CAJ | 학술논문

目的：探讨新型的自由清除剂依达拉奉( edaravone, EDA)能否保护 H9c2心肌细胞对抗阿霉素( doxorubicin, DOX)引起的损伤。方法应用 DOX (5μmol · L-1)处理H9c2心肌细胞建立DOX心肌毒性损伤模型。 CCK-8比色法测定细胞存活率；Hoechst 33258核染色法观察细胞凋亡的形态学和数量改变；双氯荧光素( DCFH-DA)染色荧光显微镜照像检测细胞活性氧(ROS)水平；罗丹明123(Rh123)染色荧光显微镜照像测定线粒体膜电位( MMP )；Western blot法测定caspase-3蛋白的表达水平。结果应用20、40、80μmol·L-1 EDA分别预处理H9c2心肌细胞60 min,可明显地抑制5μmol·L-1 DOX引起的细胞毒性,使细胞存活率升高,其中40μmol · L-1 EDA 的保护作用最大；应用40μmol·L-1 EDA分别预处理心肌细胞30、60、90、120 min,可明显地抑制DOX引起的细胞毒性,其中预处理60 min的保护作用最大；此外,在5μmol·L-1 DOX处理H9c2心肌24 h前,应用40μmol·L-1 EDA预处理60 min可明显抑制DOX引起的心肌损伤作用,表现为抑制DOX引起的细胞内ROS生成增多及抑制DOX的致细胞凋亡作用(使凋亡细胞数目减少和cleaved caspase-3表达下调)和MMP的损伤作用。结论 EDA 能保护 H9c2心肌细胞对抗 DOX 诱导的心肌毒性,此保护作用可能与其抑制ROS生成及减轻DOX对MMP的损伤有关。
목적：탐토신형적자유청제제의체랍봉( edaravone, EDA)능부보호 H9c2심기세포대항아매소( doxorubicin, DOX)인기적손상。방법응용 DOX (5μmol · L-1)처리H9c2심기세포건립DOX심기독성손상모형。 CCK-8비색법측정세포존활솔；Hoechst 33258핵염색법관찰세포조망적형태학화수량개변；쌍록형광소( DCFH-DA)염색형광현미경조상검측세포활성양(ROS)수평；라단명123(Rh123)염색형광현미경조상측정선립체막전위( MMP )；Western blot법측정caspase-3단백적표체수평。결과응용20、40、80μmol·L-1 EDA분별예처리H9c2심기세포60 min,가명현지억제5μmol·L-1 DOX인기적세포독성,사세포존활솔승고,기중40μmol · L-1 EDA 적보호작용최대；응용40μmol·L-1 EDA분별예처리심기세포30、60、90、120 min,가명현지억제DOX인기적세포독성,기중예처리60 min적보호작용최대；차외,재5μmol·L-1 DOX처리H9c2심기24 h전,응용40μmol·L-1 EDA예처리60 min가명현억제DOX인기적심기손상작용,표현위억제DOX인기적세포내ROS생성증다급억제DOX적치세포조망작용(사조망세포수목감소화cleaved caspase-3표체하조)화MMP적손상작용。결론 EDA 능보호 H9c2심기세포대항 DOX 유도적심기독성,차보호작용가능여기억제ROS생성급감경DOX대MMP적손상유관。
Aim To explore whether edaravone (EDA), a novel free radical scavenger, protects H9c2 cardiac cells against doxorubicin ( DOX )-induced car-diotoxicity. Methods H9c2 cells were treated with 5μmol·L-1 DOX to establish a model of DOX cardio-toxicity. Cell viability was examined by cell counter kit ( CCK-8 ) . Changes in morphology and amount of ap-optotic cells were detected by Hoechst 33258 staining;intracellular level of reactive oxygen species ( ROS ) was measured by DCFH-DA staining and photofluorog-raphy;mitochondrial membrane potential ( MMP) was observed by rhodamine 123 ( RH123 ) staining and photoflurograph; the expression level of caspase-3 was determined by Western blot assay. Results Pretreat-ment of H9 c2 cells with 20 , 40 and 80 μmol · L-1 EDA for 60 min markedly inhibited cytotoxicity in-duced by 5 μmol · L-1 DOX, respectively, as evi-denced by an increase in cell viability. The protective effect induced by 40 μmol · L-1 EDA was maximal. Pretreatment of H9 c2 cells with 40 μmol · L-1 EDA for 30 , 60 , 90 and 120 min significantly attenuated DOX-induced cytotoxicity, respectively, having a max-imal protection at 60 min. Furthermore, pretreatment of H9 c2 cells with 40 μmol · L-1 EDA for 60 min be-fore exposure to 5 μmol · L-1 DOX for 24 h obviously reduced cardiac injuries, as evidenced by decreases in the DOX-induced intracellular ROS generation, num-ber of apoptotic cells, and expression of cleaved caspase-3, as well as loss of MMP. Conclusions EDA can protect H9 c2 cardiac cells against DOX-in-duced cardiotoxicity, this protection may be associated with inhibition of ROS production and preservation of MMP.