摘要
目的:观察肾气丸对NRK细胞水通道蛋白2(AQP2)表达的影响,探讨肾气丸在NRK细胞"V2RcAMP-PKA-AQP2"通路上的主要作用环节。方法:首先采用H-89、SQ22536、Tolvaptan分别抑制蛋白激酶A(PKA)、环磷酸腺苷(cAMP)及V2R,再合用cAMP抑制剂SQ22536与PKA激动剂8-Br-cAMP干预NRK细胞,通过qPCR法和Western Blot法观察NRK细胞AQP2 mRNA表达水平及AQP2、p-PKA蛋白表达的变化。结果:10%肾气丸含药血清能显著上调NRK细胞AQP2 mRNA和蛋白的表达(P<0.01,P<0.05),加入PKA抑制剂H-89、cAMP抑制剂SQ22536或V2R拮抗剂Tolvaptan后,10%肾气丸含药血清促进AQP2、p-PKA表达的作用被显著抑制(P<0.01,P<0.05);SQ22536能显著抑制p-PKA、p-AQP2蛋白的表达(P<0.05),加入肾气丸含药血清或PKA激动剂8-BrcAMP后,p-PKA、p-AQP2蛋白表达均显著上调(P<0.01,P<0.05)。结论:V2R和PKA可能是肾气丸上调NRK细胞AQP2表达的重要靶点。
Objective: To study the effect of Shenqi Pills on the expression of AQP2, and explore the main role of Shenqi Pills on the V2 R-cAMP-PKA-AQP2 pathway in NRK cells. Methods: Firstly, using H-89, SQ22536 and Tolvaptan inhibited PKA,cAMP and V2 R separately, and then inhibited the activation of the V2 R-cAMP-PKA-AQP2 pathway with the cAMP inhibitor SQ22536 and agonist 8-Br-cAMP. The mRNA level of AQP2 was quantified by qPCR technique while the protein expression of AQP2 and p-PKA was detected by Western Blot. Results: The mRNA and protein expression of AQP2 significantly increased in10% Shenqi Pills medicated serum(P<0.01, P<0.05). Adding H-89, SQ22536 or Tolvaptan, the expression of mRNA and protein for AQP2 and p-PKA caused by 10% Shenqi Pills medicated serum were decreased significantly(P<0.01, P<0.05). Compared with the normal rats serum group, both of p-PKA and p-AQP2 protein expression in SQ22536 group decreased significantly(P<0.05), but the two indicators were significantly increased when 8-Br-cAMP or Shenqi Pills medicated serum was added(P<0.01,P<0.05). Conclusion: V2 R and PKA may be important targets for Shenqi Pills up-regulats the expression of AQP2 in NRK cells.
引文
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