SIRT3通过调节线粒体氧化还原平衡增加线粒体生物合成减轻肾小管细胞氧化应激损伤的机制研究

Mechanism study of SIRT3 alleviating oxidative-stress injury in renal tubular cells by promoting mitochondrial biogenesis via regulating mitochondrial redox balance

    摘要
  • 摘要:
    目的  探讨沉默调节蛋白3（SIRT3）对人肾小管上皮细胞线粒体生物合成调控的分子机制。
    方法 通过不同浓度过氧化氢（H2O2）刺激细胞，将细胞分为4组：对照组（NC组）、50 μmol/L H2O2组、110 μmol/L H2O2组、150 μmol/L H2O2组，检测H2O2对SIRT3蛋白表达的影响。使用siRNA转染细胞，敲低SIRT3基因，并将细胞分为5组：对照组（NC组）、阴性对照组（NCsi组）、SIRT3敲低组（siSIRT3组）、NCsi+H2O2组和siSIRT3+H2O2组。24 h后收集各组细胞检测细胞三磷酸腺苷（ATP）、线粒体超氧阴离子（O2•−）水平，线粒体SIRT3、过氧化物酶体增殖物激活受体γ共激活因子-1α（PGC-1α）、核呼吸因子1（NRF1）、线粒体转录因子A（TFAM）、超氧化物歧化酶2（SOD2）、乙酰化SOD2及细胞腺苷酸活化蛋白激酶α1（AMPKα1）的表达水平。
    结果 110 μmol/L和150 μmol/L H2O2能降低细胞SIRT3蛋白表达水平（均为P<0.05）。与NC组比较，NCsi组细胞ATP水平和线粒体O2•−差异均无统计学意义（均为P>0.05）。与NCsi组比较，siSIRT3组O2•−水平升高，SIRT3蛋白表达水平下降，SOD2蛋白和乙酰化SOD2蛋白表达水平升高（均为P<0.05）。与NCsi组比较，NCsi+H2O2组细胞ATP水平下降，线粒体O2•−水平升高，SIRT3、SOD2、TFAM、AMPKα1、PGC-1α和NRF1蛋白表达水平均下降（均为P<0.05）。与siSIRT3组比较，siSIRT3+H2O2组细胞ATP水平下降，线粒体O2•−水平升高，SIRT3、SOD2、TFAM、AMPKα1、PGC-1α和NRF1蛋白表达水平下降，乙酰化SOD2蛋白表达水平下降（均为P<0.05）。与NCsi+H2O2组比较，siSIRT3+H2O2组细胞ATP水平下降，线粒体O2•−水平升高，SIRT3、AMPKα1、PGC-1α和NRF1、TFAM蛋白表达水平下降，SOD2和乙酰化SOD2蛋白表达水平升高（均为P<0.05）。
    结论 SIRT3通过调控AMPK/PGC-1α/NRF1和TFAM信号通路促进肾小管上皮细胞线粒体生物合成，是SIRT3改善氧化应激线粒体功能障碍的重要分子机制。

     

    Abstract:
    Objective To elucidate the molecular mechanism of sirtuin-3 (SIRT3) in regulating mitochondrial biogenesis in human renal tubular epithelial cells.
    Methods Cells were stimulated with different concentrations of H2O2 and divided into four groups: control (NC), 50 μmol/L H2O2, 110 μmol/L H2O2 and 150 μmol/L H2O2. SIRT3 protein expression was then measured. SIRT3 was knocked down with siRNA, and cells were further assigned to five groups: control (NC), negative-control siRNA (NCsi), SIRT3-siRNA (siSIRT3), NCsi+H2O2, and siSIRT3+H2O2. After 24 h, cellular adenosine triphosphate (ATP) and mitochondrial superoxide anion (O2•−) levels were determined, together with mitochondrial expression of SIRT3, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), superoxide dismutase 2 (SOD2), acetylated-SOD2 and adenosine monophosphate activated protein kinase α1 (AMPKα1).
    Results The 110 and 150 μmol/L H2O2 decreased SIRT3 protein (both P<0.05). ATP and mitochondrial O2•− did not differ between NC and NCsi groups (both P>0.05). Compared to the NCsi group, the siSIRT3 group exhibited elevated O2•− level, decreased SIRT3 protein and increased expression levels of SOD2 and acetylated SOD2 protein (all P<0.05). Compared to the NCsi group, the NCsi+H2O2 group exhibited decreased cellular ATP levels, elevated mitochondrial O2•− levels, and reduced protein expression levels of SIRT3, SOD2, TFAM, AMPKα1, PGC-1α and NRF1 (all P<0.05). Compared with the siSIRT3 group, the siSIRT3+H2O2 group showed a decrease in cellular ATP levels, an increase in mitochondrial O2•− levels, a decrease in SIRT3, SOD2, TFAM, AMPKα1, PGC-1α and NRF1 protein expression levels and a decrease in acetylated SOD2 protein expression levels (all P<0.05). Compared with the NCsi+H2O2 group, the siSIRT3+H2O2 group showed a decrease in cellular ATP levels, an increase in mitochondrial O2•− levels, a decrease in SIRT3, AMPKα1, PGC-1α and NRF1, TFAM protein expression levels, and an increase in SOD2 and acetylated SOD2 protein expression levels (all P<0.05).
    Conclusions SIRT3 promotes mitochondrial biogenesis in tubular epithelial cells via the AMPK/PGC-1α/NRF1/TFAM axis, representing a key mechanism through which SIRT3 ameliorates oxidative stress-induced mitochondrial dysfunction.

     

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