醋柴胡小分子水溶性部位抑制HEK293-Pgp细胞中P糖蛋白的外排功能  

Micromolecular hydrophilic extract from vinegar-baked radix bupleuri weakens Pgp efflux function in HEK293-Pgp cells

作　　者：冯丽敏[1] 张娴 赵瑞芝 

机构地区：[1]广州中医药大学第二临床医学院 [2]广东省中医药科学院,广东广州510006

出　　处：《中国药理学通报》2013年第12期1684-1688,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81073063;30672668)

摘　　要：目的观察醋柴胡小分子水溶性部位(MHE)对P糖蛋白(Pgp)高表达的HEK293-Pgp细胞中Pgp的影响,分析其可能的分子机制。方法实验分人胚肾细胞HEK 293和Pgp高表达的HEK293-Pgp组。MTT法观察醋柴胡小分子水溶性部位对细胞增殖的影响;细胞分别加入秋水仙碱和罗丹明B作底物,Pgp蛋白摄取活性测定实验分别采用高效液相色谱(HPLC)法和流式细胞术(flow cytometry)检测胞内底物含量,以分析Pgp外排功能;采用Western blot法检测Pgp蛋白表达水平;实时荧光定量PCR(RT-PCR)检测Pgp mRNA水平。结果 50 g·L-1MHE作用于细胞48 h后,HEK293细胞中的Pgp外排功能略呈上调趋势,但差异无统计学意义(P>0.05);而作用于HEK293-Pgp细胞24 h后相应的蛋白表达以及mRNA水平分别降低了68.1%和38.8%(P<0.05),且HEK293-Pgp细胞对罗丹明B的摄取率升高为对照组的3.2倍(P<0.01),呈下调Pgp外排的功能。结论 MHE呈抑制Pgp高表达的细胞中Pgp外排功能的作用,这可能是通过下调Pgp mRNA和蛋白表达实现。初步暗示了MHE可能为一种潜在的Pgp抑制剂。Aim To determine the effect of MHE on Pgp overexpression in HEK293-Pgp cells, and to ex- plore the potential molecular mechanism. Methods Two groups including HEK293 and HEK293-Pgp cells were set up. Cell proliferation was measured by MTT assay. Pgp substrates eolehicine and Rhodamine B （RB） were added to cells, and then cytoplasmic sub- strates content was detected by HPLC and flow cytome- try respectively. Therefore, Pgp uptake activity and ef- flux function were determined. Pgp protein expression was detected by Western blot, and mRNA level was detected by real-time quantitative PCR （RT-PCR）. Results After treatment with MHE （50 g ·L^- 1 ） for24 h, the uptake of Rhodamine B into HEK293-Pgp cells increased by 3.2 fold （P 〈0. 01 ） compared with the control group and the protein expression and mRNA level of Pgp were down-regulated by 68. 1% （P 〈 0.05） and 38.8% （P 〈0.05） respectively. Mean- while there were no changes in FIEK293 cells. Con- elusion MHE strongly inhibits Pgp efflux function, which might be due to the down-regulation of Pgp pro- tein and mRNA level by MHE. All above indicates MHE is an inhibitor of Pgp.

关 键 词：醋柴胡小分子水溶性部位 P糖蛋白 高效液相色谱 流式细胞术 秋水仙碱 罗丹明B 

分 类 号：R284.1[医药卫生—中药学] R322.61[医药卫生—中医学]