基于PXR/CAR调控通路探讨士的宁对UGT1A4的作用

Explore the Effect of Strychnine on UGT1A4 Based on the PXR/CAR Regulatory Pathway

目的研究士的宁对人肝细胞HL-7702中葡萄糖醛酸转移酶1A4(UGT1A4)表达的影响及其与孕烷X受体(PXR)/组成型雄烷受体(CAR)调控通路的关系,明确UGT1A4与士的宁的关系及作用机制,为进一步研究士的宁代谢及合成或改造相似结构药物提供一定的实验依据。方法以人肝细胞株HL-7702为研究对象,不同浓度的士的宁(10、20、40 pmol·L^(-1))干预24 h后,RT-qPCR和Western Blot法检测士的宁对HL-7702细胞中UGT1A4、PXR和CAR mRNA和蛋白表达水平的影响。通过双荧光素酶报告基因法以及脂质体瞬时转染方法,分析UGT1A4报告基因(来自于瞬时共转染hCAR或者是hPXR之后的HL-7702细胞)的活性在各浓度士的宁(10、20、40 pmol·L^(-1))干预下的影响。利用siRNA干扰技术,探究士的宁对hPXR基因沉默后HL-7702细胞中UGT1A4蛋白表达水平的影响。结果与对照组比,20、40 pmol·L^(-1)士的宁干预后明显上调UGT1A4、PXR的mRNA和蛋白的表达水平,呈浓度依赖性(P<0.05,P<0.01,P<0.001);但对CAR mRNA和蛋白的表达无明显的影响,差异无统计学意义(P>0.05)。在瞬时共转染hPXR的HL-7702细胞中,20、40 pmol·L^(-1)士的宁干预24 h后能明显上调PXR介导UGT1A4的转录活性,呈浓度依赖性(P<0.01,P<0.001);在瞬时共转染hCAR的HL-7702细胞中,各浓度士的宁干预24 h后对CAR介导的UGT1A4转录活性无明显影响,与对照组比,差异无统计学意义(P>0.05)。士的宁对沉默hPXR的HL-7702细胞中UGT1A4蛋白的诱导作用明显减弱,与对照组比,差异无统计学意义(P>0.05)。结论士的宁对HL-7702中UGT1A4和PXR在转录和蛋白水平有明显的上调作用,对CAR在转录和蛋白水平没有明显影响。士的宁对HL-7702细胞中UGT1A4的诱导作用主要通过PXR调控通路实现。

Objective To study the effect of strychnine on the expression of UDP-glucuronosyltransferase 1A4(UGT1A4)in HL-7702 cells and its relationship with the PXR/CAR regulatory pathway,and to clarify the relationship between UGT1A4 and strychnine and its mechanism,and provide certain experimental evidence for further research on the metabolism and synthesis or modification of drugs with similar structures.Methods Human hepatocellular carcinoma cell line HL-7702 was used for the research object,different concentrations strychnine(10、20、40 pmol·L^(-1))was intervened for 24 h,RT-qPCR and Western Blot were used to detect the effect of strychnine on UGT1A4,PXR and CAR mRNA and protein in HL-7702.Liposome transient transfection and dual luciferase reporter gene method were used to investigate the effect of different concentrations of strychnine(10、20、40 pmol·L^(-1))on the UGT1A4 reporter gene in HL-7702 cells which co-transfected with hPXR or hCAR.Using siRNA interference technology,we explored the effect of strychnine on the expression levels of UGT1A4 protein in HL-7702 cells after hPXR gene-silenced.Results Compared with the control group,20,40 pmol·L^(-1) strychnine significantly increased the mRNA and protein levels of UGT1A4 and PXR,which showed concentration-dependent manner(P<0.05,P<0.01,P<0.001),but had no significant effect on the mRNA and protein levels of CAR,there is no statistical difference(P>0.05).In the HL-7702 cells transiently co-transfected with hPXR,strychnine at 20、40 pmol·L^(-1) can significantly up-regulate the PXR-mediated transcriptional activity of UGT1A4 after 24 h intervention,which showed concentration-dependent manner(P<0.01,P<0.001).In the HL-7702 cells transiently co-transfected with hCAR,strychnine at various concentrations had no significant effect on CARmediated UGT1A4 transcriptional activity after 24h intervention(P>0.05).Compared with normal HL-7702 cells,strychnine significantly weakened the induction of UGT1A4 mRNA in hPXR-silenced HL-7702 cells.There is no statistical difference(P>0.05).Conclusion Strychnine can significantly up-regulate the expression of UGT1A4 and PXR mRNA and protein in HL-7702 cells,but has no obvious effect on the expression of CAR mRNA and protein.The effect of strychnine on UGT1A4 in HL-7702 cells is mainly related to the PXR regulatory pathway.