1,25二羟维生素D3通过抑制mTOR通路而激活自噬以促进大鼠成骨细胞增殖和分化

1,25 DIHYDROXYVITAMIN D PROMOTES PROLIFERATION AND DIFFERENTIATION OF RAT OSTEOBLASTS BY ACTIVATING AUTOPHAGY THROUGH INHIBITING THE MTOR PATHWAY

目的探讨1,25二羟维生素D3[1,25(OH)2D3]对大鼠原代成骨细胞培养的第4代细胞中哺乳动物雷帕霉素靶蛋白(mTOR)的作用及其对成骨的影响。方法由新生SD大鼠颅骨获取原代成骨细胞,用培养的第4代细胞进行研究,分为对照组(control)、雷帕霉素组(rapamycin,RAPA)(20nmol/L)、MHY1485组(2nmol/L)、1,25(OH)2D3组(5nmol/L)和MHY1485+1,25(OH)2D3组,作用0、12、24、36、48、60、72、84、96、108和120h后,以CCK8检测成骨细胞增殖活性。Western blot和RT-PCR检测mTOR及下游因子真核翻译起始因子4E结合蛋白1(4E-BP1)和核糖体S6激酶1(S6K1)、自噬标志物Beclin-1、微管相关蛋白1轻链3(LC3)和成骨细胞分化相关因子碱性磷酸酶(ALP)、成骨相关转录因子(Osterix)和Runt相关转录因子2(Runx2)的表达,ALP染色观察各组细胞内ALP含量变化。以上分组条件作用21d后茜素红染色检测各组矿化情况。结果与对照组比较,1,25(OH)2D3明显促进成骨细胞增殖,逆转MHY1485抑制的成骨细胞增殖活性。Western blot和RT-PCR结果显示1,25(OH)2D3抑制mTOR、4E-BP1和S6K1的蛋白磷酸化,并使自噬标志物Beclin-1和LC3-Ⅱ、分化标志物ALP、Osterix和Runx2表达显著增加。ALP染色和茜素红染色结果显示,1,25(OH)2D3早期促进ALP的分泌,晚期促进矿化结节形成。结论 1,25(OH)2D3可以促进大鼠成骨细胞的增殖、分化与矿化,其可能与其通过抑制mTOR信号通路而激活自噬有关。[营养学报,2021,43(1):71-78,85,96]

Objective To study the effects of 1,25 dihydroxyvitamin D3[1,25(OH)2D3]on mammalian targets of rapamycin (mTOR) and osteogenesis.Methods The osteoblasts were obtained from the cultured skulls of SD rats and divided into the control group,rapamycin group (RAPA,20 nmol/L),MHY1485 group (2 nmol/L),1,25 (OH)2D3 group (5 nmol/L)and MHY1485+1,25 (OH)2 D3 group.After 0,12,24,36,48,60,72,84,96,108 and 120 h of the treatments,the proliferation activity of osteoblasts was determined by CCK8 assay.Cell cycle detection kits were used to observe the cell cycle distribution after 48 h culture.Western blot and RT-PCR were applied to detect mTOR and downstream factor e IF4E-binding protein 1 (4E-BP1) and ribosome protein subunit 6 kinase 1 (S6K1),autophagy marker Beclin-1,microtubuler associated protein 1 light chain 3(LC3),osteoblast differentiation related factors including alkaline phosphatase (ALP),osteogenesis related transcription factors (Osterix) and the expression of transcription factor 2 (Runx2).ALP content changes in cells were observed with ALP staining.Alizarin red staining was used to detect the mineralization after 21 days.Results Compared with the control group,1,25(OH)2D3 significantly promoted the proliferation of osteoblasts and reversed the proliferation activity of osteoblasts inhibited by MHY1485(P<0.01).Western blot and RT-PCR results showed that 1,25(OH)2D3 inhibited protein phosphorylation of mTOR,4E-BP1 and S6K1,but significantly increased the expressions of autophagy markers Beclin-1 and LC3-Ⅱ,differentiation markers ALP,Osterix and Runx2 (P<0.01).ALP staining and alizarin red staining showed that1,25(OH)2D3 promoted ALP secretion in the early stage and mineralized nodule formation in the late stage (P<0.01).Conclusion 1,25(OH)2D3 can significantly promote the proliferation,differentiation and mineralization of primary osteoblasts of rats,which is related to the activation of autophagy by inhibiting the mTOR signaling pathway.[ACTA NUTRIMENTA SINICA,2021,43(1):71-78,85,96]