缬沙坦对血管紧张素Ⅱ抑制klotho基因表达的影响

Effect of valsartan on angiotensin Ⅱ induced down regulation of klotho gene expression

目的：研究血管紧张素Ⅱ（AngⅡ）调控klotho基因表达的机制，探讨缬沙坦（valsartan）对其调控作用的影响。方法：将大鼠肾小管上皮细胞（NRK-52E）与干预药物共培养。（1）按AngⅡ浓度梯度0（对照组）、10^-9、10、10^-7、10^-6、10^-5mol／L和时间梯度0（对照组）、3、6、12和24h分组培养，用RT—PCR检测klotho mRNA的表达水平。（2）按Valsartan浓度梯度0（对照组）、10^-9、10^-7、10^-5和10^-3mol／L分组培养，RT—PCR检测klotho mRNA的表达。（3）设对照组、AngⅡ（10。mol／L）组、AngⅡ（10^-7mol／L）＋Valsartan（10^-5mol／L）组和Valsartan（10^-5mol／L）组，用RT—PCR和免疫组化法检测klotho mRNA和蛋白表达。结果：（1）AngⅡ对klotho mRNA表达呈量效抑制关系，但在时效关系上，klotho在3h点被AngⅡ抑制（P〈0．05），6～12hklotho mRNA表达量逐渐增加，而24h后klotho mRNA表达减少，呈明显抑制状态（P〈0．05）。（2）不同浓度Valsartan对klotho mRNA的表达无显著影响，组问差异无统计学意义（P〉0．05）。（3）AngⅡ可明显抑制klotho表达（P〈0．05），给予Valsartan阻断AngⅡ作用后，klotho表达增高（P〈0．05），而Valsartan本身对klotho表达无影响（P〉0．05）。结论：AngⅡ对klotho基因的抑制作用呈浓度依赖性，Valsartan可拮抗AngⅡ对klotho基因的抑制作用，血管紧张素Ⅱ1型受体是AngⅡ调控klotho基因表达的关键环节。

Objective ：To investigate the mechanism of angiotensin Ⅱ （Ang Ⅱ ） regulating klotho gene expression, and to elucidate the impact of valsartan on the down-regulation of klotho expression induced by Ang Ⅱ. Methodology： The rat renal tubular epithelial cells （NRK-52E） were cultured and incubated with medium containing either Ang Ⅱ or valsartan. The experimental groups were divided according to different concentration of Ang Ⅱ （0,10^-9 , 10^-8 , 10^-7 , 10^-6, 10^-5 mol/L） , and valsartan （0,10^-9,10^-7,10^-5,10^-3 mol/L）. The optimal concentration of Ang Ⅱ （ 10^-7 mol/L） was used to treat cells for 0,3,6,12,24 hours. Four groups including control, Ang Ⅱ （10^-7 mol/L） , Ang Ⅱ （10^-7 tool/L） ＋ valsartan （ 10^-5 mol/L） and valsartan （ 10^-5 mol/L） were set up to observe the effect of valsartan on the expression of klotho induced by Ang Ⅱ. Reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry was applied to evaluate the mRNA and protein expression of klotho gene. Results： We found that the Ang Ⅱ down-regulated klotho mRNA in a dose-dependent manners. The stimulating effect of Ang Ⅱ （10^-7mol./L） was beginning to strong in the first 3 hours（P 〈0. 05） , and increased in the latter hours （ 6- 12 hours）. While, on the point of 24 hours, the klotho mRNA expression was inhibited and decreased by Ang Ⅱ again （P 〈 0. 05 ）. Ang Ⅱ obviously decreased the levels of klohho mRNA and protein expression, which was reversed by valsartan, however, Valsartan had no effect on the klotho gene expression（ P 〉 0. 05 ）. The expression of klotho mRNA and protein in valsartan group was higher than those in Ang Ⅱ group（P 〈 0. 05 ）. Conclusion： Ang Ⅱ down-regulated klotho gene expression in a dose-dependent manner. The angiotensin Ⅱ type 1 receptor could ameliorate the Ang Ⅱ -induced down-expression of klotho gene, and play an important role in the regulating klotho expression by Ang Ⅱ.