大黄酚对LoVo细胞AQP2表达的调节效应

Regulating effect of aquaporin2 expression by chrysophanol on intestinal epithelial cell line LoVo

目的:探讨大黄酚对体外培养LoVo细胞AQP2表达的调节效应.方法:体外培养LoVo细胞,随机分为对照组,大黄酚10,20,40mg/L不同浓度组.间接免疫荧光定性LoVo细胞AQP2表达,WesternBlot、半定量RT-PCR检测药物处理24h后AQP2蛋白及mRNA表达.体外培养LoVo细胞后,随机又将细胞分为对照组,大黄酚40mg/L组,PKA激动剂8-Bromo-cAMP10mg/L组,大黄酚40mg/L+PKA激动剂8-Bro-mo-cAMP10mg/L组.非放射性法检测药物处理24h后LoVo细胞PKA的活性水平.结果:AQP2表达于LoVo细胞膜,药物处理24h后,大黄酚20,40mg/L组AQP2蛋白分别为(0.64±0.08),(0.46±0.09)显著低于对照组(0.83±0.05),(P<0.01);AQP2mRNA分别为(0.50±0.12),(0.39±0.09),显著低于对照组(0.73±0.08),(P<0.01).40mg/L大黄酚组PKA活性(0.54±0.08)显著低于对照组(0.78±0.10),(P<0.05).结论:大黄酚可抑制LoVo细胞AQP2基因转录与翻译.大黄酚对AQP2表达的调节效应可能与大黄泻下作用有关,其可能是通过PKA信号通路调节AQP2的表达.

AIM： To investigate the effect of chrysophanol in regulating AQP2 in LoVo cells cultured with RPMI-1640 medium containing chrysophanol. METHODS： LoVo cells cultured with RPMI-1640 medium in vitro were randomly divided into 4 groups： control group, chrysophanol treatment groups （10, 20 and 40 rag/L, respectively）. The location of AQP2 was decided by indirect immunofluorescene and the expression levels of protein and mRNA of AQP2 after a 24 h treatment were decided by Western Blot and semiquantive RT-PCR. LoVo cells cultured with RPMI-1640 medium in vitro were randomly divided into control group, 10 mg/L 8-Bromo-cAMP group, 40 mg/L chrysophanol group and 40 mg/L chrysophanol together with 10 mg/L 8-BromocAMP group. The activity of PKA following the 24 h treatment was tested in LoVo cells with non-radioactive detection method. RESULTS： AQP2 was found to be located at the cell membrane of LoVo cells. In 20 and 40 mg/L chrysophanol treatment groups,AQP2 protein were （0.64 ± 0.08 ） and （ 0.46 ± 0.09 ） respectively, significantly lower than those in control group （ 0. 83 ± 0.05 ）, （P〈0.01）, while AQP2 mRNA was（0.50 ±0.12）and（0.39 ± 0.09） respectively, significantly lower than those in control group （0.73 ± 0.08 ）, （ P 〈 0.01 ）. In 40 mg/L chrvsophanol treatment group, the expression level of PKA was （ 0.54 ± 0.08 ） , was significantly lower than that in control group （ 0.78 ± 0. 10 ）,（ P 〈 0.05 ）. CONCLUSION ： Chrysophanol inhibits the genetic transcription and the translation of AQP2 gene in LoVo cells, which demonstrates that the changes of AQP2 expression regulated by chrysophanol may be corrected with the cathartic effect of rhubarb. It is likely that the expression of AQP2 is regulated through PKA signal pathway.