葛根素对MC3T3-E1成骨前体细胞增殖能力的影响及其机制

摘要

Objective To investigate the effect of puerarin on the proliferation ability of osteoblast MC 3T3-E1 and its possible mechanism.Methods MC3T3-E1 cells in the logarithmic growth were selected and treated with different concen-trations of puerarin at different time.CCK-8 was used to detect the effect on cell proliferation.The results showed that the cell proliferation ability was the highest when the cells were treated with 0.1 μmol/L puerarin for 48 h.The Mc3t3-e1 cells were randomly divided into the blank control group, the puerarin group, and the estrogen group, respectively, and were added with the complete medium of α-MEM,0.1 μmol/L puerarin,and 0.01 μmol/L estrogen.After 48 h,we observed cell autophagy under electron microscope.Western blotting was used to detect phosphate-activated mitogen protein kinase (p-AMPK),microtubule associated protein 1 light chain 3(LC3)-Ⅱ/LC3Ⅰexpression.The effects of interfering AMPK on cell autophagy and LC3-Ⅱ/LC3-Ⅰexpression of MC3T3 E1 treated with puerarin.Results The cell organelles,nuclei and chromosome were normal in the blank control group.The cytoplasm of puerarin and oestrogens showed a large number of annular structures,and the substance had bilayer membrane structure specific to autophagy.The expression of p-AMPK and LC3-Ⅱ/LC3-Ⅰin the puerarin group and estrogen group was significantly higher than that of blank control group,and the estrogen group was significantly higher than the puerarin group(all P<0.01).Interfering AMPK expression inhibited puera-rin-mediated MC3T3-E1 cell autophagy,and decreased the LC3-Ⅱ/LC3-Ⅰexpression(all P<0.01).Conclusion Puerarin can enhance the proliferation of MC3T3-E1 cells and the activation of AMPK/LC3 signaling pathway-mediated autophagy is one of the possible mechanisms.%目的 探讨葛根素对MC3T3-E1成骨前体细胞增殖能力的影响及其可能的作用机制.方法 取对数生长期的MC3T3-E1细胞,加入不同浓度葛根素作用不同时间,采用CCK-8法检测细胞增殖能力;结果显示,葛根素浓度为0.1 μmol/L、作用时间为48 h时细胞增殖能力最强(以下实验采用葛根素浓度为0.1 μmol/L、作用时间为48 h).将MC3T3-E1细胞随机分为空白对照组、葛根素组和雌激素组,分别加入α-MEM完全培养基、0.1 μmol/L葛根素和0.01 μmol/L雌激素.作用48 h,电镜下观察细胞自噬情况,Western blotting法检测磷酸化的丝裂原活化蛋白激酶(p-AMPK)、微管相关蛋白1轻链3(LC3)-Ⅱ/LC3Ⅰ表达.观察干扰AMPK对葛根素作用后MC3T3-E1细胞自噬及LC3-Ⅱ/LC3-Ⅰ表达的影响.结果 空白对照组细胞器、细胞核和染色体形态正常;葛根素组和雌激素组细胞质内可见较多环状结构物质,该物质具有自噬体特异性的双层膜结构.葛根素组、雌激素组p-AMPK及LC3-Ⅱ/LC3-Ⅰ均明显高于空白对照组,雌激素组均明显高于葛根素组(P均<0.01).干扰AMPK表达可减轻葛根素介导的MC3T3-E1细胞自噬程度,并降低细胞 LC3-Ⅱ/LC3-Ⅰ表达(P均 <0.01).结论 葛根素可增强MC3T3-E1细胞增殖能力;激活AMPK/LC3信号通路介导的自噬水平是其可能的机制之一.