摘要
目的研究丹酚酸C对MPP^+诱导SH-SY5Y细胞损伤的保护作用。方法 SH-SY5Y细胞与丹酚酸C(0.1,1.0和5.0μmol·L^(-1))或阳性药N-乙酰半胱氨酸(NAC)5 mmol·L^(-1)孵育2 h后加入MPP^+500μmol·L^(-1),继续培养24 h。MTT法检测细胞存活率,AO/EB染色观察细胞形态及凋亡,应用DCFH-DA荧光探针检测细胞内活性氧(ROS)水平,线粒体超氧化物指示剂(Mito SOX)特异性探针检测线粒体超氧化物水平,JC-1检测线粒体膜电位,免疫荧光法测定NADPH氧化酶2(NOX2)的表达,免疫荧光和Western蛋白质印迹法检测细胞色素c,Bcl-2,Bax和活化的胱天蛋白酶3的表达水平。结果 MPP^+500μmol·L^(-1)损伤导致细胞存活率显著降低(P<0.01),丹酚酸C(1.0和5.0μmol·L^(-1))及NAC(5 mmol·L^(-1))能够逆转MPP^+损伤,显著提高细胞存活率(P<0.05,P<0.01,P<0.01)。丹酚酸C(1.0和5.0μmol·L^(-1))能够显著减少MPP^+诱导的细胞总ROS增加(P<0.01),维持Bax/Bcl-2表达平衡(P<0.01),减缓线粒体膜电位降低(P<0.05,P<0.01),减少细胞色素c的释放(P<0.01)及胱天蛋白酶3的活化(P<0.05,P<0.01)。同时,丹酚酸C(0.1,1.0和5.0μmol·L^(-1))能够明显抑制NOX2的表达(P<0.01),降低线粒体内超氧化物水平(P<0.01),改善MPP^+引起的细胞氧化应激状态。结论丹酚酸C能够抑制MPP^+诱发的SH-SY5Y细胞氧化应激及其介导的细胞凋亡。
OBJECTIVE To study the protective effect of salvianolic acid C（Sal C） on MPP＋-induced SH-SY5 Y cell injury. METHODS SH-SY5 Y cells were pre-treated with Sal C（0.1, 1.0 and5.0 μmol·L-1） or the positive drug N-acetyl-L-cysteine（NAC）（5 mmol·L^-1） for 2 h before MPP＋（500 μmol·L-1）stimulation and co-incubation for 24 h. Cell viability was detected by MTT assay. Cell morphology and apoptosis were observed by AO/EB staining. The level of reactive oxygen species（ROS） was detected by DCFH-DA fluorescent probe. Mito SOX specific probe was used to detect the levels of mitochondrial superoxide. Mitochondrial membrane potential（JC-1 probe） and nicotinamide adenine dinucleotide phosphate oxidase 2（NOX2） protein expression were detected by fluorescence assay. The levels of mitochondrial apoptosis-related proteins cytochrome c, Bcl-2, Bax and cleaved caspase 3 were detected by Western blotting or immunofluorescence assay. RESULTS MPP＋500 μmol·L-1 resulted in a significant decrease in cel viability（P〈0.01）. Sal C（1.0 and 5.0 μmol·L^-1） and the positive drug NAC（5 mmol·L^-1）reversed MPP＋-induced cell injury and increased cell viability（P〈0.05, P〈0.01, P〈0.01）. In addition, Sal C（1.0 and 5.0 μmol·L-1） could significantly reduce the increase of total ROS（P〈0.01）, maintain the Bax/Bcl-2 balance（P〈0.01）, increase mitochondrial membrane potential（P〈0.05, P〈0.01）, reduce the release of cytochrome c（P〈0.01） and the cleaved caspase 3 level（P〈0.05, P〈0.01）. Sal C（0.1, 1.0 and 5.0 μmol·L-1） inhibited the expression of NOX2（P〈0.01）, decreased the mitochondrial superoxide level（P〈0.01）, and alleviated MPP＋-induced oxidative stress in SH-SY5 Y cells. CONCLUSION Sal C can inhibit oxidative stress and apoptosis induced by MPP＋in SH-SY5Y cells.
作者
宋俊科
张雯
张雪
杨海光
周启蒙
王金华
杜冠华
SONG Jun-ke;ZHANG Wen;ZHANG Xue;YANG Hai-guang;ZHOU Qi-meng;WANG Jin-hua;DU Guan-hua(State Key Laboratory of Bioactive Substances and Functions of Natural Medicines,Beijing Key Laboratory of Drug Target and Screening Research,Institute of Materia Medica,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China)
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2018年第5期392-399,共8页
Chinese Journal of Pharmacology and Toxicology
基金
国家自然科学基金(81603100)
中国医学科学院医学与健康科技创新工程项目(2017-I2M-1-010)~~
