硫化氢对过氧化氢诱导的原代乳鼠心肌细胞氧化损伤的影响及其机制
류화경대과양화경유도적원대유서심기세포양화손상적영향급기궤제
Effect of hydrogen sulfide on H2O2-stimulated primary neonatal rat cardiomyocytes
  • 万方数据
    中华心血管病杂志 중화심혈관병잡지
    Chinese Journal of Cardiology
    2013年 4期 327-332 ,共6页

    陈玉映%胡允兆%钟健开%郑素琳%何宗云%吴焱贤%吴赛珠

    진옥영%호윤조%종건개%정소림%하종운%오염현%오새주

    肌细胞,心脏%氧化性应激%硫化氢%过氧化氢 肌細胞,心髒%氧化性應激%硫化氫%過氧化氫
    기세포,심장%양화성응격%류화경%과양화경
    Myocytes,cardiac%Oxidative stress%Hydrogen sulfide%Hydrogen peroxide
    目的 探讨硫化氡(H2S)对过氧化氢(H2O2)诱导的原代乳鼠心肌细胞氧化损伤的影响及其可能机制.方法 在常规培养3 d的原代乳鼠心肌细胞中加入不同浓度(10、100、1000 μmol/L)的H2O2处理24 h建立心肌细胞氧化损伤的模型,分别为10 μmol/L H2O2模型组、100μmol/L H2O2模型组及1000 μmol/L H2O2模型组,另以完全培养基进行常规培养作为对照组.另外,NaHS配成不同的终浓度(1、10、100μmol/L)单纯作片用或分别与100 μmol/L H2O2共同作用于心肌细胞24 h探讨NaHS对H2O2诱导的原代乳鼠心肌细胞的影响,分别为1μmol/L NaHS+100 μmol/L H2O2处理组、10 μmol/L NaHS+100 μmol/L H2O2处理组、100 μmol/L NaHS+100 μmol/LH2O2处理组、单纯10μmol/L NaHS处理组、单纯100 μmol/L NaHS处理组,另设100 μmol/L H2O2模型组和以完全培养基进行常规培养作为对照组.应用 MTT比色法检测细胞存活率,黄嘌呤氧化酶法检测超氧化物歧化酶(SOD)的活力,硫代巴比妥酸比色法检测丙二醛(MDA)含量,化学比色法测定乳酸脱氢酶(LDH)活性,流式细胞术检测细胞凋亡百分率,罗丹明123(Rh 123)染色荧光显微镜照相检测细胞线粒体膜电位(mitochondrial membrane potential,MMP),2,-7,-二氯荧光黄双乙酸盐染色荧光显微镜照相检测细胞内的活性氧簇(ROS)水平.结果 10、100、1000 μmol/LH2O2处理原代乳鼠心肌细胞24 h后,心肌细胞存活率和培养基上清液SOD活力均呈剂量依赖性地下降,细胞蛋白裂解液MDA含量、细胞培养液LDH活性则呈剂量依赖性地升高.100 μmol/L H2O2 模型组细胞存活率显著低于对照组(P=0.001),MDA含量显著高于对照组(P =0.003),10 μmol/L H2O2模型组的SOD活力及LDH活性与对照组比较差异已有统计学意义(P分别为0.013和0.028).1、10和100 μmol/L的NaHS与100μmol/L H2O2共同作用心肌细胞24 h可呈剂量依赖性地抑制H2O2诱导的细胞存活率、SOD活力的下降以及LDH活性、MDA含量的升高,其中10和100μmol/L NaHS+ 100 μmol/L H2O2处理组与100μmol/L H2O2模型组比较差异均有统计学意义(P均＜0.05).100 μmol/L H2O2模型组DCF荧光强度显著高于对照组(P=0.003),而10和100 μmol/L NaHS+ 100 μmol/L H2O2处理组DCF荧光强度均显著低于100 μmol/L H2O2模型组(p分别为0.017和0.000).此外,单纯10和100 μmol/L NaHS处理组DCF荧光强度与对照组比较差异均无统计学意义.100 μmol/L H2O2模型组MMP显著低于对照组(P =0.000),而10和100 μmol/L NaHS+100 μmol/L H2O2处理组MMP均高于100μmol/L H2O2模型组(P均＜0.05).此外,单纯10和100 μ mol/L NaHS处理组MMP与对照组比较差异均无统计学意义.10和100μmol/L NaHS+ 100 μmol/L H2O2处理组心肌细胞的凋亡率分别为(36.7±4.9)％和(20.1±1.9)％均显著低于100 μmol/L H2O2模型组[(48.9±5.6)％],p均＜0.05.结论 H2S可对抗H2O2诱导的心肌细胞氧化损伤,其机制可能与减少ROS生成、升高MMP以及降低心肌细胞凋亡率有关.
    목적 탐토류화동(H2S)대과양화경(H2O2)유도적원대유서심기세포양화손상적영향급기가능궤제.방법 재상규배양3 d적원대유서심기세포중가입불동농도(10、100、1000 μmol/L)적H2O2처리24 h건립심기세포양화손상적모형,분별위10 μmol/L H2O2모형조、100μmol/L H2O2모형조급1000 μmol/L H2O2모형조,령이완전배양기진행상규배양작위대조조.령외,NaHS배성불동적종농도(1、10、100μmol/L)단순작편용혹분별여100 μmol/L H2O2공동작용우심기세포24 h탐토NaHS대H2O2유도적원대유서심기세포적영향,분별위1μmol/L NaHS+100 μmol/L H2O2처리조、10 μmol/L NaHS+100 μmol/L H2O2처리조、100 μmol/L NaHS+100 μmol/LH2O2처리조、단순10μmol/L NaHS처리조、단순100 μmol/L NaHS처리조,령설100 μmol/L H2O2모형조화이완전배양기진행상규배양작위대조조.응용 MTT비색법검측세포존활솔,황표령양화매법검측초양화물기화매(SOD)적활력,류대파비타산비색법검측병이철(MDA)함량,화학비색법측정유산탈경매(LDH)활성,류식세포술검측세포조망백분솔,라단명123(Rh 123)염색형광현미경조상검측세포선립체막전위(mitochondrial membrane potential,MMP),2,-7,-이록형광황쌍을산염염색형광현미경조상검측세포내적활성양족(ROS)수평.