脂肪酸结合蛋白3对缺氧后心肌细胞存活和凋亡的调节作用
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摘要
目的·探讨脂肪酸结合蛋白3(fatty acid binding protein 3,FABP3)是否参与了缺氧后心肌细胞的存活及其可能的分子机制。方法·分离新生大鼠心肌细胞,使用重组人源FABP3刺激该细胞,利用锥虫蓝实验和MTT法检测缺氧状态下心肌细胞的存活和死亡情况;应用蛋白质印迹检测FABP3对凋亡相关蛋白PARP、caspase 3表达水平的影响;采用免疫荧光染色技术分析缺氧状态下细胞内活性氧(reactive oxygen species,ROS)水平和线粒体膜电位的变化。结果·使用重组人源FABP3刺激心肌细胞可显著增加心肌细胞的死亡数,缺氧后心肌细胞的存活率下降(P=0.021)。FABP3可上调cleaved PARP和cleaved caspase 3的蛋白水平(P=0.006,P=0.002),增加细胞内ROS含量(P=0.038),同时降低线粒体膜电位(P=0.002)。结论·FABP3通过诱导细胞内ROS的产生和线粒体膜电位的降低,参与了缺氧后心肌细胞的存活和凋亡。
Objective·To investigate the effects and potential mechanisms of fatty acid binding protein 3(FABP3) on cell survival under hypoxia.Methods·Neonatal rat ventricular myocytes were stimulated with recombinant human FABP3 proteins, and then the differences of cell viability and cell death between groups were verified by trypan blue assay and MTT assay. Besides, the expression of PARP and caspase 3 protein, the level of reactive oxygen species(ROS), and mitochondrial membrane potentials under hypoxia were compared between groups for more confirmation. Results·FABP3 increased cardiomyocytes' death and decreased cell viability under hypoxia(P=0.021). It was discovered that FABP3 upregulated the levels of cleaved PARP and cleaved caspase 3(P=0.006, P=0.002), increased the level of intracellular ROS(P=0.038), and declined the mitochondrial membrane potentials as well(P=0.002). Conclusion·FABP3 contributes to cell survival and apoptosis by regulating intracellular ROS and mitochondrial membrane potentials under hypoxia.
Objective·To investigate the effects and potential mechanisms of fatty acid binding protein 3(FABP3) on cell survival under hypoxia.Methods·Neonatal rat ventricular myocytes were stimulated with recombinant human FABP3 proteins, and then the differences of cell viability and cell death between groups were verified by trypan blue assay and MTT assay. Besides, the expression of PARP and caspase 3 protein, the level of reactive oxygen species(ROS), and mitochondrial membrane potentials under hypoxia were compared between groups for more confirmation. Results·FABP3 increased cardiomyocytes' death and decreased cell viability under hypoxia(P=0.021). It was discovered that FABP3 upregulated the levels of cleaved PARP and cleaved caspase 3(P=0.006, P=0.002), increased the level of intracellular ROS(P=0.038), and declined the mitochondrial membrane potentials as well(P=0.002). Conclusion·FABP3 contributes to cell survival and apoptosis by regulating intracellular ROS and mitochondrial membrane potentials under hypoxia.
引文
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[2]Del Collado M,da Silveira JC,Sangalli JR,et al.Fatty acid binding protein 3and transzonal projections are involved in lipid accumulation during in vitro maturation of bovine oocytes[J].Sci Rep,2017,7(1):2645.
[3]Haunerland NH,Spener F.Fatty acid-binding proteins:insights from genetic manipulations[J].Prog Lipid Res,2004,43(4):328-349.
[4]Smathers RL,Petersen DR.The human fatty acid-binding protein family:evolutionary divergences and functions[J].Hum Genomics,2011,5(3):170-191.
[5]Yang Y,Spitzer E,Kenney N,et al.Members of the fatty acid binding protein family are differentiation factors for the mammary gland[J].J Cell Biol,1994,127(4):1097-1109.
[6]Glatz JF,van der Vusse GJ,Simoons ML,et al.Fatty acid-binding protein and the early detection of acute myocardial infarction[J].Clin Chim Acta,1998,272(1):87-92.
[7]Reiter M,Twerenbold R,Reichlin T,et al.Heart-type fatty acid-binding protein in the early diagnosis of acute myocardial infarction[J].Heart,2013,99(10):708-714.
[8]Xu LQ,Yang YM,Tong H,et al.Early diagnostic performance of heart-type fatty acid binding protein in suspected acute myocardial infarction:evidence from a meta-analysis of contemporary studies[J].Heart Lung Circ,2018,27(4):503-512.
[9]Liou K,Ho S,Ooi SY.Heart-type fatty acid binding protein in early diagnosis of myocardial infarction in the era of high-sensitivity troponin:a systematic review and meta-analysis[J].Ann Clin Biochem,2015,52(Pt 3):370-381.
[10]Gerede DM,Gulec S,Kilickap M,et al.Comparison of a qualitative measurement of heart-type fatty acid-binding protein with other cardiac markers as an early diagnostic marker in the diagnosis of non-ST-segment elevation myocardial infarction[J].Cardiovasc J Afr,2015,26(6):204-209.
[11]Zhuang L,Li C,Chen Q,et al.Fatty acid-binding protein 3 contributes to ischemic heart injury by regulating cardiac myocyte apoptosis and MAPKpathways[J].Am J Physiol Heart Circ Physiol,2019,316(5):H971-H984.
[12]Green DR,Reed JC.Mitochondria and apoptosis[J].Science,1998,281(5381):1309-1312.
[13]Wang C,Youle RJ.The role of mitochondria in apoptosis[J].Annu Rev Genet,2009,43:95-118.
[14]Li MX,Dewson G.Mitochondria and apoptosis:emerging concepts[J].F1000Prime Rep,2015,7:42.
[15]Ly JD,Grubb D,Lawen A.The mitochondrial membrane potential(Δψm)in apoptosis;an update[J].Apoptosis,2003,8(2):115-128.
[16]Sukumar M,Liu J,Mehta GU,et al.Mitochondrial membrane potential identifies cells with enhanced stemness for cellular therapy[J].Cell Metab,2016,23(1):63-76.
[17]Gottlieb E,Armour S,Harris M,et al.Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis[J].Cell Death Differ,2003,10(6):709.
[18]Wang S,Zhou Y,Andreyev O,et al.Overexpression of FABP3 inhibits human bone marrow derived mesenchymal stem cell proliferation but enhances their survival in hypoxia[J].Exp Cell Res,2014,323(1):56-65.
[19]Zhu C,Hu DL,Liu YQ,et al.Fabp3 inhibits proliferation and promotes apoptosis of embryonic myocardial cells[J].Cell Biochem Biophys,2011,60(3):259-266.
[20]Song GX,Shen YH,Liu YQ,et al.Overexpression of FABP3 promotes apoptosis through inducing mitochondrial impairment in embryonic cancer cells[J].J Cell Biochem,2012,113(12):3701-3708.