The Study Of Effect And Mechanism Of Astragaloside IV On The Epithelial-Mesenchymal Transition Of Normal Rat Renal Tubular Epithelial Cells Induced By TGF-?1

Objective:To study the effect of Astragaloside IV on epithelial-mesenchymal transition of normal rat renal tubular epithelial cells induced by TGF-?1 and to investigate whether the mechanism is through regulating the expression of connexin43(Cx43)to regulate the activity of phosphoinositide-3 kinase-protein kinase-mammalian rapamycin target protein receptor(PI3K/AKT/mTOR)pathway.Methods: 1.The slection of optimal stimulation concentration and time of TGF-?1:NRK-52 E cells were cultured in DMEM high glucosecontaining with 5%FBS and stimulated with different concentrations of TGF-?1(0-10 ng/ml)for 24 h and 48 h;Next we employed western blot to detecte the expression levels of cells differentiation markers,such as fibronectin(FN),alpha-smooth muscle actin(?-sma),then the results were statistically analyzed to choose the most appropriate time and the best concentration of TGF-?1.2.The slection of non-toxic dose Astragaloside IV: cells were treated with different concentrations of Astragaloside IV(0-200 ?g/ml)for 48 h,and CCK-8(Cell Counting Kit-8)were used to measure cells viability.We screened the concentrations ranging from 10 to 100 ?g/ml of Astragaloside IV where no impact on proliferationas.3.Evaluation of the intervention effect ofAstragaloside IV :according to above results,cells were divided into normal control group,TGF-?1 stimulation group,low,middle and high concentration of Astragaloside IV intervention group.QuantitativePCR(qPCR)was used to detect the expression of E-cadherin,?-sma and Cx43 mRNA in each group.The expression and distribution of E-cadherin,?-sma,Cx43 protein in each group were detected by immunofluorescence.Western blot was used to detect the expression of EMT-ralated protein,Cx43 and key proteins of PI3K/AKT/mTOR pathway.Finally,the cell cycle was detected by flow cytometry and cell proliferation was analyzed.Results:1.Different concentrations of TGF-?1(0-10 ng/ml)stimulated cells for 24 h,the expression of FN increased(P<0.01)when the concentration of TGF-?1 is above 6ng/ml,and the expression of ?-sma increased(P<0.01)when the concentration of TGF-?1 is above 2 ng/ml.NRK-52 E cells were stimulated with the same concentration of TGF-?1 for 48 h.When the concentration of TGF-?1 was more than 6 ng/ml,the expression of FN and ?-sma increased(P<0.01).Based on the above results,the optimal time for TGF-?1-induced transdifferentiation of NRK-52 E cells was 48 h and the optimal stimulation concentration was 6ng/ml.2.CCK-8 assay was used to detect changes in cell viability of cells treated with different concentrations of Astragaloside IV(0-200 ?g/ml)for 48 h.When the concentration of Astragaloside IV was 0-120 ?g/ml,there was no difference in cell viability compared with the normal control group(P>0.05).When the concentration of Astragaloside IV was above 160 ?g/ml,the cell viability decreased(P<0.05).3.The expression of E-cadherin,?-sma and Cx43 mRNA were detected by quantitative PCR(q PCR).The expression of E-cadherin and Cx43 mRNA in TGF-?1 stimulation group were lower than normal control group,after Astragaloside IV intervention,the expression of E-cadherin mRNA increased in a concentration-dependent manner(P<0.01).The mRNA expression of Cx43 was increased in medium and high concentration groups(P<0.01),compared with normal control group,the expression of ?-sma mRNA in TGF-?1 stimulated group was increased,and the expression of ?-sma mRNA was decreased in a dose-dependent manner after treated with Astragaloside IV(P<0.01).4.The expression of E-cadherin,?-sma and Cx43 protein in each group was detected by immunofluorescence.The fluorescence intensity of a-sma protein was enhanced after stimulation with 6 ng/ml TGF-?1,the fluorescence intensity of ?-sma protein was weakened after treatment with different concentrations of Astragaloside IV.6ng/ml TGF-?1 stimulation,the fluorescence intensity E-cadherin and Cx43 protein are weakened,with Astragaloside IV intervention,the fluorescence intensity increased.5.Western blot showed that the expression of ?-sma and vimentin increased after TGF-?1stimulation by compared with normal control group,the expression of ?-sma decreased after middle and high concentration of Astragaloside IV treatment(P<0.05),the expression of vimentin was decreased after treated with medium and high concentrations of Astragaloside IV(P<0.01),compared with normal control group.The expression of E-cadherin and Cx43 decreased after TGF-?1stimulation by compared with normal control group,the expression of E-cadherin increased in a concentration-dependent manner(P<0.05),and the expression of Cx43 also increased in a concentration-dependent manner(P<0.05).6.The expressions of AKT,p-AKT,mTOR and p-mTOR were detected by western blot,then calculating the ratios of p-AKT/AKT and p-mTOR/mTOR.The levels of p-AKT/AKT decreased with the intervention of Astragaloside IV at high and medium concentrations(P<0.01),low,medium and high concentrations of Astragaloside IV intervention,p-mTOR/mTOR decreased in a concentration-dependent manner(P <0.01).7.Compared with the normal group,the percentage of cells in G1 phase of TGF-?1 stimulated group decreased,and the proportion of cells in G1 phase increased after treated with high concentration Astragaloside IV(P<0.01).Conclusion:1.6ng/ml TGF-?1stimulate NRK-52 E cells for 48 h can induce cells transdifferentiation;2.Astragaloside IV can inhibit TGF-?1-induced transdifferentiation of NRK-52 E cells;3.Astragaloside IV inhibits the activity of PI3K/AKT/mTOR pathway induced by TGF-?1;4.Astragaloside IV inhibits the transdifferentiation of NRK-52 E cells induced by TGF-?1,which may be related to the activation of PI3K/AKT/mTOR pathway by enhancing the expression of Cx43 protein.