결과 10、100、1000 μmol/LH2O2처리원대유서심기세포24 h후,심기세포존활솔화배양기상청액SOD활력균정제량의뢰성지하강,세포단백렬해액MDA함량、세포배양액LDH활성칙정제량의뢰성지승고.100 μmol/L H2O2 모형조세포존활솔현저저우대조조(P=0.001),MDA함량현저고우대조조(P =0.003),10 μmol/L H2O2모형조적SOD활력급LDH활성여대조조비교차이이유통계학의의(P분별위0.013화0.028).1、10화100 μmol/L적NaHS여100μmol/L H2O2공동작용심기세포24 h가정제량의뢰성지억제H2O2유도적세포존활솔、SOD활력적하강이급LDH활성、MDA함량적승고,기중10화100μmol/L NaHS+ 100 μmol/L H2O2처리조여100μmol/L H2O2모형조비교차이균유통계학의의(P균＜0.05).100 μmol/L H2O2모형조DCF형광강도현저고우대조조(P=0.003),이10화100 μmol/L NaHS+ 100 μmol/L H2O2처리조DCF형광강도균현저저우100 μmol/L H2O2모형조(p분별위0.017화0.000).차외,단순10화100 μmol/L NaHS처리조DCF형광강도여대조조비교차이균무통계학의의.100 μmol/L H2O2모형조MMP현저저우대조조(P =0.000),이10화100 μmol/L NaHS+100 μmol/L H2O2처리조MMP균고우100μmol/L H2O2모형조(P균＜0.05).차외,단순10화100 μ mol/L NaHS처리조MMP여대조조비교차이균무통계학의의.10화100μmol/L NaHS+ 100 μmol/L H2O2처리조심기세포적조망솔분별위(36.7±4.9)％화(20.1±1.9)％균현저저우100 μmol/L H2O2모형조[(48.9±5.6)％],p균＜0.05.결론 H2S가대항H2O2유도적심기세포양화손상,기궤제가능여감소ROS생성、승고MMP이급강저심기세포조망솔유관.
    Objective To investigate the effects of hydrogen sulfide (H2S) on H2O2-stimulated primary neonatal rat cardiomyocytes and related mechanism.Methods Primary neonatal rat cardiomyocytes were treated with various concentrations of H2O2 (10,100,1000 μmol/L) for 24 h to establish the oxidative stress-induced cell injury model after 3 days' conventional culture.In addition,different concentrations of NaHS (1,10,100 μmol/L) were added to cardiomyocytes in the absence and presence of 100 μmol/L H2O2 for 24 h.The viability of cardiomyocytes was measured by MTT assay.The SOD vitality was measured by xanthine oxidase method and MDA content was determined by thiobarbituric acid colorimetric method.LDH activity was measured by chemical colorimetric method.The percentage of apoptotic cells was assessed by flow cytometry (FCM).The mitochondrial membrane potential (MMP) was analyzed by rhodamine 123 (Rh123) staining and photofluorography.The level of reactive oxygen species (ROS) in cardiomyocytes was measured by DCFH-DA staining and photofluorography.Results Cell viability and SOD vitality were significantly reduced while MDA content and LDH activity were significantly increased with increasing H2O2 concentrations.These effects could be partly reduced by cotreatment with H2O2 in a concentration-dependent manner(all P ＜ 0.05).Compared with control group,the DCF fluorescence intensity significantly increased in the 100 μmol/L H2O2 group (P =0.003),which could be attenuated by NaHS in a dose-dependent manner.Compared with control group,the MMP significantly decreased in the 100 μmol/L H2O2 group (P =0.000),which could be partly reversed by cotreatment with NaHS in a dose-dependent manner.Moreover,H2O2 treatment also significantly reduced 100 μmol/L H2O2 induced apoptosis in a dosedependent manner.Conclusion H2S protects primary neonatal rat cardiomyocytes against H2O2-induced oxidative stress injury through inhibition of H2O2 induced overproduction of ROS,dissipation of MMP and apoptosis.
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