MAO-A通过激活ROS/JNK通路加重心肌细胞脂质沉积  

MAO-A Aggravated the Lipid Deposition in Cardiomyocyte by Activating the ROS/JNK Pathway

作　　者：代地林 吴园 包明威[1] DAI Dilin;WU Yuan;BAO Mingwei(Department of Cardiology,Renmin Hospital of Wuhan University,Hubei Key Laboratory of Cardiology,Hubei 430060,China)

机构地区：[1]武汉大学人民医院心内科、心血管病湖北省重点实验室,430060

出　　处：《医学研究杂志》2023年第9期23-28,34,共7页Journal of Medical Research

基　　金：国家自然科学基金资助项目(81770507,81970438)。

摘　　要：目的观察单胺氧化酶A(monoamine oxidase A,MAO-A)对与3T3-L1脂肪细胞共培养的原代新生大鼠心肌细胞(neonatal rat cardiomyocyte,NRCM)脂质沉积的影响及机制。方法选取40只出生1~3天的SD大鼠心肌细胞与3T3-L1脂肪细胞构建NRCM/3T3-L1共培养体系,并加入棕榈酸(palmitic acid,PA),模拟肥胖时心肌细胞所处的内环境。将细胞分为对照组、PA组、去甲肾上腺素(norepinephrine,NE)组、过氧化氢(hydrogen peroxide,H 2O 2)组、NE+CLG组、氯吉兰(clorgyline,CLG)组。采用α横纹肌肌动蛋白评估心肌细胞的纯度和面积,实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)测定心肌细胞内心房利钠肽(atrial natriuretic peptide,ANP)的mRNA水平,免疫荧光染色测定心肌细胞内活性氧簇水平(reactive oxygen species,ROS),油红O染色评估心肌细胞内脂质沉积,Western blot法测定心肌细胞内MAO-A、JNK的蛋白及磷酸化水平。结果与对照组比较,PA组心肌细胞面积(14.681±0.481μm^(2) vs 19.932±0.220μm^(2),P<0.01)增大,ANP的相对mRNA水平(1.006±0.017 vs 1.914±0.152,P<0.01)、MAO-A的相对蛋白水平(0.117±0.022 vs 0.430±0.092,P<0.01)、ROS水平(0.001±0.001 vs 0.004±0.002,P<0.01)、脂滴面积比(0.001±0.001 vs 0.054±0.002,P<0.01)及p-JNK的相对蛋白水平(0.032±0.007 vs 0.263±0.119,P<0.05)均增加。与PA组比较,NE明显增加心肌细胞内MAO-A的相对蛋白水平(0.430±0.092 vs 1.114±0.091,P<0.01)和ROS水平(0.004±0.002 vs 0.013±0.001,P<0.01)。另外,NE和H 2O 2均能增加心肌细胞脂滴面积比(0.054±0.002 vs 0.106±0.005,P<0.05;0.054±0.002 vs 0.092±0.002,P<0.01)和p-JNK的相对蛋白水平(0.263±0.119 vs 0.969±0.082,P<0.05;0.263±0.119 vs 0.640±0.101,P<0.05)。CLG可明显改善NE诱导的异常。结论MAO-A相对蛋白水平增加会加重与3T3-L1脂肪细胞共培养的心肌细胞内的氧化应激和脂质沉积,其机制可能与ROS/JNK信号级联反应激活有关。Objective To observe the effects and mechanisms of monoamine oxidase A(MAO-A)on lipid deposition in primary neonatal rat cardiomyocyte(NRCM)co-cultured with 3T3-L1 adipocytes.Methods Forty 1-3days old SD rats was used to establish the NRCM/3T3-L1 co-culture system with 3T3-L1 adipocytes,and treated the system with palmitic acid(PA)to mimic the internal environment of cardiomyocyte during obesity.The cells were divided into control group,PA group,norepinephrine(NE)group,hydrogen peroxide(H 2O 2)group,NE+CLG group and clogyline(CLG)group.The purity and area of NRCM was evaluated byα-sarcomeric actin,the mRNA levels of atrial natriuretic peptide(ANP)in NRCM were determined by real-time quantitative polymerase chain reaction(qPCR),the level of reactive oxygen species(ROS)in NRCM was determined by immunofluorescence staining,the content of lipid droplets in NRCM was evaluated by oil red O staining,and the protein and phosphorylation levels of MAO-A,JNK and p-JNK in NRCM were determined by Western blot.Results Compared with the control group,the area of NRCM(14.681±0.481μm^(2) vs 19.932±0.220μm^(2),P<0.01)and the relative mRNA level of ANP(1.006±0.017 vs 1.914±0.152,P<0.01),the relative protein level of MAO-A(0.117±0.022 vs 0.430±0.092,P<0.01),the ROS level(0.001±0.001 vs 0.004±0.002,P<0.01)and the area ratio of lipid droplet(0.001±0.001 vs 0.054±0.002,P<0.01),and the relative protein level of p-JNK(0.032±0.007 vs 0.263±0.119,P<0.05)were increased in PA group.Compared with the PA group,NE significantly increased the relative protein level of MAO-A(0.430±0.092 vs 1.114±0.091,P<0.01)and the level of ROS(0.004±0.002 vs 0.013±0.001,P<0.01)in NRCM.In addition,both NE and H 2O 2 could increase the lipid droplet area ratio of NRCM(0.054±0.002 vs 0.106±0.005,P<0.05;0.054±0.002 vs 0.092±0.002,P<0.01)and relative protein level of p-JNK(0.263±0.119 vs 0.969±0.082,P<0.05;0.263±0.119 vs 0.640±0.101,P<0.05)in NRCM.CLG significantly reversed the abnormalities induced by NE.Conclusion The increased relative

关 键 词：肥胖 MAO-A 氧化应激 脂质沉积 JNK信号通路 

分 类 号：R541.8[医药卫生—心血管疾病]