大黄素对LoVo细胞水通道蛋白2表达的调节效应

Regulative effects of emodin on aquaporin 2 expression in intestinal epithelial cell line LoVo

目的探讨大黄素对体外培养LoVo细胞水通道蛋白2(aquaporin 2,AQP2)表达的调节效应。方法体外培养LoVo细胞并予不同质量浓度大黄素含药培养液24 h处理及大黄素含药培养液(20 mg/L)不同时间处理,采用免疫细胞化学方法观察LoVo细胞AQP2蛋白表达位置并初步观察AQP2蛋白表达的变化趋势,采用Western blotting及半定量RT-PCR检测AQP2蛋白及mRNA的表达。结果AQP2表达于LoVo细胞的细胞膜,且与大黄素用药浓度及用药时间存在着相关性。进一步的Western blotting证实,不同质量浓度大黄素含药培养液均可抑制AQP2蛋白的表达,药物质量浓度愈大,AQP2蛋白的表达愈低,其中大黄素20 mg/L组及40 mg/L组AQP2蛋白表达显著性降低;大黄素作用3、6 h组,AQP2蛋白表达上升,但与对照组比较,无显著差异;作用12、24、48 h组,AQP2蛋白表达呈进行性下降趋势。半定量RT-PCR结果显示,随着大黄素质量浓度的增加,AQP2 mRNA表达呈进行性下降趋势;在时间-效应方面,随着用药时间的延长,AQP2 mRNA表达呈进行性下降趋势。结论大黄素可抑制LoVo细胞AQP2基因转录与翻译,这提示大黄素对AQP2表达的调节效应可能与大黄的泻下作用有关。

Objective To investigate the effects of emodin in regulating aquaporin 2 （AQP2） in LoVocells cultured with RPMI-1640 medium containing emodin. Methods LoVo ceils were cultured with RPMI-1640 medium containing emodin in different concentration for 24 h and were cultured with RPMI-1640 containing emodin （20 mg/L） for different times. The location of AQP2 protein in LoVo cells was definited by immuocytochemistry dyeing and the effects of emodin on regulating aquaporin 2 were initiallyevaluated. Western blotting and semiquantitative RT-PCR were adopted to detect the relative expression of AQP2 protein and AQP2 mRNA, respectively. Results AQP2 was located in plasma membrane of LoVo cell, and moreover, the degree of dyeing correlated with the concentration of emodin and theaffecting time. The relative expression of AQP2 protein decreased with the increasing of emodin concentration and that in 20 and 40 mg/L emodin groups decreased significantly ; The relative expression of AQP2 protein increased in 3 and 6 h groups. However there was no significant difference contrasted with that in the control group. Then the protein expression gradually decreased in 12, 24, and 48 h groups. The results of semiquantitative RT-PCR demonstrated that the relative expression of AQP2 mRNAdecreased with the increasing of emodin concentration. In the time-effect relationship, the relative expression of AQP2 mRNA also decreased with the prolongation of the culture time. Conclusion Emodin could inhibit the genetic transcription and the translation of AQP2 gene in LoVo cells, which demonstrates that the change of AQP2 expression regulated by emodin maybe correlate with the cathartic effect of rhubarb.