Mixed-lineage kinase 3 pharmacological inhibition attenuates murine nonalcoholic steatohepatitis
Kyoko Tomita, Rohit Kohli, Brittany L. MacLaurin, Petra Hirsova, Qianqian Guo, Luz H. Gutierrez Sanchez, Harris A. Gelbard, Burns C. Blaxall, Samar H. Ibrahim
Kyoko Tomita, Rohit Kohli, Brittany L. MacLaurin, Petra Hirsova, Qianqian Guo, Luz H. Gutierrez Sanchez, Harris A. Gelbard, Burns C. Blaxall, Samar H. Ibrahim
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Research Article Hepatology

Mixed-lineage kinase 3 pharmacological inhibition attenuates murine nonalcoholic steatohepatitis

Abstract

With the increase in obesity worldwide, its associated comorbidities, including nonalcoholic steatohepatitis (NASH), have become a public health problem that still lacks effective therapy. We have previously reported that mixed-lineage kinase 3–deficient (MLK3-deficient) mice are protected against diet-induced NASH. Given the critical need to identify new therapeutic agents, we sought to examine whether the small-molecule MLK3 inhibitor URMC099 would be effective in reversing diet-induced murine NASH. C57BL/6J mice were fed either a diet high in saturated fat, fructose, and cholesterol (FFC), or a chow diet for 24 weeks. Mice were treated with either URMC099 (10 mg/kg) twice daily by intraperitoneal injection or its vehicle during the last 2 weeks of the feeding study. FFC-fed mice receiving URMC099 had similar body weight, caloric intake, homeostatic model assessment of insulin resistance, metabolic phenotype, and hepatic steatosis compared with vehicle-treated mice. Furthermore, FFC-fed mice treated with URMC099 had less hepatic macrophage infiltration, activation, and proinflammatory polarization, as well as less liver injury and fibrosis when compared with vehicle-treated mice. In conclusion, URMC099 is well tolerated in mice without obvious toxicities and appears to be efficacious in reversing diet-induced NASH. Hence, URMC099 may serve as a therapeutic agent in human NASH.

Authors

Kyoko Tomita, Rohit Kohli, Brittany L. MacLaurin, Petra Hirsova, Qianqian Guo, Luz H. Gutierrez Sanchez, Harris A. Gelbard, Burns C. Blaxall, Samar H. Ibrahim

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Figure 5

URMC099-treated mice are protected against FFC diet–induced liver injury and fibrosis.

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URMC099-treated mice are protected against FFC diet–induced liver injury...
WT C57BL/6J mice were fed either chow or a diet high in saturated fat, fructose, and cholesterol (FFC) for 24 weeks, and URMC099 or vehicle (Veh) was given during the last 2 weeks. (A) Serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), bile acid, and total bilirubin were measured (n = 4–7). (B) Hepatocyte apoptosis was assessed by the TUNEL assay. Brown-stained apoptotic nuclei (black arrowheads) were quantified by counting nuclei in 10 random ×20 microscopic fields, and averaged for each animal (n = 3). Scale bars: 50 μm. (C) Huh7 cells were treated with either Veh or 20 μM lysophosphatidylcholine (LPC) with or without URMC099 for 16 hours. Apoptosis was assessed by DAPI staining and counting of apoptotic nuclei. (D) Fixed liver tissue sections were stained by Sirius red. Sirius red chromogen was quantified by morphometry using ImageJ software in 15 random ×20 microscopic fields, and averaged for each animal (n = 3.) Scale bars: 50 μm. (E) Frozen liver tissue sections were examined by second harmonic generation (SHG) microscopy using a ×25 objective. Scale bars: 50 μm. Total RNA was extracted from the liver tissues. The mRNA expression of (F) the profibrogenic markers osteopontin, collagen 1α1, and TIMP1 were evaluated by real-time PCR (n = 3–5). Fold change was determined after normalization to GAPDH mRNA expression, and expressed relative to that observed in chow-fed, vehicle-treated mice. Data represent mean ± SEM. Differences between the groups were compared using 1-way ANOVA followed by Bonferroni’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. ns, non-significant.