Shenqiwan Promotes Cell Migration Via Regulating Aquaporin 1 Expression In Renal Tubular Epithelial Cells

Objective To explore whether the Shenqiwan promotes cell migration via regulating aquaporin 1 expression in renal tubular epithelial cells.Methods The SD rats were randomly divided into the control group,Shenqiwan(1.5 g/kg)group,Shenqiwan(3.0 g/kg)group and Shenqiwan(6.0 g/kg),n=8.In addition to the normal control group,the other three groups received gavage of corresponding dose of Shenqiwan for 5 days,twice per day.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 serum.In vitro cultured NRK-52E cells and divided into five groups as follows:serum-free group,blank serum group,Sqw(1.5 g/kg)group,Sqw(3.0 g/kg)group,Sqw(6.0 g/kg).The effect on NRK-52E cells proliferation,adhesion,migration with different doses of Shenqiwan medicated serum were detected by RTCA assays.Besides,NRK-52E cells adhesion was also observed under inverted microscope and cells migration was also detected by wound healing assays.Immunocytochemistry coupled with fluorescence microscope were exerted to locate AQP1 and observe the filamentous pattern and distribution of F-actin and vimentin in NEK-52E cells.The genetic mRNA level of AQP1 were quantified by qPCR technique and the expression of AQP1,MMP2,MMP9,Vimentin protein in NRK-52E cells were detected by Western blot.AQP1 RNAi slow virus was used to infect NRK-52E cells,the genetic level of AQP1 mRNA,expression of AQP1 protein,change of cell adhesion and migration in these infection cells were detected before and after Shenqiwan intervention.Results(1)Compared with blank serum group,different doses(1.5 g/kg,3.0 g/kg,6.0 g/kg)Sqw group can improve NRK-52E cells adhesion and migration ability significantly(P<0.01 or P<0.05).Besides,the quantity of adherent cells were more than that not adherent under inverted microscope.And the result of wound healing assays also showed that Shenqiwan can promote NRK-52E cells migration.(2)The result of immunofluorescence assays suggest that AQP1 protein mainly located in cell cytoplasm and membrane,shine strong green fluorescence,and Shenqiwan can improve the expression of AQP1 protein and strengthen the fluorescence.The cytoskeleton with filaments to be organized in independent directions in control group while most of the cells displayed parallel-oriented filaments organized along the axis in Shenqiwan group.(3)Sqw(3.0 g/kg,6.0 g/kg)can improve the the genetic level of AQP1 mRNA in NRK-52E cells significantly(P<0.01 or P<0.05).(4)Sqw(3.0 g/kg,6.0 g/kg)can promote the expression of AQP1,MMP2,MMP9,vimentin protein in NRK-52E cells(P<0.01 or P<0.05).With Sqw(3.0 g/kg)from 0h to 12h,the expression of AQP1,MMP2,MMP9,vimentin protein increase gradually and reach to peck at 6h or 12h.(5)When NRK-52E cells infected by AQP1 RNAi slow virus,the genetic level of AQP1 mRNA,expression of AQP1 protein,ability of cell adhesion and migration and rate of wound healing were significant reduced.And then significant increased after Sqw(3.0 g/kg)intervention.Conclusion Shenqiwan can improve NRK-52E cells adhesion and migration ability significantly,promote the genetic level of AQP1 mRNA and the expression of AQP1,MMP2,MMP9,vimentin protein in NRK-52E cells.The decline of genetic level of AQP1 mRNA,expression of AQP1 protein,ability of cell adhesion and migration and rate of wound healing after AQP1 RNAi slow virus infect NRK-52E cells would be suppressed by Shenqiwan intervention.AQP1 play a important role in Shenqiwan promoting NRK-52E cells migration,and it maybe a internal target of renal repair by Shenqiwan.