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、10μmol／L]作用SD大鼠原代心肌细胞24h，采用噻唑蓝（MTr）法测定细胞存活率，并根据检测结果，用SPSS13．0软件计算CIT的半数抑制浓度（IC。）。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、1．650txmol／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24h，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叮共激活因子-1α（PGC-1α）、核呼吸因子1（Nrfl）、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（DCFH2一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0txmoL／LCIT组[（89．4±17．6）％]比较，2～10μmol／LCIT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24h的IC。为4．6μmol／L。高、中、低剂量组PGC—letmRNA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NIflmRNA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Nrf2mRNA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[（55．34-3．3）％、（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。
Objective To investigate the impact of citreoviridin（CIT） on mRNA expression ofmitochondrial respiratory chain synthesis related transcriptional regulation gene, mitochondrial membrane（MMP） potential and reactive oxygen species （ROS） in eardiomyocytes 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 Ixmol/L） for 24 h was determined by 3- （4,5-Dimethyhhiazol-2-yl）-2,5-diphenyhetrazolium bromide（MTF） method. Based on the MTI＇ curve, median inhibitory concentration （IC±） 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-proliferatoractivated receptor /coactivator（PGC-1α）, nuclear respiratory factor l（Nrfl） 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-dichloroflnorescein diacetate （DCFH2-DA） as fluorescent probes. Results Compared with 0 Ixmol/L CIT group [ （89.4 ± 17.6）%], viabihties of rat primary cardiomyocytes treated with 2 - 10 I±mol/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（aU P 〈 0.05）. The ICs0 value of eardiomyocytes after 24 h treatment with CIT was 4.6 p,mol/L. The PGC-lct mRNA expressions of high-, 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 Nrfl mRNA express!on 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.
出处
《中华地方病学杂志》
CAS
CSCD
北大核心
2013年第4期384-388,共5页
Chinese Journal of Endemiology
关键词
黄绿青霉素
肌细胞
心脏
线粒体
膜电位
活性氧
Citreoviridin
Myoeytes, cardiac
Mitochondria
Membrane potential
Reactive oxygen species
