Anti-Inflammatory and Therapeutic Effects of a Novel Small-Molecule Inhibitor of Inflammation in a Male C57BL/6J Mouse Model of Obesity-Induced NAFLD/MAFLD

Abstract

Purpose

Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic (dysfunction) associated fatty liver disease (MAFLD), is the most common chronic liver disease in the United States. Presently, there is an intense and ongoing effort to identify and develop novel therapeutics for this disease. In this study, we explored the anti-inflammatory activity of a new compound, termed IOI-214, and its therapeutic potential to ameliorate NAFLD/MAFLD in male C57BL/6J mice fed a high fat (HF) diet.

Methods

Murine macrophages and hepatocytes in culture were treated with lipopolysaccharide (LPS) ± IOI-214 or DMSO (vehicle), and RT-qPCR analyses of inflammatory cytokine gene expression were used to assess IOI-214’s anti-inflammatory properties in vitro. Male C57BL/6J mice were also placed on a HF diet and treated once daily with IOI-214 or DMSO for 16 weeks. Tissues were collected and analyzed to determine the effects of IOI-214 on HF diet-induced NAFL D/MAFLD. Measurements such as weight, blood glucose, serum cholesterol, liver/serum triglyceride, insulin, and glucose tolerance tests, ELISAs, metabolomics, Western blots, histology, gut microbiome, and serum LPS binding protein analyses were conducted.

Results

IOI-214 inhibited LPS-induced inflammation in macrophages and hepatocytes in culture and abrogated HF diet-induced mesenteric fat accumulation, hepatic inflammation and steatosis/hepatocellular ballooning, as well as fasting hyperglycemia without affecting insulin resistance or fasting insulin, cholesterol or TG levels despite overall obesity in vivo in male C57BL/6J mice. IOI-214 also decreased systemic inflammation in vivo and improved gut microbiota dysbiosis and leaky gut.

Conclusion

Combined, these data indicate that IOI-214 works at multiple levels in parallel to inhibit the inflammation that drives HF diet-induced NAFLD/MAFLD, suggesting that it may have therapeutic potential for NAFLD/MAFLD.

Keywords:

• high fat diet
• toll-like receptor 4
• IOI-214
• metabolomics
• gut microbiome
• lipopolysaccharide

Abbreviations

NAFLD, non-alcoholic fatty liver disease; MAFLD, metabolic (dysfunction) associated fatty liver disease; IOI-214, inhibitor of inflammation-214; HF, high fat; RT-qPCR, reverse transcription-quantitative polymerase-chain reaction; LPS, lipopolysaccharide; TG, triglyceride; LPB, LPS binding protein; IR, insulin resistance; T2DM, type 2 diabetes mellitus; FFA, free fatty acid; VLDL, very low-density lipoprotein; NASH, nonalcoholic steatohepatitis; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; DMEM, Dulbecco’s Modified Eagle Medium; DMEM-F12, DMEM/Nutrient Mixture F-12; IPGTT, intraperitoneal glucose tolerance test; IPITT, intraperitoneal insulin tolerance test; HOMA-IR, homeostatic model assessment of insulin resistance; H&E, hematoxylin and eosin; LC/MS/MS, liquid chromatography/tandem mass spectrometry; UC, University of California; MMPC, Mouse Metabolic Phenotyping Center; PCA, Principal Components Analysis; PLS-DA, partial least squares-discriminant analysis; ELISA, enzyme-linked immunosorbent assay; ANOVA, analysis of variance; HSD, honestly significant difference; TLR4, toll-like receptor 4; Tnf/TNFα, tumor necrosis factor alpha; Ifnb1, interferon beta 1; Mcp1/MCP1, monocyte chemoattractant protein 1; Il1b/IL1β, interleukin 1 beta; Il6/IL6, interleukin 6; Plin5, perilipin 5; Actb/βActin, beta actin; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; CD14, cluster of differentiation 14; Irg1, immune response gene 1; ORO, Oil Red O; OCLN, occludin; ZO1, zona-occludin; GLP-1, glucagon-like peptide-1.

Acknowledgments

The authors would like to thank Metabolon Inc. (Durham, NC, USA) for the generation of the metabolomics data and the UC Davis MMPC (Davis, CA, USA) for the generation of the gut microbiome data. The authors would also like to thank Macie Matta for her assistance with the cell culture experiments involving IOI-214 and Samantha Shaw for her assistance with the gut microbiome study. The authors would also like to thank the Ohio University Heritage College of Osteopathic Medicine Histological Core Services for their assistance with the liver tissue preparation and staining. University Research was supported by NIH grant U24-DK092993 [UC Davis Mouse Metabolic Phenotyping Center (RRID:SCR 015361)]. This research was also supported by the Ohio University Heritage College of Osteopathic Medicine. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Disclosure

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ohio University owns two patents on IOI-214. Kelly D McCall, Frank L Schwartz, Stephen C Bergmeier and Douglas J Goetz are inventors on the patents. Kelly D McCall, Douglas J Goetz and Jean R Thuma report grants from UC Davis Mouse Metabolic Phenotyping Center, during the conduct of the study. All other authors declare that they have no conflicts of interest regarding the publication of this paper.