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目的探讨中药单体薯蓣皂苷的次皂苷元B(DRG)体外诱导HCT-15细胞凋亡的分子作用机制。方法采用Westernblotting、体外Bcl-xL蛋白竞争结合检测实验及逆转录PCR(RT-PCR)对DRG诱导细胞凋亡的机制进行了研究。结果在HCT-15细胞中,DRG促发了线粒体调控的凋亡途径,细胞色素c呈时效性地从线粒体中释放到胞质中,同时Bax与Bcl-2蛋白表达的相对比例上调,caspase-9、caspase-3和PARP等被剪切成具有活性的片段,但是caspase-7却未见剪切,同时还不影响P53和Bcl-xL的表达;而与死亡受体途径相关的FADD表达无明显变化,caspase-8酶原也未见剪切;另外,DRG也不抑制BH3短肽与Bcl-xL的结合;RT-PCR检测结果表明,DRG诱导HCT-15细胞凋亡相关基因Bcl-2和Bax的mRNA水平的改变,进而导致Bcl-2蛋白表达水平下降,而Bax蛋白表达水平则明显增加,说明DRG诱发的细胞凋亡中涉及到Bcl-2和Bax基因水平的变化。结论 DRG诱发HCT-15细胞凋亡的分子机制在于从基因水平影响Bax和Bcl-2靶基因的转录表达,上调Bax/Bcl-2蛋白表达水平比例,并最终通过激活经典的线粒体途径而不是死亡受体途径来发挥其HCT-15细胞凋亡诱导作用,且对P53呈非依赖型。
Objective To investigate the molecular mechanism of apoptosis induced by diosgenin B (DRG), a traditional Chinese medicine monosaccharide, in HCT-15 cells in vitro. Methods Western Blotting, in vitro Bcl-xL protein competition assay and reverse transcription PCR (RT-PCR) were used to study the mechanism of DRG-induced apoptosis. Results In HCT-15 cells, DRG evoked a mitochondria-mediated apoptotic pathway. Cytochrome c released into the cytoplasm from mitochondria in time-dependent manner. At the same time, the relative proportion of Bax and Bcl-2 protein was up-regulated, while caspase- 9, caspase-3 and PARP were cleaved into active fragments, but no cleavage of caspase-7, while not affecting the expression of P53 and Bcl-xL; and death receptor pathway-related FADD expression In addition, DRG did not inhibit the binding of BH3 peptide to Bcl-xL. RT-PCR results showed that DRG induced the apoptosis-related gene Bcl-2 And Bax mRNA levels, leading to a decrease in Bcl-2 protein expression and a significant increase in Bax protein expression, indicating that DRG-induced apoptosis involves changes in Bcl-2 and Bax gene levels. Conclusions The molecular mechanism of DRG-induced apoptosis in HCT-15 cells lies in the gene transcription level of Bax and Bcl-2 genes, the increase of Bax / Bcl-2 protein expression level, and finally the activation of the classical mitochondrial pathway instead of death Receptor pathway to play a role in the induction of apoptosis of HCT-15 cells, and P53-independent.