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  

Gut Microbiota Metabolite Trimethylamine N-oxide Promotes Atherosclerosis by Inhibiting the Keap1/Nrf2 Signaling Pathway

作　　者：单思阳 郭凡[2] 杨浩[1] 马洁 张裕祥[1] SHAN Siyang(The Sixth Affiliated Hospital of Xinjiang Medical University,Xinjiang Urumqi 830092,China)

机构地区：[1]新疆医科大学第六附属医院,新疆乌鲁木齐830092 [2]新疆医科大学第三附属医院检验科,新疆乌鲁木齐830054

出　　处：《河北医学》2024年第6期892-899,共8页Hebei Medicine

基　　金：新疆维吾尔自治区卫生健康委员会青年项目,(编号:WJWY-XCZX202217)。

摘　　要：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 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(100μg/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 thes

关 键 词：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 

分 类 号：R543.5[医药卫生—心血管疾病]