川西獐牙菜醇提物上调HepG2细胞膜转运蛋白MRP3、核转录因子SP1及核受体CPF、PXR表达

Alcohol extract of Swertia mussitii Franch upregulates membrane protein MRP3,nuclear transcription factor SP1 and nuclear receptors PXR and CPF in HepG2 cells

目的体外培养HepG2细胞观察川西獐牙菜醇提物(Swertia mussitii Franch)对多药耐药相关蛋白3(multidrug resistance protein 3,MRP3)、核转录因子SP1(specificity protein 1 transcription factor,SP1)及核受体孕烷X受体(pregnane X receptor,PXR)、CYP7A启动子结合因子(CYP7A promoter-binding factor,CPF)表达的影响。方法用MTT比色法检测川西獐牙菜醇提物的最佳作用浓度,再分别于0、12、24、48、72h刺激HepG2细胞,抽提细胞的RNA、总蛋白和核蛋白,采用荧光定量PCR和蛋白免疫印迹技术检测膜转运蛋白MRP3、转录因子SP1及核受体PXR、CPF在转录与蛋白水平的表达变化。每个时间点设立阴性对照DMSO组和阳性对照熊去氧胆酸(ursodeoxycholic acid,UDCA)组。结果川西獐牙菜醇提物可显著诱导HepG2细胞膜转运蛋白MRP3的mRNA和蛋白水平的高表达,其作用强于UDCA(P<0.05)。川西獐牙菜醇提物也可明显上调核转录因子SP1及核受体PXR的mRNA和蛋白表达水平,作用强于UDCA。对CPF表达的上调作用与UDCA相当(P<0.05)。结论川西獐牙菜醇提物可刺激HepG2细胞膜转运蛋白MRP3上调,且有可能是通过核转录因子SP1及核受体PXR、CPF上调其表达。

Objective To observe the impact of alcohol extract Swertia mussitii Franch on the expres- sion of muhidrug resistance protein 3 （ MRP3 ）, specificity protein 1 transcription factor （ SP1 ）, pregnane X receptor （PXR） and CYPTA promoter-binding factor （CPF） in HepG2 cells. Methods The optimal concen- tration of the alcohol extract used to stimulate HepG2 cells was determined by MTY assay. Then, total RNA, membrane protein and nuclear protein were extracted from HepG2 cells at 0, 12, 24, 48 and 72 h after co-cul- tured with the alcohol extract. The expression of MRP3, SP1, PXR and CPF at mRNA and protein levels were detected using real-time PCR and Western blotting, respectively. Results The upregulation of MRP3 was significantly induced by the alcohol extract at mRNA and protein levels, which was more efficient than that by ursodeoxycholic acid （UDCA）. The expression of SP1 and PXR at mRNA and protein levels was elevated by the alcohol extract, which was also more efficient than that by UDCA. The expression of CPF was also increased by the alcohol extract at mRNA and protein levels, which was similar to that of UDCA. Conclusion Our alcohol extract upregulates the expression of MR_P3 in HepG2 cells, possibly involving the activities of SP1, PXR and CPF.