柯里拉京对胶质瘤U251细胞及胶质瘤干细胞NF-κBP65、IKB-α表达的影响

Effect of corilagin on IKB-α and nuclear factor-κB P65 in U251 glioma cells and glioma stem cells

目的探讨柯里拉京对胶质瘤U251细胞及胶质瘤干细胞增殖及核因子-κB（NF-κB）P65、NF-κB抑制蛋白（IκB-α）表达的影响。方法采用免疫磁珠法从胶质瘤U251细胞中分选、培养胶质瘤干细胞。使用不同浓度（0、25、50、100肛勘nL）柯里拉京处理胶质瘤U251细胞及胶质瘤干细胞48h。显微镜下观察其形态变化，采用CCK-8法检测细胞存活率，采用双荧光素酶报告法检测细胞中P65基因启动子表达情况，采用Westernblotting检测细胞浆中IKB-α蛋白及细胞核中NF-κBP65蛋白表达情况。结果（1）镜下观察显示：柯里拉京干预胶质瘤U251细胞及胶质瘤干细胞后，细胞出现皱缩，细胞密度降低，结构不完整，并可见较多散在的细胞碎片。（2）CCK-8法检测显示：随着柯里拉京浓度f0、25、50、100μg／mL）的增加，胶质瘤U251细胞及胶质瘤干细胞的存活率逐渐降低，各浓度间差异均有统计学意义（P〈0．05）；在相同浓度条件下，胶质瘤干细胞存活率明显低于胶质瘤U251细胞，差异均有统计学意义（P〈0．05）。（3）双荧光素酶报告法检测显示：与0μg／mL柯里拉京组相比，25μg／mL柯里拉京干预后胶质瘤U251细胞及胶质瘤干细胞P65基因启动子表达明显增多，差异均有统计学意义（P〈0．05）；但50、100μg／mL柯里拉京组P65基因启动子表达逐渐减少。各浓度间差异均有统计学意义（P〈0．05）。（4）Westernblotting检测显示：随着柯里拉京浓度（0、25、50、100μg/mL）的增加，胶质瘤U25l细胞及胶质瘤干细胞细胞浆中IKB-α蛋白表达逐渐降低，而细胞核中NF-κBP65蛋白表达逐渐增多，各浓度间差异均有统计学意义（P〈0．05）。结论柯里拉京能抑制1965基因启动子表达，诱导IKB-α蛋白表达，减少活化的NF—κBP65蛋白进入细胞核内，进而产生抑制NF-κB信号通路的作用，这可能是其抑制胶质瘤细胞及胶质瘤干细胞增殖的重要机制之一。

Objective To explore the effect of corilagin on proliferation of glioma U251 cells and glioma stem cells and IKB-α and nuclear factor （NF）-κB P65 protein expressions in these cells. Methods The glioma stem cells were isolated from glioma U251 cells by using immune magnetic beads. The cells were intervened by different corilagin concentrations （0, 25, 50 and 100 μg/mL） for 48 h, respectively. Cell morphology changes were observed by microscope; cell counting kit （CCK）-8 assay was used to detect the cell proliferation; dual-luciferase reporter assay was employed to detect the 1965 gene promoter expression; Western blotting was used to investigate the protein expressions of IκB-α in cytoplasm and NF-κB P65 in nucleus. Results （1） Cell morphology observation results showed that the cells became shrunken, cell density was decreased, and cell structure was destroyed with a great deal of cell debris. （2） CCK-8 assay results showed that as compared with those in the 0 μg/mL corilagin group, the survival rates of U251 glioma cells and glioma stem cells were significantly decreased in the 25, 50 and 100 txg/mL corilagin groups （P〈0.05）; while in the presence of the same corilagin concentration, the survival rate of U251 glioma cells was significantly higher than that of glioma stem cells （P〈0.05）. （3） Dual-luciferase reporter assay results showed that as compared with the 0 μg/mL group, the P65 gene promoter expressions of U251 glioma cells and glioma stem cells in the 25 μg/mL corilagin group were significantly increased （P〈0.05）, but with increasing concentrations of corilagin, the expressions were gradually decreased. （4） Western blotting results showed that the IκB-α expressions in cytoplasm of U251 cells and U251 stem cells were significantly increased, but the NF-κB P65 expression in nucleus of U251 cells and U251 stem cells was significantly decreased with increasing concentrations of corilagin （0, 25, 50 and 100 μg/mL）, with signficant differences between each two groups （P〈0.05）. Conclusion Corilagin could inhibit the expression of P65 gene promoter, promote the IKB-α protein expression in cytoplasm, reduce NF-κB P65 protein into the nucleus, thereby to inhibit the NF-κB signaling pathway, and it is likely to be one of the important mechanisms to inhibit the proliferation of glioma cells and glioma stem cells.