摘要
目的探讨新型硫化氢供体8L对氧化应激诱导心肌细胞损伤的保护作用及NF-E2相关因子2(Nrf2)有关的分子机制。方法用活性氧供体过氧化氢(H2O2)处理培养的大鼠H9c2心肌细胞,建立心肌细胞损伤的体外模型以模拟急性缺血再灌注诱导的心肌损伤;在H2O2处理前给予8L预处理观察其对心肌细胞的保护作用。为了明确Nrf2的作用,在H2O2处理或8L预处理前给予其选择性抑制剂鸦胆苦醇预处理。细胞计数试剂盒8比色法检测细胞存活率,试剂盒法检测乳酸脱氢酶(LDH)的释放,罗丹明123染色结合荧光照相术检测线粒体膜电位(MMP),Western Blot法检测核内Nrf2的表达。结果 H9c2心肌细胞经0~600μmol/L H2O2处理6 h可浓度依赖性地降低细胞存活率,且半数有效浓度约为400μmol/L。400μmol/L H2O2处理H9c2心肌细胞6 h可使LDH释放增加,MMP降低,并增加细胞核内Nrf2的表达(P均<0.01)。在用400μmol/L H2O2处理前,先用50、100和200μmol/L 8L预处理1 h,可将细胞存活率从(52.6±4.3)%分别提高至(72.5±6.3)%、(83.1±5.2)%和(85.7±4.9)%。200μmol/L 8L预处理1 h还可明显抑制H2O2诱导的LDH释放(P<0.01)及MMP受损(P<0.05),但可易化H2O2诱导的Nrf2表达上调(P<0.01)。另外,10μmol/L鸦胆苦醇预处理1 h不但可加重H2O2诱导的心肌细胞损伤(P<0.01),还可拮抗8L的心肌细胞保护作用(P<0.01)。结论硫化氢供体8L可减轻氧化应激诱导的H9c2心肌细胞损伤,其机制可能与上调Nrf2有关。
Aim To explore the effects of a novel H2 S donor( 8L) on oxidative stress-induced damage and the mechanisms underlying NF-E2-related factor 2( Nrf2) in H9c2 cardiomyocytes. Methods H9c2 cardiomyocytes were treated with exogenous reactive oxygen species,hydrogen peroxide( H2O2),to set up an oxidative injury to mimic the in vitro status induced by acute myocardial ischemia-reperfusion. Prior to the treatment with H2O2,the cells were treated with 8L and then its protective action was investigated. In order to test the roles of Nrf2,its selective inhibitor brusatol( BR) was used before H2O2 or 8L. Cell counting kit-8 was used to measure cell viability. Lactate dehydrogenase( LDH) release was assessed by a commercial kit,mitochondrial membrane potential( MMP) was observed by rhodamine123 staining followed by photofluorography and nuclear Nrf2 expression was examined by Western Blot assay. Results Exposure of H9c2 cardiomyocytes to H2O2 ranging from 0 to 600 μmol / L decreased cell viability in a concentration-dependent manner with a lethal concentration of 400 μmol / L. Treatment of the cells with 400 μmol / L H2O2 for 6 h increased LDH release and nuclear Nrf2 expression,while decreased MMP( P < 0. 01). Before the treatment with 400 μmol / L H2O2,the cells were preconditioned with 8L at 50,100 and 200 μmol/L for 1 h and the cell viability were enhanced to( 72. 5 ± 6. 3) %,( 83. 1 ± 5. 2) % and( 85. 7 ± 4. 9) % from( 52. 6 ± 4. 3) %,respectively. The pretreatment with 200μmol / L 8L for 1 h significantly attenuated H2O2-induced LDH release( P < 0. 01) and MMP loss( P < 0. 05),while facilitated H2O2-induced upregulation of nuclear Nrf2 expression( P < 0. 01). Pretreatment with 10 μmol / L BR for 1 h not only aggravated H2O2-induced cell injury( P < 0. 01) but also partially blocked 8L-induced cell protection( P < 0. 01).Conclusion The novel H2 S donor 8L mitigates oxidative stress-induced injury and the mechanisms may be associated with the activation of Nrf2.
出处
《中国动脉硬化杂志》
CAS
北大核心
2015年第6期561-566,共6页
Chinese Journal of Arteriosclerosis
基金
广东省科技计划项目(2012A030400033)
