益母草碱调节Akt/MDM2/p53信号通路对脑胶质瘤细胞恶性生物学行为的影响

The effect of Leonurine regulation on the Akt/MDM2/p53 signaling pathway on the malignant biological behavior of glioma cells

目的观察益母草碱调节蛋白激酶B(Akt)/双微体同源基因2(MDM2)/p53信号通路对脑胶质瘤细胞恶性生物学行为的影响。方法2022年3—9月于湖北省十堰市太和医院实验室进行实验。采用CCK-8法测定不同浓度(0、0.4、0.8、1.2、1.6、2.0 mmol/L)益母草碱处理的小鼠脑胶质瘤细胞GL261存活率并筛选出其最佳作用浓度。体外培养GL261细胞并随机分为对照组、益母草碱组、益母草碱+SC79(Akt激活剂)组,以益母草碱1.6 mmol/L和5μmol/L的SC79分组处理后,采用蛋白免疫印记法检测各组细胞Akt/MDM2/p53通路相关蛋白表达;采用CCK-8法、流式细胞术、细胞划痕实验和Transwell实验分别检测各组细胞增殖、凋亡、迁移、侵袭情况。构建颅内胶质瘤小鼠模型30只并随机分为对照组、益母草碱低剂量组、益母草碱中剂量组、益母草碱高剂量组及益母草碱高剂量+SC79组,分组处理后以TUNEL染色检测各组小鼠颅内胶质瘤细胞凋亡情况;以免疫印记法检测各组胶质瘤组织Akt/MDM2/p53通路相关蛋白表达。结果(1)细胞实验:与对照组比较,益母草碱组细胞凋亡率、p53蛋白表达显著升高(P<0.05),存活率、迁移率、侵袭数、p-Akt/Akt与p-MDM2/MDM2降低(P<0.05);与益母草碱组比较,益母草碱+SC79组细胞凋亡率、p53蛋白表达降低(P<0.05),存活率、迁移率、侵袭数、p-Akt/Akt与p-MDM2/MDM2升高(P<0.05)。(2)动物实验:与对照组比较,益母草碱组胶质瘤组织细胞凋亡率、p53蛋白表达升高(t/P=20.076/<0.001、7.486/<0.001),p-Akt/Akt与p-MDM2/MDM2降低(t/P=11.769/<0.001、7.579/<0.001);与益母草碱组比较,益母草碱+SC79组胶质瘤组织细胞凋亡率、p53蛋白表达降低(t/P=18.328/<0.001、7.359/<0.001),p-Akt/Akt与p-MDM2/MDM2升高(t/P=9.640/<0.001、5.529/<0.001)。结论益母草碱可通过抑制Akt/MDM2/p53信号而抑制脑胶质瘤细胞增殖、迁移、侵袭及在小鼠体内生长,并诱导其凋亡,最终抑制其恶性进展。

Objective To observe the effect of Leonurine regulated protein kinase B(Akt)/bimicrosomal homologous gene 2(MDM2)/p53 signaling pathway on the malignant biological behavior of glioma cells.Methods The experiment was conducted in the laboratory of Taihe Hospital in Shiyan City,Hubei Province from March to September 2022.The CCK-8 method was used to determine the survival rate of mouse glioma cells GL261 treated with Leonurine at different concentrations(0,0.4,0.8,1.2,1.6,2.0 mmol/L)and screen for their optimal concentration of action.GL261 cells were cultured in vitro and randomly divided into control group,Leonurine group,and Leonurine+SC79(Akt activator)group,with 1.6 mmol/L Leonurine and 5μmol/L SC79 after grouping,protein immunoblotting was used to detect the expression of Akt/MDM2/p53 pathway related proteins in each group of cells;CCK-8 method,flow cytometry,cell scratch assay,and Transwell assay were used to detect the proliferation,apoptosis,migration,and invasion of cells in each group.30 intracranial glioma mouse models were constructed and randomly divided into a control group,a low-dose group of Leonurine,a medium dose group of Leonurine,a high-dose group of Leonurine,and a high-dose+SC79 group of Leonurine.After grouping,the apoptosis of intracranial glioma cells in each group of mice was detected using TUNEL staining;Immunoblotting was used to detect the expression of Akt/MDM2/p53 pathway related proteins in glioma tissues of each group.Results(1)Cell experiment:Compared with the control group,the Leonurine group showed an increase in cell apoptosis rate and p53 protein expression(P<0.05),while survival rate,migration rate,invasion number,p-Akt/Akt,and p-MDM2/MDM2 decreased(P<0.05).Compared with the Leonurine group,the Leonurine+SC79 group showed a decrease in cell apoptosis rate and p53 protein expression(P<0.05),while survival rate,migration rate,invasion number,p-Akt/Akt,and p-MDM2/MDM2 increased(P<0.05).(2)Animal experiment:Compared with the control group,the Leonurine group showed an increase in cell apoptosis rate and p53 protein expression in glioma tissue(t/P=20.076/<0.001,7.486/<0.001),while p-Akt/Akt and p-MDM2/MDM2 decreased(t/P=11.769/<0.001,7.579/<0.001);Compared with the Leonurine group,the Leonurine+SC79 group showed a decrease in cell apoptosis rate and p53 protein expression in glioma tissue(t/P=18.328/<0.001,7.359/<0.001),while p-Akt/Akt and p-MDM2/MDM2 increased(t/P=9.640/<0.001,5.529/<0.001).Conclusion Leonurine can inhibit the proliferation,migration,invasion,and growth of glioma cells in mice by inhibiting Akt/MDM2/p53 signaling,and induce apoptosis,ultimately inhibiting their malignant progression.