“Sirt1-自噬”在低氧诱导小鼠成牙骨质细胞凋亡中的作用

Role of "Sirt1-autophagy" on hypoxia-induced apoptosis in mice cementoblasts

摘要:

目的： 初步研究“Sirt1-自噬”在低氧诱导小鼠成牙骨质细胞OCCM-30凋亡中的作用。方法： 体外培养小鼠成牙骨质细胞，随机分为对照组、低氧组、低氧+Sirt1激活剂白藜芦醇组、低氧+自噬抑制剂3-MA组、低氧+白藜芦醇+Sirt1抑制剂ex527组、低氧+白藜芦醇+3-MA组，MDC染色观察自噬溶酶体变化；LC3 Ⅱ免疫荧光检测自噬体变化；透射电镜观察自噬体；Western blot检测Sirt1、LC3Ⅱ、p62等蛋白表达；流式细胞仪检测细胞凋亡情况；不同组间结果数据采用单因素方差分析，组间采用LSD-t分析。结果： 低氧组较对照组自噬溶酶体含量（P=0.000）及自噬体数量（P=0.000）明显增加，LC3Ⅱ蛋白表达明显增加（P=0.001），p62蛋白表达明显减少（P=0.000），细胞凋亡率增加（P=0.000）；低氧+白藜芦醇组较低氧组自噬溶酶体（P=0.019）及自噬体数量（P=0.000）、LC3 Ⅱ蛋白表达（P=0.049）、Sirt1蛋白表达（P=0.000）明显增加，p62蛋白表达明显减少（P=0.000），细胞凋亡率减少（P=0.021）；低氧+白藜芦醇+ex527组较低氧+白藜芦醇组自噬溶酶体（P=0.000）及自噬体数量（P=0.000）、LC3 Ⅱ蛋白表达（P=0.000）、Sirt1蛋白表达（P=0.000）明显减少，p62蛋白表达明显增加（P=0.000），细胞凋亡率增加（P=0.000）；低氧+白藜芦醇+3-MA组较低氧+白藜芦醇组自噬溶酶体（P=0.000）及自噬体数量（P=0.000）、LC3 Ⅱ蛋白表达明显减少（P=0.000），p62蛋白表达明显增加（P=0.000），细胞凋亡率增加（P=0.000），Sirt1蛋白表达减少（P=0.146），差异无统计学意义。结论： 自噬相关分子可能是低氧环境下Sirt1的下游作用分子，自噬在低氧诱导小鼠成牙骨质细胞（OCCM-30）凋亡中起保护作用。

Abstract:

Objective: To investigate the role of "Sirt1-autophagy" on the hypoxia-induced apoptosis in OCCM-30 cementoblasts of mouse. Methods: OCCM-30 cells were cultured in vitro and were randomly divided into the control group, the hypoxia group, the hy-poxia + resveratrol group, the hypoxia + 3-MA group, the hypoxia + resveratrol + ex527 group and the hypoxia + resveratrol + 3-MA group. Changes of autophagy lysosomes in each group were observed by MDC staining and changes of autophagosome light chain Ⅱ (LC3 Ⅱ) were detected by immunofluorescence assay. The expression of Sirt1, LC3 Ⅱ and p62 proteins was detected by Western blot. Cell apoptosis was detected by flow cytometry. Results of different groups were analyzed by one-way ANOVA and different groups were analyzed by LSD-t. Results: Compared with the control group, the content of autophagy lysosomes (P=0.000) and the number of autophagosomes (P=0.000) in the hypoxia group were significantly increased, the expression of LC3 Ⅱ (P=0.001) protein was signifi-cantly increased, the expression of p62 protein was significantly decreased (P=0.000), and the apoptosis rate was increased (P=0.000). Compared with the hypoxia group, the number of autophagy lysosomes (P=0.019) and autophagosomes (P=0.000), and the expression of LC3 Ⅱ protein (P=0.000) and Sirt1 protein (P=0.049, P=0.000) in the hypoxia + resveratrol group were significantly increased, the expression of p62 protein (P=0.000) was signifi-cantly decreased, and the apoptosis rate was decreased (P=0.021). Compared with the hypoxia + resveratrol group, the number of autophagy lysosomes (P=0.000) and autophagosomes (P=0.000), and the expression of LC3 Ⅱ (P=0.000) and Sirt1 (P=0.000) in the hypoxia + resveratrol + ex527 group were sig-nificantly decreased, the expression of p62 protein (P=0.000) was significantly increased, and the apoptosis rate was increased (P=0.000). Compared with the hypoxia + resveratrol group, the num-ber of autophagy lysosomes (P=0.000) and autophagosomes (P=0.000), and the expression of LC3 Ⅱ (P=0.000) protein expression in the hypoxia + resveratrol + 3-MA group were significantly decreased, the expression of p62 protein (P=0.000) was increased, the apoptosis rate was increased (P=0.000), and the expression of Sirt1 protein (P=0.146) was decreased, with no statistically significant difference. Conclusion: Autophagy-related molecules may be downstream acting molecules of Sirt1 in the hypoxic environment and autophagy plays a protective role in hypoxia-induced apoptosis of OCCM-30.