Nitro-fatty acids protect against steatosis and fibrosis during development of nonalcoholic fatty liver disease in mice

Abstract

Background: Nonalcoholic fatty liver disease (NAFLD) and resulting nonalcoholic steatohepatitis (NASH) are reaching global epidemic proportions. Lack of non-invasive diagnostic tools and effective therapies constitute two of the major hurdles for a bona fide treatment and a reversal of NASH progression and/or regression of the disease. Nitro-oleic acid (OA-NO₂) has been proven effective in multiple experimental models of inflammation and fibrosis. Thus, the potential benefit of in vivo administration of OA-NO₂ to treat advanced NAFLD was tested herein in a model of long-term NASH diet-induced liver damage.

Methods: Non-invasive imaging (e.g. photoacustic-ultrasound (PA-US)) was pursued to establish advanced experimental model of NASH in mice in which both steatosis and fibrosis were diagnosed prior experimental therapy with OA-NO₂. Experimental controls included equimolar amounts of the non-nitrated oleic acid (OA). CLAMS and NMR-based analysis was used for energy metabolism.

Findings: CLAMS and NMR-based analysis demonstrates that OA-NO₂ improves body composition and energy metabolism and inhibits hepatic triglyceride (TG) accumulation. Photoacoustic-ultrasound imaging revealed a robust inhibition of liver steatosis and fibrosis by OA-NO₂. RNA-sequencing analysis uncovered inflammation and fibrosis as major pathways suppressed by OA-NO₂ administration, as well as regulation of lipogenesis and lipolysis pathways, with a robust inhibition of SREBP1 proteolytic activation and subsequent lipogenesis gene expression by OA-NO₂. These results were further supported by histological analysis and quantification of lipid accumulation, lobular inflammation (F4/80 staining) and fibrosis (collagen deposition, αSMA staining) as well as established parameters of liver damage (ALT). In vitro studies indicate that OA-NO₂ inhibits TG biosynthesis and accumulation in hepatocytes and inhibits fibrogenesis in human stellate cells.

Interpretation: OA-NO₂ improve steatohepatitis and fibrosis and may constitute an effective therapeutic approach against advanced NAFLD that warrants further clinical evaluation.

Keywords: Nitro-fatty acids; Non-alcoholic Steatohepatitis; Non-alcoholic fatty liver disease; Non-invasive liver imaging, liver fibrosis.