Effects And Mechanism Of Shenwei Compound On TGF-β1-induced Renal Tubular Epithelial-mesenchymal Transition

Objective: To observe the effects of serum containing shenwei compound on TGF-β1-induced epithelial mesenchymal transition and the secretion of extracellular of renal tubular epithelial cells(NRK-52E).To explore the effects and possible mechanism of shenwei compound in preventing renal interstitial fibrosis.Methods:1.Preparetion of medicine serum: SD rats were divided into blank group and shenwei compound group.Rats in shenwei compound group were given shenwei compound with 10 times of adult dosage,rats in blank group were given same dose of normal saline.2.The effects of medicine serum on the regular growth of NRK-52 E cell: NRK-52 E cell were divided into five groups,which were normal control group,blank serum group and serum containing shenwei compound(low/middle/high-dose).The cell growth condition were observed under inverted phase contrast microscope and the cell viability were tested by CCK8 method.3.The effects of medicine serum on EMT and Smads protein of NRK-52 E cell induced by TGF-β1: NRK-52 E cell were divided into six groups,which were normal control group(5% fetal bovine serum),blank serum group(5% normal rat serum),TGF-β1 group(10ng/ml TGF-β1 plus 5%normal rat serum),serum containing shenwei compound group(low/middle/high-dose)(10ng/ml TGF-β1 respectively plus 1.25%,2.5%,5% serum containing shenwei compound).The cell regular growth condition were observed under inverted phase contrast microscope.The expression of α-SMA、E-cadherin、Smad3、p-Smad3、Smad7 protein were tested by Western Blot,the secretion of fibronectin、laminnin protein were assesed by immunofluorescence.The mRNA of α-SMA and E-cadherin were evaluated by Real time-PCR.Results: 1.The effects of medicine serum on the regular growth of NRK-52 E cell: Cells in blank serum group and serum containing shenwei conpound group were oval or polygonal,intercellular tight junctions.Cells had the morphology of typical epithelial cells and growed adhering to the plate.CCK8 revealed that cell viability increased after 72 hours in both blank serum group and serum containing drug group.2.The effect of medicine serum on EMT of NRK-52 E cell induced by TGF-β1:(1)Observed under inverted phase contrast microscope,most of the cells elongated to be long spindle shape after TGF-β1 induced.After shenwei compound intervention,cell changed from long spindle to oval shape.(2)Western Blot showed :Compared with blank serum group,the expression of E-cadherin protein was decreased and α-SMA protein was obviously increasd in TGF-β1group(P<0.01).After shenwei compound intervention,the expression of E-cadherin protein was increased and α-SMA protein was decreasednearly to normal,(P<0.01),which were dose dependent.(3)Real-Time PCR showed: Compared with blank serum group,the expression of E-cadherin mRNA was decreased and α-SMA mRNA was obviously increasd in TGF-β1 group(P < 0.01).After intervention of shenwei compound,the expression of E-cadherin mRNA was increased andα-SMA mRNA was decreased compared with TGF-β1 group(P<0.01),which was dose dependent.3.The effect of medicine serum on the secretion of Fibronectin and Laminin protein of NRK-52 E cell induced by TGF-β1: Immunofluorescence showed that compared with blank serum group,the expression of Fibronectin and Laminin protein were obviously increasd in TGF-β1 group(P<0.01).After intervention of the serum containing shenwei compound,the expression of Fibronectin and Laminin protein were decreased gadually.4.The effects of medicine serum on Smads protein of NRK-52 E cell induced by TGF-β1:(1)There were no obvious difference of total Smad3 protein expression among all groups.Compared with blank serum group,the expression of p-Smad3 protein were obviously increased in TGF-β1 group(P < 0.01).After intervention of the serum containing shenwei compound,the expression of p-Smad3 protein were decreased.(2)Compared with blank serum group,the expression of Smad7 protein were obviously decreasd in TGF-β1 group(P<0.01).After intervention of the serum containing shenwei compound,the expression of Smad7 protein were increased,compared with TGF-β1 group(P<0.01),which was dose dependent.Conclusions: 1.Shenwei compound could maintain the normal shape and regular growth of NRE-52 E cell and had no poisonous on NRK-52 E cells.2.Shenwei compound could suppress the expression of α-SMA,Fibronectin and Laminin protein induced by TGF-β1.3.Shenwei compound could recovery the decrease of epithelial cell marker protein E-cadherin,decline the level of phosphorylated Smad3 and increase the expression of Smad7 protein in TGF-β/Smad pathway,which effectively inhibited renal tubular EMT and interstitial fibrosis.