摘要
目的探讨薯蓣皂苷元对人骨肉瘤细胞自噬的影响。方法MTT法检测薯蓣皂苷元对人骨肉瘤MG63和U2OS细胞增殖的影响,透射电镜技术观察细胞超微结构,免疫荧光法观察细胞自噬蛋白Beclin1表达,Western blot法检测细胞自噬分子标记物LC3、Beclin1和PI3K/Akt/mTOR信号通路相关蛋白表达。薯蓣皂苷元联合PI3K通路抑制剂LY294002处理MG63和U2OS细胞,RT-qPCR法检测细胞Beclin1 mRNA表达;联合自噬抑制剂3-methyladenine(3-MA)处理MG63和U2OS细胞,MTT法检测细胞增殖抑制率,Western blot法检测细胞cleaved-caspase3蛋白表达,RT-qPCR法检测细胞caspase3 mRNA表达。结果薯蓣皂苷元能抑制骨肉瘤MG63和U2OS细胞增殖,促使自噬体生成,增加LC3Ⅱ、Beclin1蛋白表达(P<0.05,P<0.01),降低LC3 I蛋白表达(P<0.01),可抑制PI3K/Akt/mTOR通路蛋白磷酸化水平(P<0.05,P<0.01);联合自噬抑制剂干预后,薯蓣皂苷元对骨肉瘤细胞的增殖抑制作用降低,并下调了细胞caspase3 mRNA、cleaved-caspase3蛋白表达(P<0.01)。结论薯蓣皂苷元能抑制骨肉瘤细胞增殖,诱导细胞产生自噬,可能与激活PI3K/Akt/mTOR信号通路以及上调LC3Ⅱ和Beclin1蛋白表达有关,同时其诱导的自噬可促使骨肉瘤细胞死亡,诱导自噬性凋亡。
AIM To investigate the effects of diosgenin on autophagy of human osteosarcoma cells.METHODS Human osteosarcoma MG63 and U2OS cells with or without exposure to diosgenin had their proliferation detected by MTT assay,their ultrastructure observed by transmission electron microscopy,their expression of autophagy protein Beclin1 observed by immunofluorescence staining,and their expressions of autophagy molecular markers LC3,Beclin1 and PI3K/Akt/mTOR signaling pathway related proteins detected by Western blot.The MG63 and U2OS cells cotreated with diosgenin and PI3K pathway inhibitor LY294002 had the expression of Beclin 1 mRNA detected by RT-qPCR.The MG63 and U2OS cells cotreated with autophagy inhibitor 3-methyladenine(3-MA)had their inhibition rate of proliferation detected by MTT assay,their expression of cleaved-caspase3 protein detected by Western blot,and their expression of caspase 3 mRNA detected by RT-qPCR.RESULTS Upon osteosarcoma MG63 and U2OS cells,diosgenin inhibited their proliferation,promoted the generation of autophagosomes,increased the protein expression of LC3Ⅱand Beclin1(P<0.05,P<0.01),reduced the protein expression of LC3 I(P<0.01),and inhibited the protein phosphorylation level of PI3K/Akt/mTOR pathway(P<0.05,P<0.01),whose effects were offset by the intervention with autophagy inhibitors in terms of the reduced proliferation inhibition and down-regulated expressions of caspase 3 mRNA and cleaved-caspase3 protein(P<0.01).CONCLUSION Diosgenin can inhibit the proliferation of osteosarcoma cells and induce their autophagy leading to their death and autophagy apoptosis,which may be related to the activation of PI3K/Akt/mTOR signaling pathway and up-regulation of the expression of LC3Ⅱand Beclin1 proteins.
作者
聂超
黄华明
侯保权
周杰
张蕾
NIE Chao;HUANG Hua-ming;HOU Bao-quan;ZHOU Jie;ZHANG Lei(Jiangsu Health Vocational College,Nanjing 211800,China;Xishan People’s Hospital of Wuxi,Wuxi 214015,China;Nanjing DIMA Biotechnology Co.,Ltd.,Nanjing 210033,China)
出处
《中成药》
CAS
CSCD
北大核心
2024年第1期100-106,共7页
Chinese Traditional Patent Medicine
基金
江苏省自然科学基金(BK20191499)
江苏高校青蓝工程团队(苏教师函[2020]10号)
江苏高校自然科学基金(18KJB320003)
江苏卫生健康职业学院重大项目(JKA201901)。
