CAJ | 학술논문

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（Yincheng extract, 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。
목적：채용분자생물학기술방법탐토담홍소유도신경모류세포（SH-SY5Y）조망화인진수제물（Yincheng extract, YCE）적보호궤제。방법새서람（MTT）관찰인진제취물대SH-S5Y5세포증식영향，Western blotting관찰NF-KB단백핵질분포，면역형광현미경관찰JC-1탐침표기선립체전위변화，류식세포의Annexin-FITC/PI관찰세포조망，형광정량PCR관찰IL-1β、TNF-α변화。결과인진제취물농도적시간의뢰성제고담홍소대세포증식적억제작용（p<0.05）。담홍소촉진NF-KB핵전이，인진제취물억제NF-KB핵전이。면역형광현미경관찰도담홍소처리조여정상조대비선립체막전위명현강저（p<0.05）표현조기조망특성。인진제취물억제선립체막전위강저（p<0.05）。류식세포Annexin-FITC/PI결과현시，담홍소유도세포조망，YCE보호담홍소유도적조망。형광정량PCR결과현시인진제취물억제NF-KB전록인자조공적염증상관단백IL-1β、TNF-α기인적표체（p<0.05）。결론담홍소명현유도SH-S5Y5세포조망，YCE능유효억제NK-KB격활화억제선립체막전위변화，정현다파점억제담홍소유도적세포조망。
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 detect 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 inhibited NF-KB nuclear transfer that was promoted by bilirubin. On immunofluorescence microscopy, mitochondrial mem?brane potentials decreased significantly in the bilirubin treated group compared with the control group (p<0.05), consis?tent with early apoptosis, which was seen reversed by YCE treatment (p<0.05). Flow cytometry annexin-FITC/PI results showed that bilirubin induced apoptosis was countered by YCE. Fluorescence quantitative PCR showed that the expression of inflammation-related 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 acti?vation and mitochondrial membrane potential changes, and thus inhibit apoptosis induced by bilirubin at multiple target points.