Aramchol attenuates fibrosis in mouse models of biliary fibrosis and blocks the TGFβ-induced fibroinflammatory mediators in cholangiocytes

Aseem, S. O.; Way, G.; Wang, J.; Zhao, D.; Tai, Y.-L.; Gurley, E.; Zeng, J.; Wang, X.; Hylemon, P. B.; Huebert, R. C.; Sanyal, A. J.; Zhou, H.

doi:10.1101/2024.11.06.621880

bioRxiv · 10.1101/2024.11.06.621880

Aramchol attenuates fibrosis in mouse models of biliary fibrosis and blocks the TGFβ-induced fibroinflammatory mediators in cholangiocytes

Aseem, S. O.·Way, G.·Wang, J.·Zhao, D.·Tai, Y.-L.·Gurley, E.·Zeng, J.·Wang, X.·Hylemon, P. B.·Huebert, R. C.·Sanyal, A. J.·Zhou, H.

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Abstract

BackgroundFibroinflammatory cholangiopathies, such as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), are characterized by inflammation and biliary fibrosis, driving disease-related complications. In biliary fibrosis, cholangiocytes activated by transforming growth factor-{beta} (TGF{beta}) release signals that recruit immune cells to drive inflammation and activate hepatic myofibroblasts to deposit the extracellular matrix (ECM). TGF{beta} regulates stearoyl-CoA desaturase (SCD), an enzyme that catalyzes the synthesis of monounsaturated fatty acids, in stimulating fibroinflammatory lipid signaling. However, the role of SCD or its inhibitor, Aramchol, has not been investigated in biliary fibrosis or TGF{beta}-mediated cholangiocyte activation. Method10-16-week-old multi-drug resistance 2 knockout (Mdr2-/-) and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC) diet-fed mice were orally gavaged daily with Aramchol at 12.5 mg/kg/day for 4 and 3 weeks, respectively. Liver and serum were harvested for the assessment of fibrosis and inflammation. Transformed human cholangiocyte cells (H69) and mouse large biliary epithelial cells (MLEs) were used to test the effects of the SCD inhibitor, Aramchol, at varying doses on TGF{beta}-mediated expression of fibroinflammatory signals and were confirmed in PSC-derived cholangiocytes (PSC-Cs) using ELISA, qPCR, and Western blot analyses. ResultsAramchol treatment of Mdr2-/- mice with established biliary fibrosis (treatment) and DDC diet-induced (prevention) models of cholestatic injury and fibrosis demonstrated significant reductions in both measures of ECM synthesis (mRNA expression of ECM components in the liver), collagen content of the liver (picrosirius red staining and hydroxyproline content) and myofibroblast activation (SMA staining). Il6 and Tnfa were also reduced with Aramchol in the liver. RNA-seq analysis of H69 cells showed that Aramchol co-treatment led to significant inhibition of TGF{beta}-induced hepatic fibrosis pathways while upregulating peroxisome proliferator-activated receptor (PPAR) signaling. SCD expression was significantly increased in TGF{beta}-treated H69 cells (2-fold, p<0.05). Aramchol in a dose-dependent manner significantly attenuated the increased expression of the fibrotic marker, plasminogen activator inhibitor-1 (PAI-1/SERPINE1), and hepatic stellate cell-activating genes (VEGFA and PDGFB) in TGF{beta}-activated H69 and MLEs. Aramchol also markedly reduced the expression of the inflammatory cytokine, interleukin 6 (IL6). SCD siRNA knockdown produced similar results in H69 cells. Furthermore, in PSC-Cs, the expressions of SCD, VEGFA and IL6 were significantly reduced with Aramchol. The expression of the anti-fibroinflammatory factors PPAR and -{gamma} were modestly increased in cholangiocyte cell lines with increased expression of PPAR-responsive genes and increased nuclear binding of DNA PPAR response elements with Aramchol co-treatment compared to TGF{beta} only. ConclusionAramchol, an SCD inhibitor, both attenuates and prevents biliary fibrosis in mouse models of cholestatic injury and fibrosis. This effect is partially due to Aramchol inhibiting TGF{beta}-induced fibroinflammatory mediators in cholangiocytes by upregulating PPAR and -{gamma} expression and activity. These findings, along with Aramchols excellent safety profile in clinical trials, provide the rationale for assessing Aramchol in further clinical studies in patients with biliary fibrosis, particularly PSC, where a treatment is desperately needed.

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Aseem, S. O., Way, G., Wang, J., Zhao, D., Tai, Y.-L., Gurley, E., Zeng, J., Wang, X., Hylemon, P. B., Huebert, R. C., Sanyal, A. J., Zhou, H.. 2024-11-06. Aramchol attenuates fibrosis in mouse models of biliary fibrosis and blocks the TGFβ-induced fibroinflammatory mediators in cholangiocytes. https://doi.org/10.1101/2024.11.06.621880

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