Hua Chen1, Tian Yang1, Min-Chang Wang2, Dan-Qian Chen1, Yang Yang1, Ying-Yong Zhao3. 1. Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, School of Life Science, Northwest University, No. 229 Taibai North Road, Xi'an, Shaanxi 710069, China. 2. Xi'an Modern Chemistry Institute, Xi'an, Shaanxi 710065, China. 3. Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, School of Life Science, Northwest University, No. 229 Taibai North Road, Xi'an, Shaanxi 710069, China. Electronic address: zyy@nwu.edu.cn.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
BACKGROUND: 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. METHODS: 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. RESULTS: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. CONCLUSIONS: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.
Authors: Ya-Long Feng; Gang Cao; Dan-Qian Chen; Nosratola D Vaziri; Lin Chen; Jun Zhang; Ming Wang; Yan Guo; Ying-Yong Zhao Journal: Cell Mol Life Sci Date: 2019-05-30 Impact factor: 9.261
Authors: Ming Wang; Dan-Qian Chen; Lin Chen; Gang Cao; Hui Zhao; Dan Liu; Nosratola D Vaziri; Yan Guo; Ying-Yong Zhao Journal: Br J Pharmacol Date: 2018-05-22 Impact factor: 8.739