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黄绿青霉素对心肌细胞线粒体呼吸链合成相关转录调节基因mRNA表达、膜电位及活性氧的影响

Impact of citreoviridin on mRNA expression of mitochondrial respiratory chain synthesis related transcriptional regulation gene, membrane potential and reactive oxygen species in rat cardiomyocytes

摘要:

目的 观察黄绿青霉素(CIT)对SD大鼠原代心肌细胞线粒体呼吸链合成相关转录调节基因mRNA表达、膜电位及活性氧的影响.方法 用不同浓度CIT[0、1、2、3、4、5、6、7、8、9、10 μmol/L]作用SD大鼠原代心肌细胞24 h,采用噻唑蓝(MTT)法测定细胞存活率,并根据检测结果,用SPSS 13.0软件计算CIT的半数抑制浓度(IC50).进一步选择心肌细胞存活率为99%、95%、90%时的CIT水平(0.715、1.234、1.650 μmol/L)作为低、中、高剂量组,同时以未加CIT作为对照组,分别作用于心肌细胞24 h,采用反转录聚合酶链反应(RT-PCR)检测心肌细胞中过氧化物酶体增殖物受体γ共激活因子1α(PGC-1α)、核呼吸因子1(Nrf1)、核呼吸因子2(Nrf2)mRNA表达;分别以阳离子荧光羰花青染色剂(JC-1)、2',7'-二氯荧光素(DCFH2-DA)作为荧光探针,用全能酶标仪检测心肌细胞线粒体膜电位(MMP)和细胞内活性氧(ROS)的变化.结果 与0μmol/L CIT组[(89.4±17.6)%]比较,2~ 10 μmol/L CIT组原代心肌细胞存活率[(80.2±20.2)%、(74.4±18.7)%、(63.2±8.9)%、(51.5±18.8)%、(39.0±15.7)%、(22.6±10.5)%、(19.9±4.9)%、(20.7±4.8)%、(18.5±3.3)%]明显下降(P均< 0.05).CIT作用于心肌细胞24 h的IC50为4.6 μmol/L.高、中、低剂量组PGC-1α mRNA表达(0.431±0.041、0.619±0.031、0.653±0.037)较对照组(0.776±0.081)明显降低(P均<0.05);高剂量组Nrf1mRNA表达(0.358±0.051)明显低于对照组(0.580±0.098,P<0.05);高、中剂量组Nrf2 mRNA表达(0.352±0.041、0.472±0.011)明显低于对照组(0.667±0.091,P均<0.05).与对照组[(100.00±0.00)%、(100.00±0.00)%]比较,高、中、低剂量组MMP水平[(55.3±3.3)%、(69.8±4.7)%、(81.8±2.7)%]显著降低(P均<0.05),ROS水平[(606.0±46.3)%、(275.0±53.5)%、(158.9±29.5)%]显著升高(P均<0.05).结论 CIT可对原代心肌细胞的线粒体呼吸链生物合成产生抑制作用,并诱导细胞发生氧化应激,通过线粒体途径介导心肌细胞死亡,从而引起心肌损伤.

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abstracts:

Objective To investigate the impact of citreoviridin(CIT) on mRNA expression of mitochondrial respiratory chain synthesis related transcriptional regulation gene,mitochondrial membrane(MMP) potential and reactive oxygen species (ROS) in cardiomyocytes of rat.Methods Viability of rat primary cardiomyocytes treated with different concentrations of CIT (0,1,2,3,4,5,6,7,8,9,10 μmol/L) for 24 h was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.Based on the MTT curve,median inhibitory concentration(IC50) was calculated using SPSS 13.0.High-,medium-and low-dose groups of CIT(1.650,1.234,0.715 μmol/L) were defined corresponding to 99%,95% and 90% of cardiomyocyte viability,respectively.CIT was not added in as the control group.After 24 hours,the mRNA expression levels of peroxisome-proliferator-activated receptor γcoactivator(PGC-1α),nuclear respiratory factor 1 (Nrf1) and nuclear respiratory factor 2(Nrf2) in cardiomyocytes were detected by reverse transcriptase polymerase chain reaction(RT-PCR).Changes of MMP and intracellular ROS were determined by a fluorescence microplate reader using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) and 2,7-dichlorofluorescein diacetate (DCFH2-DA) as fluorescent probes.Results Compared with 0 μmol/L CIT group [(89.4 ± 17.6)%],viabilities of rat primary cardiomyocytes treated with 2-10 μmol/L CIT groups[(80.2 ± 20.2)%,(74.4 ± 18.7)%,(63.2 ± 8.9)%,(51.5 ± 18.8)%,(39.0 ± 15.7)%,(22.6 ± 10.5)%,(19.9 ± 4.9)%,(20.7 ± 4.8)%,(18.5 ± 3.3)%] decreased significantly(all P < 0.05).The IC50 value of cardiomyocytes after24 h treatment with CIT was 4.6 μmol/L The PGC-1α mRNA expressions ofhigh-,medium-and low-dose groups(0.431 ± 0.041,0.619 ± 0.031,0.653 ± 0.037) were significantly lower compared to that of the control group(0.776 ± 0.081,all P < 0.05).The Nrf1 mRNA expression of high-dose group(0.358 ± 0.05) was significantly lower compared to that of the control group(0.580 ± 0.098,P < 0.05).Nrf2 mRNA expressions of the high-and medium-dose groups(0.352 ± 0.041,0.472 ± 0.011) were significantly lower than that of the control group (0.667 ± 0.091,all P< 0.05).Compared with the control groups[(100.00 ± 0.00)%,(100.00 ± 0.00)%],the MMP levels of high-,medium-and low-dose groups[(55.3 ± 3.3)%,(69.8 ± 4.7)%,(81.8 ± 2.7)%] were significantly lower and the ROS levels[(606.0 ± 46.3)%,(275.0 ± 53.5)%,(158.9 ±29.5)%] were significantly higher(all P < 0.05).Conclusions CIT inhibits the biosynthesis of mitochondria in primary cardiomyocytes and induces oxidative stress.Myocardial injury is caused by cardiomyocyte apoptosis through mitochondrial pathway,which leads to myocardial injury.

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