**Background:** Cardiovascular disease (CVD) is a leading cause of death globally, and the gut microbiota-derived metabolite trimethylamine-N-oxide (TMAO) has emerged as a key risk factor. TMAO is produced from dietary L-carnitine through a meta-organismal pathway involving gut bacteria and host liver enzymes, and it promotes atherosclerosis. Ginger (Zingiber officinale) and its essential oil (GEO) have shown cardioprotective and anti-inflammatory properties, but their effects on TMAO-mediated atherosclerosis were unknown. This study aimed to investigate the anti-atherosclerotic effects and mechanisms of GEO and its major bioactive compound citral in a mouse model of diet-induced atherosclerosis.
**Methods:** Eight-week-old female ApoE−/− mice were fed either a control diet (10 kcal% fat) or a Gubra Amylin NASH (GAN) diet (40 kcal% fat, 20 kcal% fructose, 2% cholesterol) with 1.3% L-carnitine in drinking water (GC group) for 16 weeks. The GC-fed mice were further divided into four groups (n=8 each): GC alone, GC + low-dose GEO (50 mg/kg bw/day), GC + high-dose GEO (100 mg/kg bw/day), and GC + citral (20 mg/kg bw/day). GEO was extracted by steam distillation (citral content ~31%). After 16 weeks, aortic atherosclerotic lesions were quantified by oil red O staining. Plasma lipids, glucose, insulin, hepatic enzymes (AST, ALT), and inflammatory cytokines (TNF-α, IL-6, IL-1β) were measured using commercial kits. Plasma TMA, TMAO, γ-butyrobetaine (γ-BB), and carnitine were quantified by LC-MS/MS. Fecal gut microbiota was analyzed by V3-V4 16S rRNA sequencing using the QIIME2 pipeline and SILVA database.
**Key Results:** The GC diet successfully induced atherosclerosis, with aortic lesion area increasing by 212% (13.1±2.4%) compared to controls (4.2±1.2%, p<0.0001). Low-dose GEO, high-dose GEO, and citral significantly reduced aortic lesions by 23% (p=0.0311), 20% (p=0.0610), and 29% (p=0.0043), respectively. GC feeding elevated plasma total cholesterol, HDL-C, and LDL-C (all p<0.001 vs. CON), while GEO and citral increased HDL-C (high-dose GEO p=0.0015; citral p=0.0054). GC also increased plasma glucose (p=0.0048) and HOMA-IR (p=0.0554), which were significantly improved by GEO and citral (e.g., citral reduced glucose p=0.0007, HOMA-IR p=0.0018). Hepatic enzymes AST and ALT were elevated in GC (p<0.0001 and p=0.0319, respectively), and ALT was reduced by citral (p=0.0491). Inflammatory cytokines TNF-α (p<0.0001), IL-6 (p=0.0149), and IL-1β (p=0.0638) were higher in GC; citral significantly reduced TNF-α (p=0.0212) and IL-1β (p=0.0520), while GEO reduced IL-1β (low-dose p=0.0413). Plasma TMA, TMAO, γ-BB, and carnitine were markedly increased in GC (TMA: 11.2-fold, p=0.0032; TMAO: 4.1-fold, p=0.0135; γ-BB: 43.3-fold, p<0.0001; carnitine: 1.9-fold, p<0.0001). Both GEO doses and citral significantly reduced TMA and TMAO levels (p=0.0398 and p=0.0202 for low and high GEO, respectively). Gut microbiota analysis revealed that GC reduced α-diversity (observed ASVs p=0.0003) and shifted β-diversity (ANOSIM R=0.6812, p<0.001). GC enriched CVD-related genera (Enterorhabdus, Romboutsia, Proteus, Escherichia-Shigella) and decreased beneficial bacteria (Bifidobacterium, Alistipes). GEO and citral partially reversed these changes, increasing beneficial genera such as Allobaculum, Akkermansia, and Dubosiella. Spearman correlation showed that Enterohabdus was positively correlated with plasma TMAO, while Bifidobacterium and Alistipes were negatively correlated with aortic lesions.
**Clinical Implications:** This study demonstrates that GEO and citral can ameliorate atherosclerosis in a mouse model by suppressing the gut microbiota-host TMAO pathway, reducing systemic inflammation, improving insulin resistance, and favorably modulating gut microbiota composition. These findings suggest that GEO and citral have potential as dietary supplements for the prevention of cardiovascular disease, particularly in individuals with high dietary L-carnitine intake. However, human studies are needed to confirm efficacy and safety.