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 6

URMC099 reduces macrophage activation and migration under lipotoxic conditions.

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URMC099 reduces macrophage activation and migration under lipotoxic cond...
(A) Bone marrow–derived macrophages (BMDMs) (n = 3) were incubated with either vehicle (Veh), 10 μM lysophosphatidylcholine (LPC), or 100 ng/ml lipopolysaccharide (LPS), with or without 100 nM URMC099. (B) Kupffer cells (n = 6–9) and (C) THP-1 cells (n = 3) were treated with either Veh or LPC (10 μM) with or without 100 nM URMC099. After a 4- to 6-hour treatment, total RNA was extracted and mRNA expression for IL-1β, TNF-α, and MCP1 was assessed by quantitative real-time PCR. Fold change was determined after normalization to GAPDH expression and expressed relative to that observed in vehicle-treated condition. (D) After a 30-minute 10 μM LPC treatment, nuclear localization of p65 in BMDMs was assessed by immunocytochemistry and confocal microscopy. Scale bars: 50 μm. The p65-positive nuclei were quantified in 10 random microscopic fields. (E) Primary mouse hepatocytes (PMHs) or Huh7 cells were treated overnight with either Veh, LPC, with or without 1 μM URMC099, and the treatment media were replaced with serum-free media in the morning. Fluorescently labeled BMDMs or THP-1 cells were seeded on the cell culture inserts; migrated cells were quantified by the Celigo Imaging Cytometer (n = 3). 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.