Nikolina Canová ^1* Jana Šípková ¹ Mahak Arora ¹ Zuzana Pavlíková ² Tomáš Kučera ² Ondřej Šeda ³ Tijana Šopin ³ Tomáš Vacík ³ Ondřej Slanař ¹

• ¹ Institute of Pharmacology, First Faculty of Medicine, Charles University, Prague, Czechia
• ² Institute of Histology and Embryology, First Faculty of Medicine, Charles University, Prague, Prague, Czechia
• ³ Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University, Prague, Prague, Czechia

Background: The complexity of the galaninergic system is still not fully understood, especially under specific pre-existing comorbidities related to metabolic dysfunction. A plant-derived triterpenoid celastrol was demonstrated to exert a complex effect on the galaninergic system and to have hepatoprotective and anti-obesity properties. However, the exact molecular mechanisms, responsible for these effects remain unclear. Specifically, there are no data on the impact of celastrol on the heart and liver galaninergic system. Therefore, the study aimed to investigate celastrol effects on the galaninergic system expression in the heart and liver of mice suffering from diet-induced obesity and metabolic dysfunction-associated steatotic liver disease and steatohepatitis (MASLD/MASH). Methods: The male mice C57BL/6J were fed a western-type high-fat diet for 16 and 20 weeks to induce obesity and MASLD/MASH. Celastrol was administered along with a specific diet for the last four weeks to evaluate its impact on the progression of these conditions. Moreover, the inhibitor of sterol regulatory element-binding protein 1/2 (SREBP1/2), fatostatin, was also tested to compare its influence on the galaninergic system with celastrol. Results: The study demonstrates that celastrol treatment was safe and led to a reduction in food and energy intake, body, fat and liver weights, MASLD-to-MASH progression, and improved glucose tolerance, serum biochemistry markers, and hepatic lipid peroxidation in mice. Quantitative gene expression originally showed significant regulation of galanin and all its three receptors (GalR1/2/3) in the heart ventricles and only GalR2 in the liver of obese mice. Celastrol influenced the gene expression of galanin receptors: it down-regulated Galr1 in the heart and up-regulated Galr2 in the liver and Galr3 in heart ventricles, potentially affecting energy metabolism, oxidative stress, and inflammation. Fatostatin suppressed gene expression of all detected members of the galaninergic system in heart ventricles depicting the role of SREBP in this process. Conclusion: These findings suggest that celastrol may beneficially modulate the galaninergic system under obesity and MASLD-to-MASH progression, indicating its potential as a therapeutic agent for disorders associated with metabolic dysfunction.