目的:研究去甲斑蝥素(norcantharidin,NCTD)抑制小鼠肺纤维瘤(L929)细胞的作用机制.方法:用MTT法测定细胞生长抑制率.采用Hoechst 33258染色、DNA凝胶电泳和乳酸脱氢酶(LDH)活力检测细胞凋亡.用免疫印迹法(Western blot)分析了细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)和磷酸化ERK、c-Jun N-末端激酶(C-Jun N-terminal kinase,JNK)和磷酸化JNK蛋白表达的变化.结果:NCTD诱导L929细胞凋亡.JNK抑制剂(SP600125)与ERK抑制剂(PD98059),可明显抑制NCTD对细胞的杀伤作用.同时磷酸化ERK和磷酸化JNK表达增加.结论:NCTD对L929细胞的细胞毒作用是通过诱导其凋亡而发生的,且呈时间剂量依赖性.这种作用可能与JNK,ERK通路激活有关.
Purpose:To study the mechanism of norcantharidi n (NCTD)-induced apoptosis on L929 cell lines. Methods:Inhibitory effects of NCTD on L929 cell lines were meas ured by MTT test. Morphological changes were observed by Hoechst 33258 staining. DNA gel electrophoresis and LDH activity test were used to determine apoptosis. The mechanism of NCTD-induced apoptosis was analyzed by addition of several ki nds of mitogen activated protein kinase (MAPK) inhibitors and by Western blot an alysis. Results:NCTD induced L929 cell death in a time and dose depende nt manner. The cytotoxic effect of NCTD on L929 cell lines were induced mainly t hrough apoptotic pathways. Membrane blebbing and chromatin condensation were obs erved in NCTD-treated L929 cells. DNA fragments were also detected by agarose g el electrophoresis. By adding of MAPK (JNK or ERK) inhibitors, a distinct inhibi tion of apoptosis was detected. Western blot analysis showed that the expression of ERK proteins was decreased whereas the expression of phosphorylated form of ERK was increased depending on the time period. At the same time, the expression of JNK showed no change, while the expression of phosphorylated JNK was increas ed. Conclusions:The cytotoxic effect of NCTD on L929 cell lines was mainly due to apoptosis. ERK and JNK MAPKs might be involved in the apoptotic p athways of NCTD-treated L929 cells.