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.

Keywords: NAFLD; eugenol; gut microbiota; olfactory receptor; volatile oils.

Figure 1

SAVO reduces hepatic steatosis in HFD‐fed mice. A) Experimental outline. C57BL/6 J mice were randomly divided into the NC, HFD, silybin, and SAVO‐treated groups (n = 8/group). B) Weight gain rate (grams). C) Food intake was measured once a week. D) Liver weight and the ratio of liver weight to body weight. E) Serum AST, ALT, TG, and TC contents. F) Serum HDL and HDL contents. G) Hepatic TG and TC contents. H) Serum inflammatory factors. I) Representative H&E and Oil Red O staining images of liver sections (original magnification 100x and 200x). B–H, Statistical analysis was performed using one‐way analysis of variance (ANOVA) followed by Dunnett's test. The data are shown as means ± SEMs (n = 8). ^* p < 0.05, ^** p < 0.01. NC, negative control; HFD, high‐fat diet; SAVO‐H, high dose of Syzygium aromaticum volatile oil (2.5 µL/10 g); SAVO‐L, high dose of Syzygium aromaticum volatile oil (1.25 µL/10 g). TNF‐α, tumor necrosis factor‐alpha; IL‐6, interleukin‐6; IL‐1β, interleukin‐1 beta.

Figure 2

Mechanisms of NAFLD suppression by SAVO in mice. A) GO term analysis of all differentially expressed genes based on RNA‐sequencing data. B) Volcano plot analysis of all differentially expressed genes. Pparγ, Olfr544, CPT‐1, and SCD‐1 were labeled. C) mRNA expression of olfactory receptors in the liver (n = 6). D) Representative immunoblotting of Olfr544, PKA, CREB, and pCREB in livers from different groups expressed as fold‐change relative to GAPDH. E) Relative quantification of (D). F) Liver Olfr544 immunohistochemical staining. The red arrow indicates Olfr544 on the cell membrane. Scale bars: 5 and 25 µm. G,H) Relative gene expression in the livers (n = 6). I) GC‐MS total ion flow diagram of volatile components contained in SAVO. J) Mass spectrometry imaging revealed the EUG content in mouse livers at different time points following SAVO inhalation, where EUG is the primary component of SAVO. Data are presented as means ± SEMs (n = 8). ^* p < 0.05, ^** p < 0.01, ns, not significant. NC, negative control; HFD, high‐fat diet; SAVO‐H, high dose of Syzygium aromaticum volatile oil (2.5 µL/10 g); SAVO‐L, high dose of Syzygium aromaticum volatile oil (1.25 µL/10 g).

Figure 3

Olfr544 activation is crucial for EUG to reduce FFA‐induced lipid deposition in Hepa1c1c‐7 cells. A) Intracellular Olfr544 gene expression after treatment with different components. B) Intracellular TG, TC, GSH, and SOD levels. C) Hepa1c1c‐7 cell ORO staining (original magnification 200). D) Intracellular cAMP levels in Hepa1c1c‐7 cells were determined by ELISA. SQ22536 is an inhibitor of adenylyl cyclase, and forskolin is an activator of adenylyl cyclase. E) Representative immunoblotting of Olfr544, PKA, CREB, and pCREB in Hepa1c1c‐7 cells. F) Relative gene expression in Hepa1c1c‐7 cells. G,H) Measurement of lipolysis (G) and FAO activity (H) in Hepa1c1c‐7 cells. I) Olfr544 knockdown in Hepa1c1c‐7 cells transfected with shRNA against Olfr544. The protein level of Olfr544 was determined by immunoblotting. Scr, scrambled shRNA. J) Intracellular cAMP levels in Hepa1c1c‐7 cells. K) Hepa1c1c‐7 cell ORO staining (original magnification 20). L) Relative gene expression in Hepa1c1c‐7 cells with Olfr544 gene knockdown. Data are presented as means ± SEMs (n = 6). ^* p < 0.05, ^** p < 0.01, ns, not significant. NC, negative control; FFA, free fatty acid; EUG‐H, high‐dose EUG (50 µM); EUG‐L, low‐dose EUG (25 µM).

Figure 4

Olfr544 is a receptor of EUG. Bar graphs A) showing the response of Olfr544 to EUG or inhibitor and dose‐response curves of ORs to EUG B). C) Close‐up view of the EUG‐binding site in Olfr544. D) RMSD of EUG‐Olfr544 complexes. E) Morphological changes in EUG‐Olfr544 composite structures during molecular dynamics simulations. F) Rg over the entire simulation. G) RMSF values of the α carbon over the entire simulation. H) Total number of H‐bonds counted over the entire simulation. I) Bar graphs showing the response of Olfr544 and Olfr544 with a point mutation to EUG. J) Dose‐dependent SPR response following EUG injection at varying concentrations. (A, I) The respective highest response value was normalized to 1. Data are presented as means ± SEMs; n = 4.

