ObjectiveThe gut microbiota-derived metabolite, trimethylamine N-oxide (TMAO) plays an important role in cardiovascular disease (CVD). The fasting plasma TMAO was shown as a prognostic indicator of CVD incident in patients and raised the interest of intervention targeting gut microbiota. Here we develop a clinically applicable method called oral carnitine challenge test (OCCT) for TMAO-related therapeutic drug efforts assessment and personalising dietary guidance.DesignA pharmacokinetic study was performed to verify the design of OCCT protocol. The OCCT was conducted in 23 vegetarians and 34 omnivores to validate gut microbiota TMAO production capacity. The OCCT survey was integrated with gut microbiome, host genotypes, dietary records and serum biochemistry. A humanised gnotobiotic mice study was performed for translational validation.ResultsThe OCCT showed better efficacy than fasting plasma TMAO to identify TMAO producer phenotype. The omnivores exhibited a 10-fold higher OR to be high TMAO producer than vegetarians. The TMAO-associated taxa found by OCCT in this study were consistent with previous animal studies. The TMAO producer phenotypes were also reproduced in humanised gnotobiotic mice model. Besides, we found the faecal CntA gene was not associated with TMAO production; therefore, other key relevant microbial genes might be involved. Finally, we demonstrated the urine TMAO exhibited a strong positive correlation with plasma TMAO (r=0.92, p<0.0001) and improved the feasibility of OCCT.ConclusionThe OCCT can be used to identify TMAO-producer phenotype of gut microbiota and may serve as a personal guidance in CVD prevention and treatment.Trial registration numberNCT02838732; Results.

Cardiovascular disease (CVD) is strongly associated with the gut microbiota and its metabolites, including trimethylamine-N-oxide (TMAO), formed from metaorganismal metabolism of ʟ-carnitine. Raw garlic juice, with allicin as its primary compound, exhibits considerable effects on the gut microbiota. This study validated the benefits of raw garlic juice against CVD risk via modulation of the gut microbiota and its metabolites. Allicin supplementation significantly decreased serum TMAO in ʟ-carnitine-fed C57BL/6 J mice, reduced aortic lesions, and altered the fecal microbiota in carnitine-induced, atherosclerosis-prone, apolipoprotein E-deficient (ApoE−/−) mice. In human subjects exhibiting high-TMAO production, raw garlic juice intake for a week reduced TMAO formation, improved gut microbial diversity, and increased the relative abundances of beneficial bacteria. In in vitro and ex vivo studies, raw garlic juice and allicin inhibited γ-butyrobetaine (γBB) and trimethylamine production by the gut microbiota. Thus, raw garlic juice and allicin can potentially prevent cardiovascular disease by decreasing TMAO production via gut microbiota modulation.

Diet and gut microbiota contribute to non-alcoholic steatohepatitis (NASH) progression. High-fat diets (HFDs) change gut microbiota compositions and induce gut dysbiosis and intestinal barrier leakage, which facilitates portal influx of pathogen-associated molecular patterns [e.g., lipopolysaccharides (LPS)] to the liver and triggers inflammation in NASH. The current therapeutic drugs for NASH have adverse side effects; however, several foods and herbs have exhibited hepatoprotection. We investigated ginger essential oil (GEO) against palm oil-containing HFDs in LPS-injected murine NASH model. GEO prevented NASH development by increasing plasma alanine aminotransferase levels; increasing antioxidant catalase, glutathione reductase, and glutathione levels; and reducing hepatic pro-inflammatory cytokine levels. GEO alleviated hepatic inflammation through mediated NLR family pyrin domain-containing 3 inflammasome and LPS/Toll-like receptor 4 signaling pathways. GEO further increased beneficial bacterial abundance and reduced NASH-associated bacterial abundance. GEO may, therefore, prevent NASH by inhibiting hepatic inflammation and modulating the gut microbiota and its metabolite-related pathway.

This study demonstrates that even with limited exposure in early life, a residual dose of tylosin might cause long-lasting metabolic disturbances by altering the gut microbiota and its metabolites. Our findings reveal that the gut microbiota is susceptible to previously ignored environmental factors.

