二甲双胍通过激活自噬促进MC3T3-E1细胞系成骨分化的研究

Research on metformin promotes osteoblastic differentiation of MC3T3-E1 cells by activating autophagy

目的探讨二甲双胍通过激活自噬促进MC3T3-E1细胞系成骨分化的作用。方法用含有不同浓度二甲双胍的成骨诱导剂处理MC3T3-E1细胞,其中二甲双胍的浓度分别为0(对照)、200、400、800μmol/L。利用碱性磷酸酶(ALP)染色检测二甲双胍促进MC3T3-E1细胞成骨的最适浓度,采用免疫印迹法(Western blotting)和免疫荧光法检测自噬相关蛋白,并选择出二甲双胍的最佳干预浓度。对照组、二甲双胍400μmol/L组、二甲双胍400μmol/L+3-甲基腺嘌呤(3-MA,5 mmol/L)组和3-MA 5 mmol/L组分别干预MC3T3-E1细胞4 h后,采用Western blotting法、免疫荧光法、透射电镜检测自噬指标,并利用ALP染色、半定量RT-PCR和Western blotting检测成骨能力。结果二甲双胍能够剂量相关性地促进MC3T3-E1细胞的ALP活性,并且400μmol/L二甲双胍是促进MC3T3-E1细胞成骨的最适浓度。二甲双胍0(对照)、200、400μmol/L组的LC3 Ⅱ/Ⅰ比值逐渐增大,P62/β-actin比值逐渐减小,LC3荧光强度逐渐增强;800μmol/L二甲双胍组的LC3 Ⅱ/Ⅰ值减小,P62/β-actin比例增大,LC3荧光强度下降;且与对照组比较,400μmol/L二甲双胍组的LC3Ⅱ/Ⅰ比值明显增大,P62/β-actin比值明显降低(P<0.05),LC3荧光强度最强。与对照组比较,二甲双胍400μmol/L组中LC3 Ⅱ/Ⅰ的比值增大,P62蛋白表达减少(P<0.05),LC3荧光强度增强,自噬体数量增多;ALP活性增强,成骨相关基因和蛋白OCN,COL1的表达增强(P<0.001、0.05、0.01)。二甲双胍中加入3-MA 5 mmol/L后,LC3 Ⅱ/Ⅰ的比值减小,P62蛋白表达增多,LC3荧光强度减弱,自噬体数量减少;ALP活性减弱,成骨相关基因和蛋白OCN,COL1的表达减弱。结论二甲双胍能通过激活MC3T3-E1细胞系的适度自噬促进其成骨分化。

Objective To investigate the effects of metformin promoting osteoblastic differentiation of MC3T3-E1 cells by activating autophagy. Methods MC3T3-E1 cells were treated with osteogenic inducers containing different concentrations of metformin, and the concentrations of metformin were 0(control), 200, 400, and 800 μmol/L, respectively. The optimal concentration of metformin in MC3T3-E1 cells was determined by ALP staining. Western blotting and immunofluorescence were used to detect autophagy-related proteins, and the optimal intervention concentration of metformin was selected. The MC3T3-E1 cells in control group, metformin 400μmol/L group,metformin 400 μmol/L + 3-MA 5 mmol/L group, and 3-MA 5 mmol/L group were interfered for 4 h, respectively.Autophagy indexes were detected by Western blotting, immunofluorescence, and transmission electron microscopy methods. And the ALP staining, semi-quantitative RT-PCR, and Western blotting methods were used to detect the osteogenic ability. Results Metformin could promote the ALP activity of MC3T3-E1 cells in a dose-dependent manner, and 400μmol/L was the optimum concentration for promoting the osteogenesis of MC3T3-E1 cells. In the metformin 0(control), 200, and 400 μmol/L group, the LC3 Ⅱ/Ⅰ ratio was gradually increased, the P62/β-actin ratio was gradually decreased, and the LC3 fluorescence intensity was gradually increased. In the 800 μmol/L metformin group, LC3 Ⅱ/Ⅰ ratio was decreased, P62/β-actin ratio was increased, and LC3 fluorescence intensity was gradually decreased. Compared with the control group, the LC3 Ⅱ/Ⅰ ratio in the 400 μmol/L metformin group was significantly increased, but the P62/β-actin ratio was significantly decreased(P < 0.05). And the LC3 had the strongest fluorescence intensity. Compared with the control group, the ratios of LC3 Ⅱ/Ⅰ were significantly increased in the 400 μmol/L metformin group, the expression of P62 protein was decreased(P < 0.05), the fluorescence intensity of LC3 was increased, and the number of autophagosome was increased. The activity of ALP was enhanced, and the osteogenesis related genes and protein expression of OCN and COL1 were increased(P < 0.001, 0.05, 0.01). After 3-MA 5 mmol/L was added to metformin, the ratio of LC3 Ⅱ/Ⅰ was decreased,and expression of P62 protein was increased, but the fluorescence intensity of LC3 and the number of autophagosomes were decreased. The activity of ALP and the osteogenesis related genes and protein expression of OCN and COL1 were decreased.Conclusion Metformin can promote osteogenic differentiation of MC3T3-E1 cell line by activating moderate autophagy.