1091013);暨南大学附属第一医院科研培育专项基金

摘　　要：目的研究狼毒大戟提取物对潜伏HIV激活的影响,探讨其激活潜伏HIV可能的机制以及清除HIV的作用。方法取新鲜狼毒大戟植物根部组织,液氮速冻并粉碎成末,冷冻干燥3 d后称取少量,采用丙酮一步法提取并浓缩。以甲醇-水为流动相,采用HPLC反相C18柱分离,并通过MS鉴定后,得到狼毒大戟提取物(EFE)。以潜伏HIV细胞系J-Lat 10.6为激活模型,以TNF-α(10 ng/m L)作为阳性对照,EFE(50μg/m L)处理24 h后,采用FACS分析GFP阳性率以观测激活活性。采用不同浓度NF-κB抑制剂(Bay 11-7082)抑制EFE活性,并用WB检测2 h内p65入核水平,以及24 h内HIV蛋白p24随时间点变化情况。结果丙酮一步法可简便快速从狼毒大戟植物中提取得到EFE,经鉴定含prostratin及其类似物,分离后测得其中prostratin浓度为0.53 mmol/L。EFE(50μg/m L)作用24 h可产生约50%激活潜伏HIV活性,同时HIV蛋白p24水平随时间显著增加。进一步研究发现NF-κB通路抑制剂(Bay 11-7082)可对EFE活性产生浓度依赖抑制作用,且2 h内胞核p65水平在EFE作用下增加明显。结论狼毒大戟提取物EFE含有效成分prostratin及其类似物,可产生较强激活潜伏HIV活性,其机制为通过激活NF-κB通路促进p65入核从而发挥作用。Objective To investigate the effect of Euphorbia fischeriana extract on latent HIV reactivation and the pathway involved in this process and discuss the value of Euphorbia fischeriana extract in eliminating HIV. Methods Fresh tissues of Euphorbia fischeriana root were crushed into powder after quick freezing with liquid nitrogen and extracted with acetone followed by a three-day vacuum freeze-drying for dehydration of the extract. The extract（EFE） was separated using RP-C18 column with high-performance liquid chromatography（HPLC） and identified with mass spectrometry（MS）. The activity of reactivated latent HIV was analyzed by fluorescence-activated cell sorting in a J-Lat 10.6 cell model treated with EFE（50 μg/m L） for 24 h, using TNF-α（10 ng/m L） as the positive control. The effect of a NF-κB pathway inhibitor（Bay 11-7082） on EFE activity was tested. The changes in P65 expression in the cell nuclei within 2 h and HIV protein p24 expression within 24 h were analyzed by Western blotting in cells treated with EFE. Results EFE was obtained by one-step acetone extraction, and the concentration of prostratin in the extract was around 0.53 mmol/L. About 50% of the cells showed HIV reactivation after treatment with 50 μg/m L EFE for24 h accompanied by a significantly increased p24 expression. The activity of EFE in reactivating latent HIV was inhibited by Bay 11-7082 in a concentration-dependent manner, and p65 accumulation was detected in the cell nuclei within 2 h. Conclusion EFE we obtained contains the active compounds of prostratin and its analogues and shows a strong capacity to reactivate latent HIV through classical NF-κB pathway.

关 键 词：NF-ΚB prostratin 潜伏HIV 狼毒大戟 

分 类 号：R285[医药卫生—中药学]