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目的采用天然药物提取物水飞蓟宾作用于人表皮癌A431细胞,考察药物诱导细胞内生成活性氮(reactive nitrogen species,RNS)的来源及其在诱导细胞凋亡过程中所起的调控作用。方法采用噻唑蓝比色法、流式细胞术及一氧化氮(nitric oxide,NO)试剂盒检测法等方法考察药物对细胞的生长抑制、RNS生成、线粒体损伤及凋亡等影响。结果及结论水飞蓟宾可浓度依赖性诱导A431细胞生长抑制;在此过程中细胞内RNS及NO水平增高,一氧化氮合酶(nitric oxide synthase,NOS)抑制剂或NO抑制剂的加入可逆转这一现象的发生;同时,N-硝基-L-精氨酸甲酯(Nnitro-L-arginine methyl ester,L-NAM E)及一氧化氮清除剂PTIO可显著逆转水飞蓟宾诱导的细胞凋亡。说明水飞蓟宾可能是通过激活内皮型一氧化氮合酶(endothelial nitric oxide synthase,e NOS)而诱导细胞内NO水平增高,并通过线粒体途径促进了细胞凋亡的发生。
Objective To investigate the origin of reactive nitrogen species (RNS) induced by drug-induced intracellular epidermal carcinoma (A431) cells and the regulatory effect of silybin on the apoptosis of A431 cells. Methods Thiazolyl blue colorimetric assay, flow cytometry and nitric oxide (NO) kit assay were used to investigate the effects of drugs on cell growth inhibition, RNS production, mitochondrial damage and apoptosis. RESULTS AND CONCLUSION Silibinin induces the growth inhibition of A431 cells in a concentration-dependent manner. During this process, intracellular levels of RNS and NO are increased. Inhibitors of nitric oxide synthase (NOS) or NO inhibitors Reversing the occurrence of this phenomenon; at the same time, Nnitro-L-arginine methyl ester (L-NAM E) and nitric oxide scavenger PTIO significantly reversed the induction of silybin Apoptosis. Silybin may induce the increase of intracellular NO level by activating endothelial nitric oxide synthase (eNOS) and promote the apoptosis through mitochondrial pathway.