【摘要】 目的采用分子生物学技术方法探讨胆红素诱导神经母瘤细胞(SH-SY5Y)凋亡和茵陈水提物(Yinchengextract,YCE)的保护机制。方法噻唑蓝(MTT)观察茵陈提取物对SH-S5Y5细胞增殖影响,Western blotting观察NF-KB蛋白核质分布,免疫荧光显微镜观察JC-1探针标记线粒体电位变化,流式细胞仪Annexin-FITC/PI观察细胞凋亡,荧光定量PCR观察IL-1β、TNF-α变化。结果茵陈提取物浓度的时间依赖性提高胆红素对细胞增殖的抑制作用(p<0.05)。胆红素促进NF-KB核转移,茵陈提取物抑制NF-KB核转移。免疫荧光显微镜观察到胆红素处理组与正常组对比线粒体膜电位明显降低(p<0.05)表现早期凋亡特性。茵陈提取物抑制线粒体膜电位降低(p<0.05)。流式细胞Annexin-FITC/PI结果显示,胆红素诱导细胞凋亡,YCE保护胆红素诱导的凋亡。荧光定量PCR结果显示茵陈提取物抑制NF-KB转录因子调控的炎症相关蛋白IL-1β、TNF-α基因的表达(p<0.05)。结论胆红素明显诱导SH-S5Y5细胞凋亡,YCE能有效抑制NK-KB激活和抑制线粒体膜电位变化,呈现多靶点抑制胆红素诱导的细胞凋亡。更多还原

【Abstract】 Objective To investigate the mechanism of protection by capillary artemisia aqueous extract(Yincheng ex-tract, YCE) against bilirubin induced neuroblastoma apoptosis. Methods MTT was used to assess SH-S5Y5 cells prolifera-tion. Western blotting was employed to examine NF- KB protein expression in the nucleus and cytoplasm. Changes of JC-1 probe labeled mitochondrial potential were examined by immunofluorescence microscopy. Annexin-FITC/PI was used to de-tect cell apoptosis with flow cytometry. IL-1β and TNF- alpha changes were tested using quantitative fluorescence PCR. Results YCE improved concentration and time dependent inhibition of bilirubin’s effects on cell proliferation(p<0.05). YCE in-hibited NF-KB nuclear transfer that was promoted by bilirubin. On immunofluorescence microscopy, mitochondrial membranepotentials decreased significantly in the bilirubin treated group compared with the control group(p<0.05), consistent with earlyapoptosis, which was seen reversed by YCE treatment(p<0.05). Flow cytometry annexin-FITC/PI results showed that bilirubininduced apoptosis was countered by YCE. Fluorescence quantitative PCR showed that the expression of inflammation-relat-ed proteins IL-1 beta and TNF- alpha(regulated by the transcription factor NF-KB) was decreased by YCE(p<0.05). Conclusion While bilirubin can induce apoptosis of SH-S5Y5 cells, YCE can effectively reduce NF-KB activation and mitochon-drial membrane potential changes, and thus inhibit apoptosis induced by bilirubin at multiple target points.更多还原