大黄素对体外培养NRK细胞水通道蛋白2表达的影响

作者	单位
刘青	第四军医大学西京医院中医科暨全军中医内科中心
李锋	第四军医大学西京医院中医科暨全军中医内科中心
任秦有	第四军医大学唐都医院中医科
王文	第四军医大学西京医院中医科暨全军中医内科中心
张鹏	第四军医大学西京医院肾脏内科

摘要点击次数: 1451

全文下载次数: 570

中文摘要:

目的探讨大黄素对体外培养NRK细胞水通道蛋白2(AQP2)表达的调节效应及机制。方法体外培养NRK细胞,分为两步实验:第一步随机分为对照组和5、10、20mg/L大黄素处理组,采用间接免疫荧光法定性NRK细胞AQP2表达,Westen blot、半定量RT-PCR检测药物处理24h后AQP2蛋白及mRNA的表达;第二步随机分为对照组,20mg/L大黄素组,10mg/L8-Bromo-cAMP组、20mg/L大黄素加10mg/L8-Bromo-cAMP组,采用非放射性法检测药物处理24h后NRK细胞蛋白激酶A(PKA)活性水平。结果 AQP2表达于NRK细胞的细胞膜,进一步的Western blot及半定量RT-PCR结果显示,10、20mg/L大黄素培养液均可抑制NRK细胞的AQP2蛋白及mRNA的表达(P<0.05)。PKA活性结果显示,20mg/L大黄素能降低NRK细胞的PKA磷酸化水平(P<0.05)。结论大黄素可抑制NRK细胞AQP2基因转录与翻译,提示大黄的利尿作用可能与其调节AQP2表达有关,而且有可能是通过PKA通路调节AQP2的表达。

英文摘要:

Objective To investigate the effect and mechanism of emodin for regulating aquapoin-2(AQP2) in NRK cells cultured in vitro.Methods Experiments on NRK cells cultured with α-DMEM medium in vitro were conducted in two steps.(1) Cells were randomly divided into 4 groups:the control group,and the three emodin treated groups treated with different dosages of emodin(5,10 and 20 mg/L) respectively.After 24 h treatment,the location of AQP2 was decided by indirect immunofluorescene,and the AQP2 protein and mRNA expression levels were detected by Western blot and semiquantive RT-PCR.(2) Cells were randomly divided into 4 groups,the control group,and the three treated groups treated respectively with 10 mg/L 8-Bromo-cAMP,20 mg/L emodin,and 20 mg/L emodin +10 mg/L 8-Bromo-cAMP.The activity of protein kinase A(PKA) in NRK cells after 24 h treatment was determined with non-radioactive detecting method.Results AQP2 was located at the cell membrane of NRK cells.Western blot and semiquantitive RT-PCR found that AQP2 protein and mRNA expressions were significantly decreased in NRK cells of groups treated by 10 mg/L and 20 mg/L emodin(P < 0.05) .PKA activity determination showed significantly decreased phosphorylation level of PKA in NRK cells of groups treated with 20 mg/L emodin group(P <0.05) .Conclusion Emodin can inhibit the genetic transcription and the translation of AQP2 gene in NRK cells,which demonstrates that the change of AQP2 expression regulated by emodin may be correlated with the diuresis effect of rhubarb,and it is likely that the regulation is going through PKA signal pathway.

关闭