The Study Of Zhenwutang On Regulating On NRK-52E Cells "AVP-V2R-AQP2" Pathway

Objective To screen for optimum medicated serum preparation strategy,different time courses and daily frequencies of Zhenwutang administration were set The NRK-52E cells were exposed to medicated serum which prepared in the best way,observe the effect of Shenqiwan on protein related with "AVP-V2R-AQP2" pathway and the changes of cytoskeletal,investigate the mechanism and pathway targets of Zhenwutang on "AVP-V2R-AQP2" pathway.)Methods The 48 normal adult male SD rats were stochastically partitioned into 8 groups(n=6),respectively,ld2c(sterile water,twice per day,1 day),1d2Z(Zhenwutang twice per day,1 day),3d2c(sterile water,twice per day,3 days),3d2Z(Zhenwutang twice per day,3 days),5d2c(sterile water,twice per day,5 days),5d2Z(Zhenwutang,twice per day,5 days),7d2c(sterile water,twice per day,7 days),7d2Z(Zhenwutang,twice per day,7 days)groups.Each group received gavage of corresponding drugs(10 ml/kg),and the dose of Zhenwutang was 22.68 g/(kgd)(according to the amount of total crude drug).Before the last administration,rats underwent fasting without deprivation of water,and were intraperitoneally anesthetized by 0.3%pentobarbital sodium solution one hour after the last administration.The blood was taken from the heart and centrifuged for medicated serumIn vitro cultured NRK-52E cells were treated with the prepared medicated serum and dDAVP.Immunocytochemistry coupled with fluorescence microscope were exerted to locate AQP2 in the NRK-52E cells.The expression of AQP2 in NRK-52E cells were detected by Western blot and the genetic mRNA level were quantified by real time PCR technique.Results(1)The gradient concentration dDAVP were acted on NRK-52E cells for 1h and 24h,in comparison with the corresponding control groups,it is noted that 10-7M dDAVP can significantly reduce the expression of V2R,increasing the expression of AQP2.With 10"7M of dDAVP acting on NRK-52E cells for 0-60min,the results showed at 30min,dDAVP can significantly increase the expression of AQP2 and AQP3(2)After treatment with Zhenwutang medicated serum of varied days and time interval for 24h,compared with the control group,the serum of normal rats had no significant effect on the protein;compared with normal rat serum group,Zhenwutang medicated serum Z7d2 group can significantly increase the amount of V2R,AQP2 and AQP3 protein expression and mRNA levels of AQP2 and AQP3(3)After treatment with Zhenwutang medicated serum and dDAVP together,7d2+dDAVP group can significantly increase the expression of V2R on the basilar membrane of NRK-52E cell and AQP2,AQP3's protein expression.Conclusion Administration with Zhenwutang twice per day continuously for 7 days is preferential for medicated serum preparation;Zhenwutang medicated serum can extend V2R duration action on the NRK-52E cell membrane,increase the expression of AQP2 and regulate the transport processes of water molecules in the intracellular and extracellular;"AVP-V2R-AQP2" pathway may underlied the mechanism of regulating water transport balance,affecting parts of the mechanism of water metabolism of Zhenwutang.