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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、10-8、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klothomRNA的表达。(3)设对照组、AngⅡ(10-7mol/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),6~12h klotho 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,组间差异无统计学意义(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 (AngⅡ) regulating the expression of klotho gene and to explore the effect of valsartan on its regulation. Methods: Rat renal tubular epithelial cells (NRK-52E) were co-cultured with the interventions. (1) AngⅡ concentration gradient 0 (control group), 10-9,10-8,10-7,10-6,10-5 mol / L and time gradient 0 (control group), 3,6,12 and 24h Groups were cultured and the expression level of klotho mRNA was detected by RT-PCR. (2) The Valsartan concentration gradient 0 (control group), 10-9, 10-7, 10-5 and 10-3mol / L groups were cultured and the expression of klothomRNA was detected by RT-PCR. (3) The control group was treated with AngⅡ (10-7mol / L), AngⅡ (10-7mol / L) + Valsartan (10-5mol / L) and Valsartan (10-5mol / Immunohistochemistry was used to detect klotho mRNA and protein expression. The results showed that: (1) Ang Ⅱ inhibited the expression of klotho mRNA in a dose-dependent manner. However, the klotho mRNA was inhibited by AngⅡ at 3h (P <0.05), while the klotho mRNA increased gradually after 6 ~ 12h The expression was decreased and was significantly inhibited (P <0.05). (2) There was no significant difference in the expression of klotho mRNA between different concentrations of Valsartan (P> 0.05). (3) Ang Ⅱ significantly inhibited the expression of klotho (P <0.05), and increased the expression of klotho by Valsartan (P <0.05), while Valsartan itself had no effect on klotho expression (P> 0.05). CONCLUSION: Ang II inhibits klotho gene in a concentration-dependent manner. Valsartan can antagonize the inhibitory effect of AngⅡ on klotho gene. Angiotensin II type 1 receptor is the key factor that AngⅡ regulates klotho gene expression.