自噬对糖皮质激素作用下MC3T3-E1成骨细胞增殖的影响

Effect of autophagy on the proliferation of MC3T3-E1 osteoblasts induced by glucocorticoid

背景:目前,糖皮质激素(GCs)作用下成骨细胞自噬与其增殖能力的关系尚存争议。目的:探讨自噬对GCs作用下MC3T3-E1成骨细胞增殖能力的影响。方法:不同浓度(0、10-8 M、10-6 M、10-4 M)的地塞米松(DXMS)分别作用于MC3T3-E1成骨细胞,CCK-8检测不同时间(12 h、24 h、48 h)的细胞增殖能力;以Western Blot、细胞免疫荧光法检测细胞培养24 h的Beclin-1和LC-3表达水平,透射电镜法检测自噬小体数目,明确不同浓度DXMS作用下成骨细胞自噬变化特点;在10-6 M DXMS培养的成骨细胞中加入自噬激活剂雷帕霉素(RAP),以上述同样的技术检测自噬及细胞增殖能力的变化,以明确GCs作用下自噬对MC3T3-E1成骨细胞增殖能力的影响。结果:CCK-8结果示,DXMS浓度越高,对MC3T3-E1成骨细胞增殖能力的抑制作用越强,其中10-8M DXMS对细胞增殖能力无明显影响(P>0.05),10-6M DXMS对细胞增殖能力抑制较明显,10-4M DXMS对细胞增殖能力抑制作用最为明显。Western Blot、细胞免疫荧光法及透射电镜法检测结果示,随着DXMS浓度的增加,Beclin-1和LC-3B的表达水平均明显提高,自噬小体数量明显增加;在10-6M DXMS培养的成骨细胞中加入自噬激活剂RAP后,Beclin-1和LC-3B的表达水平及自噬小体数量进一步提高,而成骨细胞增殖能力变强。结论:GCs作用下自噬对成骨细胞增殖起保护作用,随着GCs浓度的增高自噬水平明显增加但相对不足,不能抵抗高浓度GCs对细胞增殖的抑制作用,提高自噬水平有助于促进成骨细胞增殖。

Background:The relationship between autophagy and proliferation of osteoblasts under the action of glucocorti⁃coid is still controversial.Objective:To investigate the effect of autophagy on the proliferation of MC3T3-E1 osteoblasts under the action of glucocorticoid.Methods:Different concentrations of DXMS(0,10-8 M,10-6 M,10-4 M)were used to treat MC3T3-E1 osteoblasts,and CCK-8 was used to detect the proliferation of osteoblasts at different times(12 h,24 h,48 h).Western blot and cell immunofluorescence were used to detect the expression of Beclin-1 and LC-3 at 24 hours,and the number of autopha⁃gosomes was detected by transmission electron microscope to determine the characteristics of autophagy of osteoblasts treated with different concentrations of DXMS.Autophagy activator Rapamycin was added to osteoblasts cultured with 10-6 M DXMS to detect the changes of autophagy and cell proliferation by the same technique mentioned above,and to clarify the effect of au⁃tophagy on the proliferation of MC3T3-E1 osteoblasts under the action of glucocorticoid.Results:CCK-8 results showed that the higher the concentration of DXMS was,the stronger its inhibiting effect on MC3T3-E1 was.DXMS of 10-8 M had no effect on the proliferation of MC3T3-E1 osteoblasts(P>0.05);DXMS of 10-6 M showed significant effect(P<0.05);DXMS of 10-4 M showed most significant effect(P<0.05).Western blot,cell immunofluorescence and transmission electron microscope showed that with the increase of the concentration of DXMS,the expression levels of Beclin-1 and LC-3B increased consequently and the number of autophagosomes also significantly increased.The expression levels of Beclin-1 and LC-3B and the number of autophagosomes further increased after the adding of Rapamycin into osteoblasts cultured by DXMS of 10-6 M,and the prolifer⁃ation of osteoblasts also increased.Conclusions:Autophagy has a protective effect on the proliferation of osteoblasts under glu⁃cocorticoid.With the increase of glucocorticoid concentration,the level of autophagy increased but insufficient,which cannot resist the inhibitory effect of high glucocorticoid concentration on cell proliferation.Increasing the level of autophagy is helpful to promote the proliferation of osteoblasts.