南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (12): 1816-1820.doi: 10.12122/j.issn.1673-4254.2020.12.18

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自噬在脂多糖诱导成牙本质细胞凋亡中的作用

熊华翠，陈 柯，李美美   

  • 出版日期:2020-12-20 发布日期:2020-12-28

Role of autophagy in lipopolysaccharide-induced apoptosis of odontoblasts

  • Online:2020-12-20 Published:2020-12-28

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摘要： 目的 探讨自噬在脂多糖（LPS）诱导成牙本质细胞（mDPC-23）凋亡中的作用。方法 将5 μg/mL脂多糖作用于成牙本质细胞0、6、12、24 h后，CCK8检测细胞活力，TUNEL染色检测细胞凋亡。5 μg/mL脂多糖作用细胞24 h后，用Western blot检测自噬标志蛋白LC3、Beclin1和Atg5以及自噬相关通路AKT、p-AKT、mTOR和p-mTOR的表达水平。以脂多糖（5 μg/mL）和脂多糖（5 μg/mL）+3-甲基腺嘌呤（3-MA，5 mmol/L）为实验组，不加脂多糖或3-MA为空白对照组，作用细胞24 h后，Western blot检测凋亡蛋白Caspase 3和Bax的表达水平。结果 脂多糖作用mDPC-23细胞6、12 h，细胞增殖能力和凋亡无明显变化；作用24 h后，其增殖能力较对照组显著降低（P<0.05），凋亡水平较作用0 h明显增加（P<0.05）。且脂多糖作用mDPC-23细胞24 h后，自噬标志蛋白LC3、Beclin1和Atg5表达较对照组均明显增加（P<0.05），通路相关蛋白p-AKT和p-mTOR较对照组表达下降（P<0.05）。脂多糖组凋亡蛋白Caspase 3和Bax显著高于对照组（P<0.05）；脂多糖+3-MA组中凋亡蛋白明显低于脂多糖组（P<0.05）。结论 脂多糖可通过诱导mDPC-23自噬发生以促进细胞凋亡；而抑制自噬，可减弱脂多糖的促凋亡作用，提示在炎性牙髓组织损伤中，自噬发挥重要作用。

关键词: 自噬；凋亡；脂多糖

Abstract: Objective To investigate the role of autophagy in lipopolysaccharide (LPS)-induced apoptosis of murine odontoblasts. Methods Murine odontoblasts (mDPC-23 cells) were treated with 5 μg/mL LPS for 6, 12 and 24 h, and the changes in cell viability was examined using CCK8 kit and cell apoptosis was detected by TUNEL staining. The changes in the protein levels of LC3, Beclin1, Atg5, AKT, p-AKT, mTOR and p-mTOR were detected using Western blotting. The effect of 3-MA treatment for 24 h on LPS-induced apoptosis of mDPC-23 cells was evaluated by detecting the expressions of apoptosis-related proteins caspase-3 and Bax using Western blotting. Results Stimulation with LPS for 6 and 12 h did not cause significant changes in the proliferation or apoptosis of mDPC-23 cells, but LPS treatment for 24 h significantly suppressed cell proliferation (P<0.05) and promoted cell apoptosis as shown by TUNEL assay (P<0.05). Stimulation with LPS for 24 significantly increased the expression levels of LC3, Beclin1 and Atg5, decreased the expressions of p-AKT and p-mTOR (P<0.05), and obviously upregulated the expressions of caspase-3 and Bax (P<0.05). Treatment with 3-MA markedly lowered caspase-3 and Bax protein expressions in LPS-stimulated cells (P<0.05). Conclusion LPS stimulation induces autophagy to promote apoptosis of mDPC-23 cells, and suppression of autophagy attenuates LPS-induced apoptosis. Autophagy may play an important role in the injury of inflamed pulp tissues.

Key words: autophagy; apoptosis; lipopolysaccharide