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目的研究异甘草素(ISL)对人胶质瘤细胞SHG44增殖、自噬、凋亡及相关分子信号的影响。方法应用细胞计数试剂盒(CCK-8)法检测ISL在不同时段、不同浓度下对人胶质瘤细胞SHG44的增殖抑制情况。用流式细胞术Annexin V-IP标记检测ISL(0,20,40,60,80μmol/L)作用于SHG44细胞48 h后细胞凋亡的情况,透射电镜下观测ISL对SHG44细胞内部超微结构的改变,应用免疫荧光染色法观测细胞内部及其周边自噬及凋亡相关蛋白的分布,Western blot检测ISL对SHG44细胞中Caspase-3,Bcl-2以及LC3B等蛋白表达的影响。结果在ISL作用下,SHG44细胞的增殖过程呈现时间及剂量依赖性抑制,ISL(80μmol/L)组在作用72 h后对SHG44细胞的抑制率超过了半数;流式细胞术检测ISL作用SHG44细胞48 h后,细胞凋亡率分别为(1.54±0.85)%,(6.72±2.81)%,(11.82±3.21)%,(16.61±3.79)%,(26.15±3.82)%;透射电镜下可见SHG44细胞内部出现自噬小泡及自噬溶酶体;在荧光显微镜下可见细胞内出现自噬及凋亡相关蛋白的免疫荧光颗粒;Western blot显示ISL作用于SHG44细胞48 h后,LC3-Ⅱ/Ⅰ比值以及Caspase-3的表达呈上升趋势,而Bcl-2表达呈下降趋势。结论 ISL能有效抑制人胶质瘤细胞SHG44的增殖,诱导SHG44细胞发生自噬和凋亡,并且抑制了抗凋亡分子Bcl-2的表达,增加了凋亡相关分子Caspase-3以及自噬相关分子LC3-Ⅱ/Ⅰ的比值。
Objective To study the effects of isoliquiritigenin (ISL) on the proliferation, autophagy, apoptosis and related molecular signaling in human glioma SHG44 cells. Methods The proliferation inhibition of human glioma SHG44 cells was detected by CCK-8 at different time and different concentrations. The apoptosis of SHG44 cells treated with ISL (0, 20, 40, 60 and 80 μmol / L) was detected by flow cytometry with Annexin V-IP, and the ultrastructure of SHG44 cells was observed under transmission electron microscope The changes of autophagy and apoptosis related proteins in and around the cells were observed by immunofluorescence staining. The protein expressions of Caspase-3, Bcl-2 and LC3B in SHG44 cells were detected by Western blot. Results Under the action of ISL, the proliferation of SHG44 cells was inhibited in a time and dose-dependent manner. The inhibitory rate of SHG44 cells in ISL (80μmol / L) group was more than 50% after 72 h treatment. Flow cytometry was used to detect the effect of ISL on SHG44 cells After 48 h, the apoptotic rates were (1.54 ± 0.85)%, (6.72 ± 2.81)%, (11.82 ± 3.21)%, (16.61 ± 3.79)%, (26.15 ± 3.82)%, respectively. Autophagy vesicles and autophagy lysosomes were found in the cells. Immunofluorescent particles with autophagy and apoptosis-related proteins appeared in the cells under the fluorescence microscope. Western blot showed that after treated with ISL for 48 h, LC3-Ⅱ / Ⅰ ratio and the expression of Caspase-3 showed an upward trend, while the expression of Bcl-2 showed a downward trend. Conclusion ISL can effectively inhibit the proliferation of human glioma SHG44 cells and induce autophagy and apoptosis in SHG44 cells, and inhibit the expression of anti-apoptotic molecule Bcl-2 and increase the expression of apoptosis-related molecules Caspase-3 and autophagy Molecular LC3-Ⅱ / Ⅰ ratio.