摘要： 目的 探讨藁本内酯对骨肉瘤细胞增殖、迁移、凋亡的影响及其作用机制。方法 DMEM培养基培养骨肉瘤MG-63细胞，采用细胞毒性实验观察藁本内酯对MG-63细胞增殖的影响；细胞划痕实验检测藁本内酯对骨肉瘤细胞迁移的影响；流式细胞仪检测不同浓度藁本内酯对骨肉瘤细胞周期的影响。明确藁本内酯作用MG-63细胞的浓度，将细胞分为对照组、顺铂（DDP）组，藁本内酯联合顺铂（DDP+LIG）组，顺铂联合自噬抑制剂（DDP+3MA）组。Western blotting检测自噬和凋亡相关蛋白LC3B、Beclin1、Bcl-2、Bax，细胞色素C以及通路蛋白ULK1、ATG3的表达情况。免疫荧光检测各组细胞LC3B蛋白的表达情况。结果 与对照组比较，20、40、80、160、320、640 μmol/L藁本内酯作用下骨肉瘤细胞增殖均被抑制（均P < 0.05）；与对照组比较，160 μmol/L藁本内酯作用下骨肉瘤细胞迁移能力显著降低（P < 0.01）。与对照组比较，40、80、160、320、640 μmol/L藁本内酯作用下骨肉瘤细胞G2/M期细胞均显著阻滞（均P < 0.05）。与DDP组比较，DDP+LIG组Bax表达升高（P < 0.01），抗凋亡蛋白Bcl-2表达降低（P < 0.05），细胞色素C表达升高（P < 0.05），自噬蛋白LC3B、Beclin1，通路蛋白ULK1、ATG3表达均降低（均P < 0.05）；与DDP组比较，DDP+3MA组自噬荧光体数量减少。结论 藁本内酯能够抑制骨肉瘤细胞增殖，降低细胞迁移能力，导致骨肉瘤细胞G2/M期阻滞。与单独使用顺铂比较，藁本内酯联合顺铂可使骨肉瘤细胞凋亡明显增加，其作用机制可能是通过抑制自噬ULK1/ATG3信号通路实现的。

藁本内酯对骨肉瘤细胞凋亡的影响及其作用机制

于冬冬

辽宁中医药大学附属医院骨一科, 沈阳 110033

收稿日期:2022-06-23出版日期:2023-04-30发布日期:2023-04-15
通讯作者:于冬冬E-mail:dongdongyu10256@163.com
作者简介:于冬冬(1983-),男,主任医师,博士.
基金资助:辽宁省教育厅科学技术研究项目（L201912）；沈阳市科学技术计划（20-205-4-040）；辽宁中医药大学中医脏象理论及应用国家教育部重点实验室开放基金项目（zyzx1908）


关键词: 骨肉瘤, 藁本内酯, 顺铂, 自噬, 凋亡
Abstract: Objective To investigate the effect of ligustilide on proliferation, migration, and apoptosis of osteosarcoma cells and its mechanism of action. Methods Osteosarcoma MG-63 cells were cultured in DMEM medium. The effect of ligustilide on the proliferation and migration of MG-63 cells was observed by cytotoxicity assay and cell scratch assay, respectively. The effect of different ligustilide concentrations on the cell cycle was assessed by flow cytometry. Cells were divided into four groups:control, cisplatin (DDP), ligustilide combined with cisplatin (DDP + LIG), and cisplatin combined with autophagy inhibitor (DDP + 3MA). Western blotting was used to detect the expression of autophagy and apoptosis-related proteins LC3B, Beclin1, Bcl-2, Bax, and cytochrome c, as well as pathway proteins ULK1 and ATG3. Immunofluorescence was used to detect changes in LC3B expression in each group. Results Compared with the control group, the proliferation of osteosarcoma cells was inhibited when treated with 20, 40, 80, 160, 320, and 640 μmol/L ligustilide (P < 0.05). The migration ability of osteosarcoma cells was significantly reduced by treatment with 160 μmol/L ligustilide compared with the control group (P < 0.01). Significantly more cells were arrested in G2/M phase in response to 40, 80, 160, 320, and 640 μmol/L ligustilide compared with the control group (P < 0.05). Compared with the DDP group, the addition of ligustilide (DDP + LIG group) increased Bax (P < 0.01) and cytochrome c expression (P < 0.05), and decreased expression of anti-apoptotic protein Bcl-2, autophagic proteins LC3B and Beclin1, and autophagic pathway proteins ULK1 and ATG3 (all P < 0.05). Compared with the DDP group, the number of autophagic fluorescent bodies was decreased in the DDP + 3MA group. Conclusion Ligustilide inhibited proliferation, reduced cell migration, and led to G2/M phase arrest of osteosarcoma cells. Compared with cisplatin alone, ligustilide combined with cisplatin increased the apoptosis rate of osteosarcoma cells, and its mechanism of action may be through inhibition of the ULK1/ATG3 autophagy pathway.
Key words: osteosarcoma, ligustilide, cisplatin, autophagy, apoptosis
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