CAJ | 학술논문

目的：探讨虾青素对体外氧化应激诱导人外周血内皮祖细胞凋亡的影响及其机制。方法体外培养人外周血单核细胞源的内皮祖细胞，分为正常对照组、模型组（给予叔丁基过氧化氢100μmol·L-1）、预处理组（给予叔丁基过氧化氢100μmol·L-1+虾青素0.1，1.0，10.0 nmol·L-1预处理24 h）。噻唑蓝（ MTT）法检测细胞存活率；4′，6-二脒基-2-苯基吲哚（ DAPI）染细胞检测细胞凋亡率；2′，7′-二氯荧光黄双乙酸盐（ DCFH-DA）法检测细胞内活性氧（ ROS）水平；比色法检测半胱氨酸天冬氨酸蛋白酶（ caspase-3）活性；阳离子脂质JC-1法测定线粒体膜电位。结果 MTT检测发现，随着虾青素浓度的增加，内皮祖细胞的存活率也增加。与模型组[（48.5±4.3）％]比较，虾青素+叔丁基过氧化氢组细胞存活率明显升高[（57.6±8.2）％，（77.6±7.5）％，和（85.3±6.1）％，P﹤0.05]；DAPI染细胞检测发现，预处理组凋亡细胞明显减少，模型组凋亡小体形成率（27.8±3.2）％，3个预处理组凋亡小体形成率分别为[（20.4±2.9）％，（14.9±1.7）％和（7.8±0.7）％，P﹤0.05]；caspase-3活性检测表明，与模型组（0.345±0.018）比较，预处理组活性随虾青素剂量增大而依次降低[（0.291±0.013），（0.209±0.004），（0.169±0.013），P﹤0.05]；DCHF-DA分子探针检测细胞内ROS发现，与模型组比较，预处理组细胞内ROS水平呈剂量依赖性降低，荧光强度明显减弱。JC-1法线粒体膜电位检测显示，与模型组比较，预处理组线粒体膜电位丢失被明显抑制。结论虾青素通过减少细胞内ROS，保护线粒体膜电位，下调caspase-3活性，最终起到抗氧化应激诱导的EPCs凋亡。
목적：탐토하청소대체외양화응격유도인외주혈내피조세포조망적영향급기궤제。방법체외배양인외주혈단핵세포원적내피조세포，분위정상대조조、모형조（급여숙정기과양화경100μmol·L-1）、예처리조（급여숙정기과양화경100μmol·L-1+하청소0.1，1.0，10.0 nmol·L-1예처리24 h）。새서람（ MTT）법검측세포존활솔；4′，6-이미기-2-분기신타（ DAPI）염세포검측세포조망솔；2′，7′-이록형광황쌍을산염（ DCFH-DA）법검측세포내활성양（ ROS）수평；비색법검측반광안산천동안산단백매（ caspase-3）활성；양리자지질JC-1법측정선립체막전위。결과 MTT검측발현，수착하청소농도적증가，내피조세포적존활솔야증가。여모형조[（48.5±4.3）％]비교，하청소+숙정기과양화경조세포존활솔명현승고[（57.6±8.2）％，（77.6±7.5）％，화（85.3±6.1）％，P﹤0.05]；DAPI염세포검측발현，예처리조조망세포명현감소，모형조조망소체형성솔（27.8±3.2）％，3개예처리조조망소체형성솔분별위[（20.4±2.9）％，（14.9±1.7）％화（7.8±0.7）％，P﹤0.05]；caspase-3활성검측표명，여모형조（0.345±0.018）비교，예처리조활성수하청소제량증대이의차강저[（0.291±0.013），（0.209±0.004），（0.169±0.013），P﹤0.05]；DCHF-DA분자탐침검측세포내ROS발현，여모형조비교，예처리조세포내ROS수평정제량의뢰성강저，형광강도명현감약。JC-1법선립체막전위검측현시，여모형조비교，예처리조선립체막전위주실피명현억제。결론하청소통과감소세포내ROS，보호선립체막전위，하조caspase-3활성，최종기도항양화응격유도적EPCs조망。
Objective To investigate the effect of astaxanthin( ASX)on endothelial progenitor cells( EPCs)injury induced by oxidative stress in vitro and to explore its underlying mechanism. Methods Cultured EPCs isolated from peripheral blood were randomly divided into 5 groups:normal control,model group[ tert-butyl hydroperoxide( tBHP)100μmol·L-1 ],and ASX+tBHPgroups(thecellswerepreconditionedwithASX0.1,1.0,and10.0nmol·L-1,respectively).Thecellviabilitywas measured by MTT method. The level of reactive oxygen species( ROS)was determined by DCFH-DA method. The changes of mitochondrial membrane potential( MMP)and apoptosis ratio were detected by JC-1 method and DAPI method,respectively. caspase-3 activity changes of EPCs were detected. Results The cell viability of EPCs was improved with the increasing concentration of ASX. Compared with the model group[(48. 5±4. 3)%],0. 1,1. 0,10. 0 nmol·L-1 ASX significantly increased the cell viabilities[(57. 6±8. 2)%,(77. 6±7. 5)%,and(85. 3±6. 1)%,P﹤0. 05]. The results of DAPI staining revealed that ASX pretreatment could significantly reduce the apoptotic rate of EPCs. The apoptotic rate of the model group was( 27. 8 ± 3. 2)%,while that of ASX+tBHP groups was[(20. 4±2. 9)%,(14. 9±1. 7)%,and(7. 8±0. 7)%,P﹤0. 05],respectively. The data from caspase-3 activity assay indicated that ASX precondition could also remarkably decrease the caspase-3 activity for EPCs. The caspase-3 activity of the model group was(0. 345±0. 018),while that of the ASX+tBHP group were[(0. 291± 0. 013),(0. 209±0. 004),and(0. 169±0. 013),P﹤0. 05],respectively. In addition,treatment with tBHP resulted in an increase of DCF fluorescence,while ASX precondition could decrease the DCF fluorescence,which suggested the accumulation of intercellular ROS for EPCs. Injury of michondrial membrane resulted in the loss of mitochondrial membrane potential( MMP). The MMP detected by JC-1 method revealed that compared with model group,pretreatment of ASX inversed the reduction of MMP. Conclusion Astaxanthin inhibits endothelial progenitor cell apoptosis induced by oxidative stress through inhibiting ROS production,improving the mitochondrial function and down-regulating caspase-3 activity.