自噬
碘化丙啶
细胞凋亡
膜联蛋白
活力测定
肿瘤坏死因子α
化学
程序性细胞死亡
细胞生物学
成骨细胞
MTT法
癌症研究
下调和上调
分子生物学
骨细胞
ATG5型
坏死
生物
免疫学
体外
生物化学
基因
遗传学
作者
Liwen Zheng,Wanchun Wang,Jiangdong Ni,Xinzhan Mao,Deye Song,Tang Liu,Jianwei Wei,Hua-ying Zhou
出处
期刊:Journal of Investigative Medicine
[BMJ]
日期:2017-08-01
卷期号:65 (6): 1014-1020
被引量:38
标识
DOI:10.1136/jim-2017-000426
摘要
The aim of this study is to investigate the role of tumor necrosis factor-α (TNF-α) in apoptosis and autophagy of mouse osteoblast MC3T3-E1 cells, as well as the crosstalk between autophagy and apoptosis. Mouse osteoblast MC3T3-E1 cells were cultured in vitro and treated with 5-fluorouracil (5-FU), rapamycin, 3-methyl adenine (3-MA) and TNF-α either alone or in combination, respectively. MTT assays were used to monitor the cell viability upon different treatments. Annexin-V-FITC/propidium iodide (PI) staining was used to detect the apoptotic rate of osteoblasts. Autophagic structure and apoptotic bodies were visualized by transmission electron microscopy (TEM). Western blot analysis was performed to detect the autophagic marker LC3-II/I, p62 and apoptotic marker cleaved caspase-3. TNF-α inhibits MC3T3-E1 cell viability in a dose-dependent and time-dependent manner. Annexin-V-FITC/PI staining, coupled with TEM, showed that TNF-α induced cell apoptosis and autophagy in MC3T3-E1 cells. The autophagy inducer rapamycin ameliorated TNF-α-induced apoptosis. In contrast, 3-MA, which is an autophagy inhibitor, caused an exaggerated induction of TNF-α-induced apoptosis. TNF-α upregulated autophagy marker LC3-II/I, but downregulated p62 in osteoblasts. Combined treatment of rapamycin and TNF-α further exaggerated this effect, whereas co-treatment of 3-MA and TNF-α decreased LC3-II/I, but increased p62 compared with TNF-α alone. In addition, TNF-α caused an induction of apoptotic marker cleaved caspase-3. TNF-α-mediated induction of cleaved caspase-3 was downregulated by rapamycin, but upregulated by 3-MA, respectively. TNF-α induced both autophagy and apoptosis in osteoblasts, and upregulated autophagy protects the cell by reducing TNF-α-induced apoptosis.
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