京尼平对缺氧/复氧心肌细胞氧化应激损伤的影响

Effect of Genipin on Hypoxia / Reoxygenation Induced Oxidative Stress Injury in H9c2 Cell Line

目的研究京尼平(genipin)对缺氧/复氧心肌细胞氧化应激损伤的影响。方法模拟缺血再灌注损伤模型,体外培养H9c2心肌细胞,建立缺氧/复氧模型。实验分为6组:对照组、缺氧/复氧组、京尼平预处理后缺氧/复氧模型组、缺氧/复氧+0.5μmol·L^(-1)京尼平组、缺氧/复氧+1.25μmol·L^(-1)京尼平组、缺氧/复氧+2.5μmol·L^(-1)京尼平组、缺氧/复氧+10μmol·L^(-1)京尼平组。CCK-8检测H9c2心肌细胞存活率;酶标仪检测细胞培养上清液中乳酸脱氢酶(LDH)水平及细胞内的丙二醛(MDA)含量和细胞内超氧化物歧化酶(SOD)的活力;激光共聚焦检测细胞内线粒体活性氧(ROS)的产生和线粒体钙离子含量及线粒体膜电位变化;Western-blot检测细胞中细胞色素C蛋白表达。结果与对照组比较,缺氧/复氧组显著降低H9c2心肌细胞存活率,并呈现时间依赖性(r=-0.82,P<0.01);低浓度(1.25~40μmol·L^(-1))的京尼平可提高缺氧/复氧处理后心肌细胞存活率(P<0.05),高浓度(80~320μmol·L^(-1))的京尼平对细胞有明显的毒性作用(P<0.05);与缺氧/复氧组比较,缺氧/复氧+1.25μmol·L^(-1)京尼平组及缺氧/复氧+2.5μmol·L^(-1)京尼平组和缺氧/复氧+10μmol·L^(-1)京尼平组可显著降低乳酸脱氢酶水平、减少细胞内线粒体活性氧产生和线粒体钙离子含量及丙二醛的含量、明显提高细胞内超氧化物歧化酶活力、减少线粒体膜电位去极化比例及细胞色素C蛋白表达(P<0.01或P<0.05)。结论低浓度的京尼平可减少缺氧/复氧后导致H9c2心肌细胞氧化应激损伤,其机制可能与平衡氧化应激产物和抗氧化应激的酶系统及改善线粒体功能有关。

OBJECTIVE To investigate the effect of genipin on hypoxia/reoxygenation induced oxidative stress injury in H9c2 cell line. METHODS For mimicking myocardial ischemia/reperfusion （I/R） injury, H9c2 cells were cultured in vitro and es- tablished hypoxia/reoxygenation induced oxidative stress injury model. H9c2 cells were divided into six groups： control group, H/R group, H/R＋0.5μmol·L-1 genipin group, H/R ＋ 1.25μmol·L-1 genipin group, H/R ＋2.5μmol·L-1 geuipin group and H/R ＋ 10 μmol·L-1 genipin group. Cell viability was detected by cell counting kit-8 assay （CCK-8）. The levels of lactate dehydrogenase （LDH）, intracellular total superoxide dismutase （T-SOD） and malondialdehyde （MDA） were assessed u- sing microplate reader. Confocal laser scanning microscope was performed to examine the production of reactive oxygen species （ROS） and the level of mitochondrial calcium （ [ Ca＋ ] m） as well as the depolarization ratio of mitochondrial membranes poten- tial（△ψm）. The protein expression of cytochrome-C was detected by Western-blot. RESULTS Comparing to control group, the cell viability of H/R group was significantly decreased in a time-dependent manner（ r=- 0.82 ,P 〈 0.01 ）. Low concentra- tion （ 1.25 - 40 μmol·L-1 ） of genipin could improve cell viability exposed to H/R treatment （ P 〈 0.05 ）, on the contrary, high concentration（80- 320μmol·L-1） of genipin remarkably reduced the cell viability （P 〈 0.05 ）. In compared with H/R group, the levels of LDH release, MDA production and ROS production, the level of [ Ca2＋ ] m, depolarization ratio of △ψm and the protein expression of cytochrome-c in H/R ＋ （ 1.25 - 10 μmol·L-1） genipin groups were notably lessened, while the level of T-SOD was increased（ P 〈 0.01 or P 〈 0.05 ）. CONCLUSION Genipin could reduce H/R-induced oxidative stress injury, the mechanism might be connected with balancing the oxidative stress products and anti-oxidation enzyme system as well as impro- ving mitochondrial dysfunction.