Astragaloside IV attenuates high glucose-induced EMT by inhibiting the TGF-β/Smad pathway in renal proximal tubular epithelial cells.

In the present study, we examined the molecular mechanism of astragaloside IV (AS-IV) in high glucose (HG)-induced epithelial-to-mesenchymal transition (EMT) in renal proximal tubular epithelial cells (PTCs). NRK-52E cell viability and apoptosis were determined by the cell counting kit-8 (CCK-8) assay and flow cytometric analysis, respectively. Expressions of E-cadherin, N-cadherin, vimentin, and occludin were measured by Western blot, and those of E-cadherin and N-cadherin were additionally measured by immunofluorescence analysis. Transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The expressions of Smad2, Smad3, phosphorylated-Smad2 (p-Smad2), and p-Smad3 were measured using Western blot. We found that AS-IV could recover NRK-52E cell viability and inhibit HG-induced cell apoptosis. TGF-β1, α-SMA, Smad2, Smad3, p-Smad2, and p-Smad3 expressions were decreased in the AS-IV-treated groups compared with the HG group. Moreover, the expressions of E-cadherin and occludin were remarkably up-regulated and those of N-cadherin and vimentin were down-regulated in the AS-IV-treated groups compared with the HG group. Interestingly, the TGF-β1 activator SRI-011381 hydrochloride had an antagonistic effect to AS-IV on HG-induced EMT behavior. In conclusion, AS-IV attenuates HG-induced EMT by inhibiting the TGF-β/Smad pathway in renal PTCs.

Wang YN ，Zhao SL ，Su YY ，Feng JX ，Wang S ，Liao XM ，Wang LN ，Li JC ，Meng P ，Li HY ，Zhang YF ... -  《-》

Novel RAS inhibitor 25-O-methylalisol F attenuates epithelial-to-mesenchymal transition and tubulo-interstitial fibrosis by selectively inhibiting TGF-β-mediated Smad3 phosphorylation.

Tubulo-interstitial fibrosis (TIF) is the common pathway in the chronic kidney disease (CKD). Epithelial-to-mesenchymal transition (EMT) is a major contributor to the TIF by the increased myofibroblasts. Renin-angiotensin system (RAS) is critical mediator on EMT in progressive CKD. Angiotensin II (ANG) mediates EMT and causes TIF by stimulating transforming growth factor-β1 (TGF-β1). RAS activation could further activate TGF-β1. Inhibition of the RAS is one of the most powerful therapies for progressive CKD. 25-O-methylalisol F (MAF) is a new tetracyclic triterpenoid compound isolated from the Alismatis rhizoma, which is extensively used for anti-hypertensive, diuretic and anti-hyperlipidemic effects. Inhibitory effect of MAF on EMT is investigated in both TGF-β1- and ANG-induced tubular epithelial cells (NRK-52E) and fibroblasts (NRK-49F). Western blot analysis, qRT-PCR, siRNA, immunofluorescence staining and co-immunoprecipitation techniques were used to evaluate the inhibition of MAF on EMT and further revealed the intervention effects on RAS, TGF-β/Smad and Wnt/β-catenin pathways. MAF treatment significantly inhibited TGF-β1 and ANG-induced expressions of collagen I, fibronectin, α-SMA, vimentin and E-cadherin at both mRNA and protein levels in the NRK-52E and NRK-49F cells. The action mechanism revealed that MAF significantly ameliorated upregulation of angiotensinogen, renin, ACE and AT1R expressions. Further, MAF attenuated upregulation of Smad3 phosphorylation and downregulation of Smad7, but did not affect the phosphorylation of Smad2, PI3K, ERK1/2 and p38 expressions and Smad4 expression in NRK-52E cells. Co-immunoprecipitation analysis indicated that MAF selectively blocked the combination of Smad3 with TGFβRI and Smad3 with SARA without interfering with the Smad2, TGFβRI and SARA interaction. Additionally, MAF suppressed the expressions of Wnt1 and β-catenin as well as its downstream target Snail1, Twist, MMP-7, PAI-1 and FSP1 expressions in NRK-52E cells. MAF simultaneously targeted multiple RAS components and it was a novel RAS inhibitor. MAF inhibited EMT by Smad3-specific signaling in the TGF-β/Smad-dependent pathway and Wnt/β-catenin pathway. MAF has an important effect on crosstalk between the TGF-β/Smad and Wnt/β-catenin pathway in EMT process by activation of RAS.

Chen H ，Yang T ，Wang MC ，Chen DQ ，Yang Y ，Zhao YY ... -  《-》

Effects of Astragaloside IV Against the TGF-β1-Induced Epithelial-to-Mesenchymal Transition in Peritoneal Mesothelial Cells by Promoting Smad 7 Expression.

