Abstract：Objective: To investigate the effects of the gut microbiota (GM) metabolite trimethylamine N-oxide (TMAO) on atherosclerosis (AS) and its related mechanisms.Methods: Male mice were randomly divided into the control group, model group, TMAO group, Keap1/Nrf2 activator RTA-408 group, and TMAO+RTA-408 group, with 12 mice in each group. The model group mice were fed a high-fat diet, while the TMAO group mice were fed a high-fat diet with 1% choline for a modeling period of 12 weeks. After modeling, mice in the RTA-408 and TMAO+RTA-408 groups received a single daily intraperitoneal injection of RTA-408 (100 μg/kg) for 14 days, while mice in other groups received equivalent amounts of saline. Biochemical analysis was used to quantify the levels of TG, TC, LDL-C, and HDL-C. Histological changes in the aorta were detected using HE, Masson trichrome, and oil red O staining. ELISA was used to detect serum levels of interleukin-1β (IL-1β), reactive oxygen species (ROS), and superoxide dismutase (SOD). Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to detect plasma TMAO levels in mice. Fluorescent probe assays were used to detect ROS fluorescence intensity in the aorta. qRT-PCR and Western blot were used to detect mRNA and protein expression levels of Keap1, Nrf2, and HO-1 in mouse aortic tissue. Immunofluorescence was used to observe Nrf2 nuclear translocation.Results: Serum TC, TG, and LDL-C concentrations were higher in AS mice compared to the control group, while HDL-C concentration was lower (P<0.01). Additionally, the model group showed extensive aortic intima thickening, significant foam cell formation, and increased collagen deposition in the arterial wall. Serum levels of IL-1β, ROS, and TMAO were significantly elevated (P<0.01), while SOD activity was significantly reduced (P<0.01). Aortic ROS content increased, Nrf2 nuclear translocation was significantly inhibited (P<0.01), and mRNA and protein expression levels of Keap1, Nrf2, and HO-1 increased (P<0.01). Compared to AS mice, TMAO treatment further aggravated the changes in these indicators (P<0.05); RTA-408, however, negated the exacerbating effects of TMAO on AS mice (P<0.05).Conclusion: TMAO may exacerbate aortic pathological changes, inflammatory responses, and endothelial injury in AS mice by inhibiting the activation of the Keap1/Nrf2 signaling pathway.
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2024, Vol. 30 
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