Inhaling Eugenol Inhibits NAFLD by Activating the Hepatic Ectopic Olfactory Receptor Olfr544 and Modulating the Gut Microbiota

Abstract Non‐alcoholic fatty liver disease (NAFLD) is a major public health threat with currently limited therapeutic options. Inhalation therapy shows promise for treating metabolic disorders due to rapid absorption and high patient adherence, though relevant medications remain scarce. Eugenol (EUG), the primary component of Syzygium aromaticum volatile oil, emerges as a promising NAFLD inhibitor from lipid‐lowering aromatic Chinese medicine screening. EUG elicited significant anti‐steatotic effects in both cultured hepatocytes and comprehensive high‐fat diet‐induced NAFLD animal models. Mechanistically, EUG targeted the activation of the hepatic ectopic olfactory receptor Olfr544 and up‐regulated its downstream cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element binding protein signaling pathway, which further promoted fat lipolysis and oxidation. This effect is prevented by Olfr544 knockdown models both in vitro and in vivo, with supporting bioinformatics analysis. Moreover, EUG reversed gut microbiota dysbiosis and enriched two probiotic strains L. ruteri XR23 and L. johnsonii XR25 , and oral gavage potently mitigated NAFLD in mice, with the key metabolites 3‐indolepropionic acid (IPA) and 5‐hydroxyindole‐3‐acetic acid (5‐HIAA) inhibiting lipid synthesis. Lower levels of 5‐HIAA and IPA are observed in patients with NAFLD. These results highlight the considerable potential of EUG as an agonist of Olfr544 for treating NAFLD by inhalation.