从“V2R-cAMP-PKA-AQP2”通路探讨肾气丸上调NRK细胞AQP2表达的作用机制
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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.
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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[10]路琼琼,韩军,曾百惠,等.基于慢性心力衰竭大鼠模型的苓桂术甘汤和肾气丸“同病异治”之内涵研究.中华中医药杂志,2019,34(2):573-576
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[15]钟杰敏,朱延涛,金蓉家,等.六味地黄丸对甲亢型肾阴虚大鼠肾组织AQP1、AQP2含量的影响.浙江中医药大学学报,2013(5):493-496
[2]Yang B,D Zhao,L Qian,et al.Mouse model of inducible nephrogenic diabetes insipidus produced by floxed aquaporin-2gene deletion.American Journal of Physiology Renal Physiology,2006,291(2):465-472
[3]Radin M J,M J Yu,L Stoedkilde,et al.Aquaporin-2 regulation in health and disease.Veterinary Clinical Pathology,2012,41(4):455-470
[4]Rai T,K Sekine,K Kanno,et al.Urinary excretion of aquaporin-2water channel protein in human and rat.Journal of the American Society of Nephrology,1997,8(9):1357-1362
[5]童骏峰,徐志伟,杨元宵,等.腺嘌呤与氢化可的松所致大鼠肾阳虚证模型比较研究.中华中医药杂志,2015,30(11):3901-3904
[6]刘长稳.金匮肾气丸.开卷有益:求医问药,2015(8):51
[7]何灵玲,林晓彤.《金匮要略》中肾气丸临床运用探析.亚太传统医药,2016,12(6):66-67
[8]金蓉家,李守业,杨元宵,等.肾气丸对结肠癌LoVo细胞水通道蛋白2表达的调节作用.中国临床药理学与治疗学,2013,18(5):481-486
[9]谢晨,徐鹏,梁星,等.肾病综合征脾阳虚证候结合模型肾脏水通道蛋白表达的改变.中华中医药杂志,2018,33(3):929-933
[10]路琼琼,韩军,曾百惠,等.基于慢性心力衰竭大鼠模型的苓桂术甘汤和肾气丸“同病异治”之内涵研究.中华中医药杂志,2019,34(2):573-576
[11]HoffertJD,TPisitkun,GWang,etal.Quantitative phosphoproteomics of vasopressin-sensitive renal cells:Regulation of aquaporin-2 phosphorylation at two sites.Proceedings of the National Academy of Sciences of the United States of America,2006,103(18):7159-7164
[12]Van Balkom B W,P J Savelkoul,D Markovich,et al.The role of putative phosphorylation sites in the targeting and shuttling of the aquaporin-2 water channel.Journal of Biological Chemistry,2002,277(44):41473-41479
[13]Kortenoeven M L,C Trimpert,d B M Van,et al.In mpkCCD cells, long-term regulation of aquaporin-2 by vasopressin occurs independent of protein kinase A and CREB but may involve Epac.American Journal of Physiology,2012,302(11):1395-1401
[14]Tamma G,D Lasorsa,C Trimpert,et al.A protein kinase A-independent pathway controlling aquaporin 2 trafficking as a possible cause for the syndrome of inappropriate antidiuresis associated with polycystic kidney disease 1 haploinsufficiency.Journal of the American Society of Nephrology,2014,25(10):2241-2253
[15]钟杰敏,朱延涛,金蓉家,等.六味地黄丸对甲亢型肾阴虚大鼠肾组织AQP1、AQP2含量的影响.浙江中医药大学学报,2013(5):493-496