肠道菌群代谢产物氧化三甲胺通过抑制Keap1/Nrf2信号通路激活发挥促动脉粥样硬化作用OACSTPCD
Gut Microbiota Metabolite Trimethylamine N-oxide Promotes Atherosclerosis by Inhibiting the Keap1/Nrf2 Signaling Pathway
目的:研究肠道菌群(GM)代谢产物氧化三甲胺(TMAO)对动脉粥样硬化(AS)的影响及其相关机制.方法:按照随机数字表法将雄性小鼠分为对照组、模型组、TMAO 组、Keap1/Nrf2 激动剂RTA-408 组和TMAO+RTA-408 组,每组12 只.其中,模型组小鼠采用高脂饲料喂养,TMAO组小鼠在高脂饲料中加1%胆碱,造模周期为12 周.造模结束后,RTA-408 组和TMAO+RTA-408 组小鼠每天腹腔单次注射RTA-408(100μg/kg),持续给药 14d,期间其他各组小鼠腹腔注射等量的生理盐水.采用生化分析法定量测定TG、TC、LDL-C和HDL-C的水平.通过HE、Masson三色和油红O染色检测主动脉的组织学改变.通过 ELISA 检测血清中白细胞介素-1β(IL-1β)、活性氧(ROS)和超氧化物歧化酶(SOD)的水平.超高液相色谱串联质谱法(UHPLC-MS/MS)检测小鼠血浆中 TMAO 含量;荧光探针法检测主动脉 ROS的荧光强度;qRT-PCR、Western blot分别检测小鼠主动脉组织中Keap1、Nrf2、HO-1 的mRNA和蛋白表达水平;免疫荧光观察Nrf2 的核易位情况.结果:AS 小鼠血清 TC、TG、LDL-C 浓度相较于对照组升高,HDL-C浓度则降低(P<0.01).此外,模型组显示广泛的主动脉内膜增厚,明显的泡沫细胞形成,动脉壁胶原沉积增加.此外,血清中 IL-1β、ROS 和 TMAO 水平显著升高(P<0.01),SOD活性显著降低(P<0.01),主动脉中ROS含量增加、Nrf2 核转位显著抑制(P<0.01),Keap1、Nrf2 和HO-1 mRNA与蛋白表达水平升高(P<0.01).与 AS 小鼠相比,TMAO 处理进一步加重对应指标上述变化趋势(P<0.05);RTA-408 则取消TMAO对AS小鼠的加重作用(P<0.05).结论:TMAO可能通过抑制Keap1/Nrf2 信号通路激活对AS小鼠的主动脉病理改变、炎症反应和内皮损伤发挥加重作用.
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.
单思阳;郭凡;杨浩;马洁;张裕祥
新疆医科大学第六附属医院,新疆 乌鲁木齐 830092新疆医科大学第三附属医院检验科,新疆 乌鲁木齐 830054
动脉粥样硬化肠道菌群代谢产物氧化三甲胺Keap1/Nrf2信号通路
AtherosclerosisGut microbiome metabolitesTrimethylamine-N-oxideKeap1/Nrf2 signaling pathway
《河北医学》 2024 (006)
892-899 / 8
新疆维吾尔自治区卫生健康委员会青年项目,(编号:WJWY-XCZX202217)
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