| Objective：1. The objective of this study was to investigated the protective effects ofSalvianolic acid-B (Sal B) on the transdifferentiation of renal tubular epithelial cellsand to elucidate the mechanism of Sal B effect on renal fibrosis.2. The objective of this study was to investigated the ability reversal of Sal B toreverse the transdifferentiation of human kidney proximal tubular epithelial cells thatwas induced by transforming growth factor-beta1.3. To investigate the alteration of microRNA(miRNA) expression patternsduring the reversal effects of salvianolic acid B (Sal B) on Epithelial-mesenchymalTransition(EMT) induced by transforming growth factor-β1(TGF-β1).4. To investigate the effects of Salvianolic acid B on epithelial-mesenchymaltransition in rat with unilateral ureteral obstruction (UUO).Methods：1. Prevent experiment：Human kidney proximal tubular cell line (HK-2) wasused as the proximal tubular cell model. For experiments, cells were divided into sixgroups as follows: control group, transforming growth factor-β1(TGF-β1)(5ng/ml)group, TGF-β1(5ng/ml) plus Sal B (0.1μmol/mL,1μmol/mL,10μmol/mL, and100μmol/mL) groups. Epithelialto-mesenchymal transition (EMT) was induced with5ng/mL of human TGF-β1. The effects of the Sal B on cell morphology was observedby phase contrast microscopy, and α-smooth muscle actin (α-SMA) and E-cadherin were studied by immunocytochemistry and RT-PCR.2. Revert experiment: HK-2cells were cultured in DMEM/F12,For experiments,cells were divided into six groups as follows: a PTEC control group, a TGF-β1(5ng/mL) induced EMT control group, and groups in which cells were exposed to bothTGF-β1(5ng/mL) plus Sal B (0.1,1,10and100mmol/L). HK-2cells were culturedfor24h after seeding in DMEM/F12+5%FCS, then for72h in fresh culturemedium containing TGF-β1. Media were changed to fresh DMEM/F12containingTGF-β1plus Sal B at different concentrations, with both positive (5%FCS+TGF-β1)and negative controls (5%FCS). Cultures were then continued for a further72h. Theeffects of Sal B on HK-2cell morphology were observed by phase contrastmicroscopy, while alpha smooth muscle actin and E-cadherin were studied byimmunocytochemistry and real-time reverse transcription polymerase chain reaction,respectively.3. In this study, we used Human kidney proximal tubular cell line (HK-2)cultured in vitro as the proximal tubular cell model. Cells were divided into threegroups as follows:①Control group，②Transforming growth factor-β1(TGF-β1)group,③Salvianolic acid B (Sal B) reversal group: Epithelial-mesenchymaltransition (EMT) was induced by human TGF-β1for48h, and then TGF-β1plus SalB for72h, the effects of the Sal B on cytomorphology were observed by invertedphase contrast microscope, and expressions of E-cadherin and miRNAs were detectedby immunocytochemistry and gene chips respectively.4. Male SD rats were divided into3groups at random:①sham operated group(8rats),②UUO group(8rats),③the treated group (8rats): UUO+Salvianolic acid Bper kg30mg/d by gastrogavage. Kidney were harvested at day9after UUO. Therenal tissues were examined by HE，PAS and Masson. Tissue TGF-β1、α-SMA andVimentin protein expression was determined by Immunohistochemistry. Results：1. Our results revealed that Sal B not only prevent the de novo expression of themyofibroblast marker α-SMA but also prohibit the loss of the epithelial markerE-cadherin in HK-2cells, and also showed a dose-dependent prevention of α-SMAincrease and E-cadherin reduction, Simultaneous incubation of Sal B with TGF-β1could protect the change to the myofibroblast phenotype and restored the epithelialmorphology of the HK-2cells. These observations strongly suggest that Sal B is apotent inhibitor of TGF-β1-induced EMT and may be a promising agent for treatingtubulointerstitial fibrosis.2. In revert experiment, Exposure of HK-2cells to TGF-β1for72h induced acomplete conversion of the epithelial cells to myofibroblasts. When HK-2cells wereco-incubated with Sal B and TGF-β1for a further72h, the morphology ofmyofibroblasts returned to that of proximal tubular epithelial cells, whereas themyofibroblast phenotype was maintained after exposure of cells to TGF-β1for144h.Sal B reduced alpha smooth muscle actin levels and increased E-cadherin levelscompared with their epithelial-to-mesenchymal transition controls. The reversal effectof Sal B was dose-dependent.3. In this study,①Compared with normal HK-2cells,72miRNAs expressionprofiles were significantly changed in TGF-β1group, of which43weredownregulated and29upregulated more than2times.②Compared with TGF-β1group,47miRNAs were showed differentially expressed profiles in Sal B reversalgroup with27downregulated and20upregulated more than2times.③Among the9miRNAs altered in both the two groups,7were markedly downregulated in TGF-β1group while upregulated in Sal B reversal group, and2dramatically upregulated inTGF-β1group while downregulated in Sal B group. parts of the miRNAs closelyassociated with TGF-β type I receptor and zinc finger protein. 4. UUO for9days significantly activated TGF-β1、α-SMA and Vimentin proteinexpression, induced interstitial fibrosis and tubulointerstitial lesion in obstructedkidney of UUO group compared with sham operated group rats. Conversely dailyadministration of Salvianolic acid B significantly inhibited TGF-β1、α-SMA andVimentin protein expression and decreased interstitial fibrosis and tubulointerstitiallesion in obstructed kidney of treated group.ConclusionSal B not only prevents TGF-β1induced EMT in HK-2cells but also reversesEMT. Sal B is a promising candidate for developing clinically relevant therapeuticstrategies for renal disease.EMT of renal tubular reversed by Sal B attaches to different expression ofmiRNAs, and parts of the miRNAs may be closely associated with the reversal effect. |
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