Hepatoprotective effect of cinnabarinic acid in metabolic dysfunction-associated steatohepatitis

Metabolic dysfunction-associated steatohepatitis (MASH) is an advanced form of metabolic dysfunction-associated steatotic liver disease (MASLD) characterized by accumulation of fats in liver, chronic inflammation, hepatocytic ballooning, and fibrosis. This study investigates role of endogenous Aryl hydrocarbon Receptor (AhR) agonist, cinnabarinic acid (CA) in protection against in vivo mouse model of MASH. To analyze the transcriptomic changes in response to CA treatment, male AhRfl/fl (AhR-floxed) mice were subjected to control diet (CD), high-fat high-fructose high-cholesterol diet (MASH diet, MD), and fed with MASH diet and treated with CA thrice a week for 20-week period. RNA sequencing revealed altered expression of multiple genes involved in fatty acid metabolism and inflammation in response to CA treatment. Gene set enrichment analysis indicated enrichment of AMPK among other kinases. Subsequent immunoblotting and immunofluorescence studies performed on CA-treated livers and primary hepatocytes confirmed activated AMPK signaling. Moreover, in a hepatocyte-specific AhR knockout mice livers (AhR-hKO, constructed by crossing AhRfl/fl with alb-Cre), CA failed to phosphorylate AMPK strongly indicating that CA-induced AMPK mediated protection against lipotoxicity in MASH is dependent on hepatic AhR signaling. Eight-week-old male AhRfl/fl (AhR-floxed) mice were subjected to control diet, CD (10 kcal% fat and matching sucrose), high-fat, high-fructose, high-cholesterol diet termed MASH diet, MD (40 kcal% fat, 20 kcal% fructose, and 2% cholesterol), and MD + cinnabarinic acid (MD + CA) for 20-weeks, ad libitum. For the MD + CA group, 12 mg/kg body weight CA (in 0.2% DMSO) was intraperitoneally injected thrice a week on Mondays, Wednesdays, and Fridays for the entire duration of the study (20-weeks). Mice in the MD group were treated with vehicle (0.2% DMSO + 98.8% saline) via i.p. three days a week (Mondays, Wednesdays, and Fridays) for 20 weeks. At end of the 20-weeks, livers were collected, frozen in liquid nitrogen, and stored at -80°C. RNA was extracted from ~50 mg snap-frozen liver tissue using Trizol and subjected to library preparation for subsequent RNA sequencing.