Figure 5

Inhaling EUG suppresses the evolution of NAFLD in HFD‐fed wild‐type, Olfr544 knockdown, and GF mice. A) Experimental outline. WT mice were randomly divided into NC, HFD, and EUG‐treated groups (n = 6/group) after 8 weeks on a high‐fat diet. B) Weight gain rate of mice. C) Changes in body weight of mice during the experimental period (n = 6/group). The numbers represent interval growth rates. D) Food intake was measured once a week. E) Energy expenditure of mice in different groups, measured using an indirect calorimetry system for 33 h. F) Representative H&E and Oil Red O staining images of liver sections (original magnification 20x and 80s). G) Lipid synthesis, lipolysis, and oxidation relative genes expression in the livers. H) Measurement of lipolysis in livers. I) ‌Fatty acid oxidation assay in hepatocytes isolated from mice treated with HFD or EUG. J) Olfr544 mRNA expression in the liver. K) AAV‐8‐TBG‐m‐shOlfr544‐mediated knockdown of Olfr544 in mouse liver. The protein level of Olfr544 was determined by WB. shScr, scrambled shRNA. L) Weight gain rate of mice. M) Lipid synthesis, lipolysis, and oxidation relative genes expression in the livers. N) Intracellular cAMP levels in mouse hepatocytes. O) Experimental outline. GF mice were randomly divided into NC, HFD, and EUG‐treated groups (n = 6/group). P) Weight gain rate of mice. Q) Serum TC and TG levels. R) Olfr544 mRNA expression in the liver. S) Relative expression of lipolysis, oxidation, and lipid synthesis genes in the liver. Data are presented as means ± SEMs (n = 6). ^* p < 0.05, ^** p < 0.01, ns, not significant.

Figure 6

EUG alters the gut microbiota. A) Shannon index and Sobs α‐diversity indexes. B) Sobs index. C) Principal coordinate analysis (PCoA) of all samples by weighted UniFrac distance. (A–C) n = 6/group. D,E) GMHI and MDI of the fecal microbiota. F) Community barplot analysis of the fecal microbiota composition in HFD and EUG‐treated mice at the genus level. G)Analysis of the relative abundance of taxa among different groups using a LEfSe cladogram. Circle sizes in the cladogram plot are proportional to bacterial abundance. From the outer to the inner circle, the circles represent species, genus, class, order, family, and phylum. LDA ≥ 3.0 and p ≤ 0.05 (by the Wilcoxon rank‐sum test) were regarded as significant. H) LDA scores representing taxonomic data with significant differences between the two groups. Only LDA scores > 3 are shown. Pink indicates taxa enriched in the EUG group. Blue indicates taxa enriched in the NAFLD group. I) Relative abundance of Lactobacillus johnsonii, Lactobacillus reuteri, Lachnospiraceae bacterium 28‐4, Lachnospiraceae bacterium DW59, Helicobacter rodentium, and Faecalibaculum rodentium in HFD and EUG‐treated groups. J) Correlation analysis of serum TG and hepatic SOD levels with the abundance of L. reuteri and L. johnsonii in EUG‐treated mice. K) Correlation analysis between clinical factors and metabolites in EUG‐treated mice using partial Spearman's correlation. ^* p < 0.05, ^** p < 0.01.

Figure 7

EUG‐enriched L. reuteri XR23 and L. johnsonii XR25 ameliorate HFD‐induced NAFLD in mice. A) Schematic of the animal experiment. C57BL/6 J mice were randomly divided into NC, HFD, and bacteria‐treated groups after 8 weeks on a high‐fat diet. Bacteria were enriched, cultured, and then administered to mice by oral gavage. NS represents normal saline. B) Grams of weight gain measured over time and the weight gain rate. C) Fasting blood glucose was measured using a blood glucose dipstick. D) Hepatic GSH content. E) Serum TG and TC contents. F) Energy expenditure of mice in different groups, measured using an indirect calorimetry system for 30 h. G) PCoA of fecal microbiota in mice of different groups. H) Shannon α‐diversity index values. I) Fecal microbiota composition in different groups of mice at the genus and species levels. J) Relative gene expression in the livers. K) Schematic of Hepac1c‐7 cells treated with bacterial supernatants. L) Intracellular TG and TC levels. M) ORO staining pf Hepa1c1c‐7 cells (original magnification 20x). N) Relative gene expression in different treatment groups of Hepa1c1c‐7 cells. Data are presented as means ± SEMs. (A–E, H–J), n = 5/group; (K,L, N), n = 6/group. ^* p < 0.05, ^** p < 0.01, ns, not significant.

Figure 8

The microbial metabolites IPA and 5‐HIAA can ameliorate NAFLD. A) Principal components analysis of fecal metabolites in HFD and bacteria‐treated mice (n = 5). Differences in beta diversity were evaluated by analysis of similarity (ANOSIM). B) Volcano plots showing altered fecal metabolites in the HFD + L. j group and the HFD + L. r group compared to the HFD group. (n = 5 per group; fold‐change < −1.3 or >1.3, p < 0.05). C) Correlation analysis between clinical factors and metabolites using partial Spearman's correlation. D) Intracellular TG and TC contents. E) Relative gene expression in Hepa1c1c‐7 cells. F) Subjects with and without NAFLD were recruited. G) Serum concentrations of 5‐HIAA and IPA in individuals (HC, healthy control, n = 28; NAFLD patients, n = 27). H) Schematic of the animal experiment. Mice were randomly divided into NC, HFD, and IPA or 5‐HIAA‐treated groups. I) Weight gain rate measured over time. J) Serum TG, TC, AST, and ALT contents. K) Relative gene expression in different treatment groups of mice. Statistical analyses were performed using a two‐tailed Wilcoxon test, and the data are presented as means ± SEMs. ^* p < 0.05, ^** p < 0.01.

Figure 9

Schematic diagram of the mechanisms of EUG in suppressing NAFLD. On one hand, inhaling eugenol can promote the decomposition and oxidation of liver fat by activating the olfr544‐cAMP‐PKA pathway. On the other hand, the improved gut microbiota, especially the enriched L. reuteri XR23 and L. johnsonii XR25 can inhibit the synthesis of lipid by producing 5‐HIAA and IPA, which form a synergistic and complementary effect.