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P-糖蛋白(P-gp)是ABC(ATP binding cassette)转运体家族重要成员,也是药物在机体内转运的重要载体。本文考察PKC/NF-κB-PXR信号途径对LS174T细胞中P-gp基因表达的调控作用。运用孕甾烷X受体(PXR)-MDR1双荧光报告基因实验探究PKC激动剂佛波酯(PMA)对LS174T细胞中MDR1荧光素酶活性的影响;分别采用real-time qPCR和Western blot检测PMA对LS174T细胞中P-gp mRNA表达、蛋白表达及NF-κB通路相关蛋白的影响。结果表明,PKC激动剂PMA能明显抑制PXR介导的P-gp荧光素酶活性、m RNA和蛋白表达,并能显著性增加胞内Rel A/p65的核转位。此外,si RNA干扰实验结果显示,PKCα siRNA、Rel A siRNA或PXR siRNA干扰均可显著削弱PMA对P-gp基因表达的下调作用。因此,PKC激动剂能显著抑制PXR介导的P-gp基因表达并伴随NF-κB激活,提示PKC/NF-κB-PXR信号通路对P-gp基因表达具有重要的调控作用。
P-glycoprotein (P-gp) is an important member of the ABC (ATP binding cassette) transporter family and an important carrier of drug transport in the body. This article investigates the regulatory effect of PKC / NF-κB-PXR signaling pathway on P-gp gene expression in LS174T cells. The effect of PKC agonist phorbol ester (PMA) on MDR1 luciferase activity in LS174T cells was investigated by dual reporter reporter assay of pregnane X receptor (PXR) -MDR1. Real-time qPCR and Western blot were used to detect PMA On P-gp mRNA expression, protein expression and NF-κB pathway related proteins in LS174T cells. The results showed that PKC agonist PMA significantly inhibited PXR-mediated P-gp luciferase activity, m RNA and protein expression, and significantly increased intracellular translocation of Rel A / p65. In addition, si RNA interference experiments showed that PKCα siRNA, Rel A siRNA or PXR siRNA could significantly reduce the down-regulation of P-gp gene expression by PMA. Therefore, PKC agonist can significantly inhibit PXR-mediated P-gp gene expression accompanied by NF-κB activation, suggesting that PKC / NF-κB-PXR signaling pathway plays an important regulatory role on P-gp gene expression.