To investigate the effect of Astragaloside IV (AS-IV) on the regulation of the TGF-β1/Smad signaling pathway in peritoneal mesothelial cells with an epithelial-to-mesenchymal transition (EMT). EMT of human peritoneal mesothelial cells (HMrSV5) was induced using 2 ng/ml TGF-β1. Cells were randomly divided into a vehicle group, a vehicle group with AS-IV, a TGF-β1 treated group, and a TGF-β1 treated group receiving varied doses of AS-IV or NAC. Real-time quantitative PCR and western blot were used to detect the expression of genes and proteins associated with the TGF-β1/Smad signaling pathway and EMT. DCFH-DA was used to detect the generation of ROS in HMrSV5 cells, and a transwell migration assay was used to verify the capacity of AS-IV to inhibit EMT in HMrSV5 cells. Lentiviruses were used as carriers for the overexpression or knockdown of the Smad7 gene. Expression levels of E-cadherin (epithelial marker) was decreased and vimentin, α-SMA (EMT markers) and collagen I (extracellular matrix protein) phospho-Smad2/3, Snail1 and Snail2 was increased significantly in the TGF-β1-treated HMrSV5 cells. AS-IV was associated with downregulated expression of vimentin and phospho-Smad2/3 in a dose-dependent manner, while the expression of Smad7 increased. Silenced or forced expression of Smad7 verified its role in the inhibitory effect of AS-IV on TGF-β1-induced EMT in HMrSV5 cells. AS-IV effectively promotes the upregulation of Smad7 in the TGF-β1/Smad signaling pathway during the EMT of HMrSV5 cells, indicating its potential therapeutic effect for the control of PF.

Zhang L ，Li Z ，He W ，Xu L ，Wang J ，Shi J ，Sheng M ... -  《-》

Simvastatin attenuates TGF-β1-induced epithelial-mesenchymal transition in human alveolar epithelial cells.

Transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to idiopathic pulmonary fibrosis (IPF). TGF-β1-induced EMT in A549 cells (a human AEC cell line) resulted in the adoption of mesenchymal responses that were predominantly mediated via the TGF-β1-Smad2/3 signaling pathway. Simvastatin (Sim), a 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitor, has been previously reported to inhibit EMT in human proximal tubular epithelial cells and porcine lens epithelial cells and to suppress Smad2/3 phosphorylation in animal models. However, whether Sim can attenuate TGF-β1-induced EMT in A549 cells and its underlying mechanisms remains unknown. Cells were incubated with TGF-β1 in the presence or absence of Sim. The epithelial marker E-cadherin (E-Cad) and the mesenchymal markers, α-smooth muscle actin (α-SMA), vimentin (Vi) and fibronectin (FN), were detected using western blotting analyses and immunofluorescence. Phosphorylated Smad2 and Smad3 levels and connective tissue growth factor (CTGF) were analyzed using western blotting. In addition, a cell migration assay was performed. Moreover, the levels of matrix metalloproteinase (MMP)-2 and -9 in the culture medium were examined using ELISA. Sim significantly attenuated the TGF-β1-induced decrease in E-Cad levels and elevated the levels of α-SMA, Vi and FN via the suppression of Smad2 and Smad3 phosphorylation. Furthermore, Sim inhibited the mesenchymal-like responses in A549 cells, including cell migration, CTGF expression and secretion of MMP-2 and -9. However, Sim failed to reverse the cell morphologial changes induced by TGF-β1 in A549 cells. Sim attenuated TGF-β1-induced EMT in A549 cells and might be a promising therapeutic agent for treating IPF.

Yang T ，Chen M ，Sun T 《-》

Alleviation of TGF-β1 induced tubular epithelial-mesenchymal transition via the δ-opioid receptor.

Renal fibrosis is a common pathological feature of progressive chronic kidney disease (CKD). It is indicated that transforming growth factor-β1 (TGF-β1) plays as a central mediator in renal fibrosis. The present study aimed to investigate the role of δ-opioid receptor (DOR) on renal fibrosis of the rat renal proximal tubular epithelial cell line (NRK-52E) induced by TGF-β1 and to elucidate its underlying mechanism, as well as its involvement in signaling pathways. Cells were treated with TGF-β1 (10 ng·mL-1 ), along with a specific DOR agonist (UFP-512) or naltrindole (a DOR antagonist). Cell viability and morphology, as well as cell migration, were measured after drug administration. Western blotting was employed to examine the extracellular matrix (ECM) protein Fibronectin, and the tubular epithelial-mesenchymal transition (EMT) markers (E-cadherin and α-smooth muscle actin (α-SMA)), signal transducer (p-Smad3), and EMT-regulatory gene (Snail). The expression level of phosphorylated Akt and p38 was also examined. Our results showed that TGF-β1 induced fibroblastic appearance and increased the expression of Fibronectin, α-SMA, P-Smad3, and Snail, while it decreased the expression of E-cadherin in NRK-52E cells. Moreover, TGF-β1 induced the activation of Akt and p38 MAPK signaling pathways. DOR activation was found to efficiently block morphological changes and cell migration, as long as the expression changes of Fibronectin, E-cadherin, α-SMA, P-Smad3, Snail, P-Akt, and P-p38 were induced by TGF-β1. These findings suggest that DOR may serve as an antifibrotic factor for renal proximal tubule cells by inhibiting the fibrosis process via TGF-β/Smad, Akt, and p38 MAPK signaling pathways.

Luo F ，Xu R ，Song G ，Xue D ，He X ，Xia Y ... -  《-》