ASK1 inhibition reduces cell death and hepatic fibrosis in an Nlrp3 mutant liver injury model
Susanne Schuster-Gaul, … , Hal M. Hoffman, Ariel E. Feldstein
Susanne Schuster-Gaul, … , Hal M. Hoffman, Ariel E. Feldstein
Published January 30, 2020
Citation Information: JCI Insight. 2020;5(2):e123294. https://doi.org/10.1172/jci.insight.123294.
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Research Article Hepatology Therapeutics Article has an altmetric score of 3

ASK1 inhibition reduces cell death and hepatic fibrosis in an Nlrp3 mutant liver injury model

Abstract

Hepatic inflammasome activation is considered a major contributor to liver fibrosis in NASH. Apoptosis signal–regulating kinase 1 (ASK1) is an apical mitogen-activated protein kinase that activates hepatic JNK and p38 to promote apoptosis, inflammation, and fibrosis. The aim of the current study was to investigate whether pharmacologic inhibition of ASK1 could attenuate hepatic fibrosis driven by inflammasome activation using gain-of-function NOD-like receptor protein 3 (Nlrp3) mutant mice. Tamoxifen-inducible Nlrp3 knock-in (Nlrp3A350V/+CreT-KI) mice and WT mice were administered either control chow diet or diet containing the selective ASK1 inhibitor GS-444217 for 6 weeks. Livers of Nlrp3-KI mice had increased inflammation, cell death, and fibrosis and increased phosphorylation of ASK1, p38, and c-Jun. GS-444217 reduced ASK1 pathway activation, liver cell death, and liver fibrosis. ASK1 inhibition resulted in a significant downregulation of genes involved in collagen production and extracellular matrix deposition, as well as in a reduced hepatic TNF-α expression. ASK1 inhibition also directly reduced LPS-induced gene expression of Collagen 1A1 (Col1a1) in hepatic stellate cells isolated from Nlrp3-KI mice. In conclusion, ASK1 inhibition reduced liver cell death and fibrosis downstream of inflammatory signaling induced by NLRP3. These data provide mechanistic insight into the antifibrotic mechanisms of ASK1 inhibition.

Authors

Susanne Schuster-Gaul, Lukas Jonathan Geisler, Matthew D. McGeough, Casey D. Johnson, Anna Zagorska, Li Li, Alexander Wree, Vivian Barry, Igor Mikaelian, Lily J. Jih, Bettina G. Papouchado, Grant Budas, Hal M. Hoffman, Ariel E. Feldstein

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Figure 3

ASK1 inhibition significantly reduced liver injury and cell death in Nlrp3-KI mice.

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ASK1 inhibition significantly reduced liver injury and cell death in Nlr...
(A) TUNEL staining showed increased percentage of TUNEL+ cells in Nlrp3-KI mice, and this was ameliorated by GS-444217 (magnification, 20×; scale bar: 100 μm) (WT + vehicle n = 5, WT + ASK1i n = 6, Nlrp3-KI + vehicle n = 5, Nlrp3-KI + ASK1i n = 8). (B)Nlrp3 mutant mice showed higher levels of ALT than WT + vehicle, indicating increased liver cell death, which was reduced by GS-444217. (WT + vehicle n = 10, WT + ASK1i n = 6, Nlrp3-KI + vehicle n = 5, Nlrp3-KI + ASK1i n = 9). (C) Immunoblot analysis of liver lysates of WT and Nlrp3-KI mice treated with vehicle and ASK1i for caspase-3 and its active cleaved form (cl–caspase-3). Data were normalized on GAPDH, and WT + vehicle was set at 1 (WT + vehicle n = 5, WT + ASK1i n = 4, Nlrp3-KI + vehicle n = 5, Nlrp3-KI + ASK1i n = 7). A dashed vertical black line is used to indicate splicing of noncontiguous lanes of the same blot. These experiments were performed at the same time and on the same gels as those shown in Figure 1. (D) H&E staining of liver sections with arrows pointing on areas with liver cell death (magnification, 10×; scale bar: 500 μm). *P < 0.05; **P < 0.01; ***P < 0.001 (1-way ANOVA with Bonferroni post hoc test unless otherwise stated).