海南医科大学学报

目的：探究圣和散（Shenghe Powder, SHP）通过FoxO3a信号通路增强人恶性胶质母细胞瘤U251细胞放疗敏感性的作用机制。方法：以U251细胞为研究对象，设置正常对照（Control）组、SHP组、阴性对照（SHP+si-NC）组、敲低FoxO3a(SHP+si-FoxO3a)组，并利用6 MV X线对细胞进行单次剂量为5 Gy照射。采用Western blot、免疫荧光染色、CCK-8、Edu、流式细胞术、肿瘤干细胞成球法进行检测。通过建立荷瘤裸鼠模型检测瘤体变化，免疫组化检测肿瘤组织相关蛋白。结果：与Control组相比，SHP组细胞的BNIP3、FoxO3a蛋白表达明显升高，p-FoxO3a蛋白表达明显降低（P<0.01）;SHP组细胞FoxO3a的红色荧光强表达于细胞核内（P<0.001）;SHP组细胞BNIP3的绿色荧光强表达在线粒体膜上（P<0.001）。SHP+si-FoxO3a组细胞活力、阳性细胞率、细胞迁移和侵袭数量、细胞成球数量明显高于SHP+si-NC组，细胞凋亡率明显低于SHP+si-NC组（P<0.01）;SHP+si-FoxO3a组细胞的Cl-caspase3、Beclin1、LC3Ⅱ/LC3Ⅰ、BNIP3 、FoxO3a蛋白表达量明显低于SHP+si-NC组，p-FoxO3a蛋白表达量明显高于SHP+si-NC组（P<0.01）。与Control组相比，SHP组的肿瘤生长缓慢；与SHP+si-NC组相比，SHP+si-FoxO3a组的肿瘤生长缓慢。与Control组相比，SHP组肿瘤体积和重量明显减少；与SHP+si-NC组相比，SHP+si-FoxO3a组肿瘤体积和重量明显增加（P<0.01）。与Control组比较，SHP组肿瘤组织中Cl-caspase3、Beclin1、BNIP3蛋白表达明显增加（P<0.01）,p-FoxO3a蛋白表达明显降低（P<0.01）；与SHP+si-NC组相比，SHP+si-FoxO3a组肿瘤组织中Cl-caspase3、Beclin1、BNIP3蛋白表达明显降低（P<0.01）,p-FoxO3a蛋白表达明显增加（P<0.001）。结论：细胞实验表明SHP通过诱导FoxO3a去磷酸化向细胞核转位，BNIP3转位至线粒体膜，增强了FoxO3a-BNIP3信号通路；敲低FoxO3a后逆转了SHP对胶质瘤U251细胞放射敏感性的增强作用；体内实验结果也验证SHP通过FoxO3a-BNIP3通路增强人恶性胶质母细胞瘤U251细胞的放疗敏感性。

Objective: To investigate the mechanism of Shenghe Powder(SHP) in enhancing the radiotherapy sensitivity of human malignant glioblastoma U251 cells through the FoxO3a signaling pathway. Methods: U251 cells were taken as the research subject, and the normal control(Control) group, SHP group, negative control(SHP+si-NC) group, and knockdown FoxO3a(SHP+si-FoxO3a) group were set up, and the cells were irradiated with a single dose of 5 Gy 6 MV X-ray. Western blot, immunofluorescence staining, CCK-8, Edu, flow cytometry, and cancer stem cell spheroidization were used for detection. Through establishing a tumor-bearing nude mouse model, tumor changes were detected, and tumor tissue-related proteins were detected by immunohistochemistry. Results: Compared to the Control group, the expressions of BNIP3 and FoxO3a proteins of cells in the SHP group were increased, and the expression of p-FoxO3a protein was decreased(P<0.01). The red fluorescence of FoxO3a in the SHP group cells was strongly expressed in the nucleus(P<0.001). The green fluorescence of BNIP3 was strongly expressed on the mitochondrial membrane in the SHP group cells(P<0.001). The cell viability, positive cell rate, number of cell migration and invasion, and spheroidal number of cells in the SHP+si-FoxO3a group were significantly higher than those in the SHP+si-NC group, and the apoptosis rate was significantly lower than that in the SHP+si-NC group(P<0.01). The expressions of Cl-caspase3, Beclin1, LC3Ⅱ/LC3Ⅰ, BNIP3, and FoxO3a proteins in the SHP+si-FoxO3a group cells were significantly lower than those in the SHP+si-NC group, and the expression of p-FoxO3a protein was significantly higher than that in the SHP+si-NC group(P<0.01). Tumors grew slowly in the SHP group compared to the Control group; Tumor growth was slower in the SHP+si-FoxO3a group compared to the SHP+si-NC group. Compared to the Control group, the volume and weight of tumors in the SHP group were significantly reduced; Compared to the SHP+si-NC group, the tumor volume and weight of the SHP+si-FoxO3a group were significantly increased(P<0.01). Compared to the Control group, the tumor tissue expression of Cl-caspase3, Beclin1, and BNIP3 proteins in the SHP group was significantly increased(P<0.01), and the expression of p-FoxO3a protein was significantly decreased(P<0.01). Compared to the SHP+si-NC group, the tumor tissue expression of Cl-caspase3, Beclin1, and BNIP3 proteins in the SHP+si-FoxO3a group was significantly reduced(P<0.01), and the expression of p-FoxO3a protein was significantly increased(P<0.001). Conclusion: Cellular experiments shows that SHP enhances the FoxO3a-BNIP3 signaling pathway by inducing FoxO3a dephosphorylation to the nucleus, and BNIP3 translocation to the mitochondrial membrane. Knockdown of FoxO3a reverses the enhanced effect of SHP on the radiosensitivity of glioma U251 cells. In vivo experiments also confirm that SHP enhances the radiotherapy sensitivity of human malignant glioblastoma U251 cells through the FoxO3a-BNIP3 signaling pathway.