Gut Microbiota Metabolite Trimethylamine N-oxide Promotes Atherosclerosis by Inhibiting the Keap1/Nrf2 Signaling Pathway
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 di-vided 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 model-ing,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.Bio-chemical 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.Re-sults: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 inti-ma 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 significant-ly 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 inhibi-ting the activation of the Keap1/Nrf2 signaling pathway.
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