A high-fat diet (HFD) animal model has an indispensable role in the research of gut-liver axis by generating an imbalance of gut microbiota and intestinal leakage, resulting in translocation of harmful bacterial metabolites and lipopolysaccharides (LPSs) to the liver, triggering immune responses, and stimulating non-alcoholic steatohepatitis (NASH). Replacing trans-fat with palm oil in HFDs is an emerging animal model with human translatability in terms of liver biopsy phenotype and transcriptome changes. We developed the NASH and fibrosis mouse model using a palm oil-containing high-fat diet (P-HFD) combined with LPS to simulate gut dysbiosis and endotoxemia and examined the role of carbon tetrachloride (CCl4) on the gut-liver axis. The mice were fed P-HFD with or without intraperitoneal LPS/CCl4 injections. NASH/fibrosis progression and gut microbiota shift were examined at 4, 8, 12, 16, 20, and 24 weeks (n = 192). The P-HFD animal model showed obesogenic and metabolic changes, hepatomegaly, NASH phenotype, increased gut dysbiosis, changes in gut microbiota and its function, reduced beneficial bacteria, increased pathogenic related microbiome, intestinal leakage, and endotoxemia. P-HFD with LPS supplementation resulted in comparable NASH phenotypes compared with the P-HFD group, but worsened the degree of intestinal microbiota dysbiosis by increasing the occurrence of pathogenic bacteria and reducing beneficial microbiota. P-HFD with CCl4 resulted in a liver fibrosis phenotype and produced a more severe gut microbiota shift. These mouse models could be informative for a researcher focused on the gut-liver axis and could be used as valuable tool for preclinical drug testing.

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Gut microbiota and its metabolites, along with host metabolism of ʟ-carnitine, play a crucial role in cardiovascular disease (CVD) development, forming Trimethylamine-N-oxide (TMAO), an atherosclerosis risk factor. TMAO promotes the formation of atherosclerotic plaques and platelet aggregation potential, causing thrombosis. A high-fat diet and carnitine administration can accelerate CVD progression. Ginger (Zingiber officinale Roscoe) essential oil (GEO) and its bioactive compound citral have lipid lowering and anti-inflammatory effects, which may prevent CVD; however, their ability to prevent atherosclerosis through gut microbiota modulation remains to be elucidated. Furthermore, the Gubra Amylin NASH (GAN) diet is a palm oil-containing high-fat diet for inducing steatohepatitis; however, the study of the GAN diet in combination with ʟ-carnitine for inducing atherosclerosis in mouse model has not been investigated yet. We examined the CVD-protecting effect of GEO and citral against the formation of aortic atherosclerosis and linked them with changes in the gut microbiota and its metabolites in the ʟ-carnitine/GAN diet-treated apolipoprotein E-deficient (ApoE−/−) mouse model. GEO and citral demonstrated CVD protective function by alleviating aortic atherosclerotic lesions. They reduced blood sugar, improved insulin resistance, decreased plasma TMAO levels, and inhibited serum inflammatory cytokines, especially interleukin-1β. Moreover, they demonstrated their ability to modulate gut microbiota diversity and composition into a favourable direction. Collectively, GEO and citral may serve as potential prebiotics for CVD prevention by improving dysbiosis.

Recently, the role of the gut microbiota in diseases, including cardiovascular disease (CVD), has gained considerable research attention. Trimethylamine-N-oxide (TMAO), which is formed during ʟ-carnitine metabolism, promotes the formation of atherosclerotic plaques, causing thrombosis. Here, we elucidated the anti-atherosclerotic effect and mechanism of ginger (Zingiber officinale Roscoe) essential oil (GEO) and its bioactive compound citral in Gubra Amylin NASH (GAN) diet with ʟ-carnitine-induced atherosclerosis female ApoE−/− mice. Treatment with GEO at both low and high doses and citral inhibited the formation of aortic atherosclerotic lesions, improved plasma lipid profile, reduced blood sugar, improved insulin resistance, decreased plasma TMAO levels, and inhibited plasma inflammatory cytokines, especially interleukin-1β. Additionally, GEO and citral treatment modulated gut microbiota diversity and composition by increasing the abundance of beneficial microbes and decreasing the abundance of CVD-related microbes. Overall, these results showed that GEO and citral may serve as potential dietary supplements for CVD prevention by improving gut microbiota dysbiosis.