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EW-7197 inhibits hepatic, renal, and pulmonary fibrosis by blocking TGF-β/Smad and ROS signaling

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Abstract

Fibrosis is an inherent response to chronic damage upon immense apoptosis or necrosis. Transforming growth factor-beta1 (TGF-β1) signaling plays a key role in the fibrotic response to chronic liver injury. To develop anti-fibrotic therapeutics, we synthesized a novel small-molecule inhibitor of the TGF-β type I receptor kinase (ALK5), EW-7197, and evaluated its therapeutic potential in carbon tetrachloride (CCl4) mouse, bile duct ligation (BDL) rat, bleomycin (BLM) mouse, and unilateral ureteral obstruction (UUO) mouse models. Western blot, immunofluorescence, siRNA, and ChIP analysis were carried out to characterize EW-7197 as a TGF-β/Smad signaling inhibitor in LX-2, Hepa1c1c7, NRK52E, and MRC5 cells. In vivo anti-fibrotic activities of EW-7197 were examined by microarray, immunohistochemistry, western blotting, and a survival study in the animal models. EW-7197 decreased the expression of collagen, α-smooth muscle actin (α-SMA), fibronectin, 4-hydroxy-2, 3-nonenal, and integrins in the livers of CCl4 mice and BDL rats, in the lungs of BLM mice, and in the kidneys of UUO mice. Furthermore, EW-7197 extended the lifespan of CCl4 mice, BDL rats, and BLM mice. EW-7197 blocked the TGF-β1-stimulated production of reactive oxygen species (ROS), collagen, and α-SMA in LX-2 cells and hepatic stellate cells (HSCs) isolated from mice. Moreover, EW-7197 attenuated TGF-β- and ROS-induced HSCs activation to myofibroblasts as well as extracellular matrix accumulation. The mechanism of EW-7197 appeared to be blockade of both TGF-β1/Smad2/3 and ROS signaling to exert an anti-fibrotic activity. This study shows that EW-7197 has a strong potential as an anti-fibrosis therapeutic agent via inhibition of TGF-β-/Smad2/3 and ROS signaling.

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Abbreviations

4-HNE:

4-Hydroxy-2,3-nonenal

α-SMA:

Alpha-smooth muscle actin

ALK5:

Activin receptor-like kinase 5

BDL:

Bile duct ligation

BLM:

Bleomycin

CCl4 :

Carbon tetrachloride

ECM:

Extracellular matrix

EW-7197:

N-[[4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl]methyl]-2-fluoroaniline

GOx:

Glucose oxidase

HSCs:

Hepatic stellate cells

IHC:

Immunohistochemistry

NOX:

Nicotinamide adenine dinucleotide phosphate oxidase

Prdx:

Peroxiredoxin

qRT-PCR:

Quantitative real-time reverse-transcriptase polymerase chain reaction

ROS:

Reactive oxygen species

TGF-β:

Transforming growth factor-beta

UUO:

Unilateral ureteral obstruction

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Acknowledgments

This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant, which is funded by the Korean government (MEST) (No. 20090093972). We thank Seung Won Kim, Sol-Ji Kim, Jung In Jee, and Min-Kyung Park for technical and administrative support.

Conflict of interest

The authors have no conflicts of interest.

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    Authors and Affiliations

    1. College of Pharmacy, Ewha Womans University, Seodaemun-gu, Seoul, 120-750, South Korea

      Sang-A Park, Min-Jin Kim, So-Yeon Park, Jung-Shin Kim, Seon-Joo Lee, Hyun Ae Woo, Dae-Kee Kim & Yhun Yhong Sheen

    2. Laboratory of Tumor Suppressor, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, 406-840, South Korea

      Jeong-Seok Nam

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    Correspondence to Yhun Yhong Sheen.

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    S.-A. Park and M.-J. Kim contributed equally to this work.

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    Park, SA., Kim, MJ., Park, SY. et al. EW-7197 inhibits hepatic, renal, and pulmonary fibrosis by blocking TGF-β/Smad and ROS signaling. Cell. Mol. Life Sci. 72, 2023–2039 (2015). https://doi.org/10.1007/s00018-014-1798-6

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