对乙酰氨基酚通过过氧化物酶体增殖物激活受体γ共激活因子1α信号通路影响HepaRG细胞线粒体新生

Effect of acetaminophen on mitochondrial biogenesis via PGC-1α pathway in HepaRG cells

目的探讨对乙酰氨基酚(APAP)对HepaRG细胞过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1α)信号通路介导的线粒体新生的影响。方法接种HepaRG细胞并给予生长培养基,待细胞长满后,替换为分化培养基进行诱导分化,每天观察细胞形态并拍照。APAP(0.125,0.25,0.5,1,2,4,8和12 mmol·L^(-1))处理诱导分化后的HepaRG细胞24和48 h,MTT法测定细胞存活率。Western蛋白印迹法检测细胞线粒体新生相关蛋白PGC-1α、核呼吸因子2(NRF-2)和线粒体转录因子A(TFAM),以及线粒体构成蛋白NADH脱氢酶亚基1(ND-1)的表达。结果诱导分化后显微镜下可见肝细胞样和胆管细胞样2种形态的细胞。与正常对照组相比,APAP作用24和48 h后,随APAP浓度的增加,细胞存活率不断降低,其IC_(50)分别5.64和2.65 mmol·L^(-1)。与正常对照组相比,APAP作用24 h,0.5,1,2和4 mmol·L^(-1)组PGC-1α表达水平显著增加(P<0.01),8 mmol·L^(-1)组显著降低(P<0.01);0.5 mmol·L^(-1)组NRF-2表达水平显著增加(P<0.05),2,4和8 mmol·L^(-1)组显著降低(P<0.01);1 mmol·L^(-1)组TFAM表达水平显著增加(P<0.05),4和8 mmol·L^(-1)组显著降低(P<0.01);0.5,1,2和4 mmol·L^(-1)组ND-1表达水平显著增加(P<0.01),8 mmol·L^(-1)组显著降低(P<0.01)。结论 APAP可诱导或抑制HepaRG细胞的线粒体新生,其机制可能与PGC-1α通路蛋白表达有关。

OBJECTIVE To observe the effect of acetaminophen（APAP） on mitochondrial biogenesis regulated by peroxisome proliferator activated receptor-γ coactivator 1α（PGC-1α） pathway in HepaRG cells.METHODS HepaRG cells were seeded and cultured with growth medium, which was replaced by differential medium after confluence.The morphology of cells was daily observed and photographed.Cell viability was tested using MTT method after cells were exposed to APAP 0.125, 0.25, 0.5, 1, 2, 4, 8and 12 mmol·L^-1for 24 and 48 h, respectively.Protein expression of PGC-1α, nuclear respiratory factor-2（NRF-2）, mitochondrial transcription factor A（TFAM） pathway and subunit NADH dehydrogenase subunit1（ND-1） of mitochondrial respiratory chain complex Ⅰ was detected by Western blotting after exposure at different concentrations for 24 h.RESULTS Two types of cells, hepatocyte-like and biliarylike cel s, were observed by microscopy.Compared with normal control, cell viability at 24 h and 48 h was inhibited in a concentration-dependent manner.IC_（50） was 5.64 and 2.65 mmol·L^-1, respectively.After24 h exposure, protein expression of PGC-1α increased significantly（P〈0.01） in APAP 0.5, 1, 2 and4 mmol·L^-1groups, but decreased significantly（P〈0.01） in 8 mmol·L^-1group.Protein expression of NRF-2 increased significantly（P〈0.05） in 0.5 mmol·L^-1groups but decreased significantly（P〈0.01） in2, 4 and 8 mmol·L^-1groups.Protein expression of TFAM increased significantly（P〈0.05） in 1 mmol·L^-1group, but decreased significantly（P〈0.01） in 4 and 8 mmol·L^-1groups.Protein expression of ND-1increased significantly（P〈0.01） in 0.5, 1, 2 and 4 mmol·L^-1groups but decreased significantly（P〈0.01）in 8 mmol·L^-1group.CONCLUSION APAP can induce or inhibit mitochondrial biogenesis, which is possibly regulated by PGC-1α pathway in successfully differentiated HepaRG cells.