山奈酚通过mTORC1信号促进牵张力下小鼠骨髓间充质细胞成骨分化机制研究

Kaempferol promotes osteogenic differentiation of mouse bone marrow mesenchymal cells under tension stress via the mTORC1 signaling pathway

目的探讨山奈酚(kaempferol,Kae)对周期性单轴牵张力下小鼠骨髓间充质细胞(bone marrow mes⁃enchymal cells,BMMCs)成骨分化过程中哺乳动物雷帕霉素靶蛋白复合物1(mammalian target of rapamycin com⁃plex 1,mTORC1)信号通路的作用。方法对体外分离培养的小鼠BMMCs施加形变量10%的单轴动态牵张力,通过细胞毒性试验筛选出合适浓度Kae,并添加工具药pp242改变内源性mTOR信号,在牵张后4 h利用化学比色法检测碱性磷酸酶(alkaline phosphatase,ALP)活性变化、ELISA法检测骨钙素(osteocalcin,OCN)表达量,流式细胞仪检测细胞内钙离子相对含量;利用Western Blot检测内源性mTORC1信号通路主要分子哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、核糖体蛋白S6激酶(ribosomal proteinS6 kinases,S6K)、4E/BP1的磷酸化表达及成骨转录因子Runx2和Osterix的表达变化,qRT⁃PCR检测上述因子mRNA表达水平。结果10μmol/L Kae对细胞的抑制作用较小,且成骨能力最强。加力结束后4 h,Kae能够有效促进BMMCs成骨分化,ALP表达为(153.04±18.72)U/mg,OCN表达为(1.64±0.25)U,成骨转录因子Runx2、Osterix的mRNA水平和蛋白水平表达上调,细胞内钙离子含量下降,同时mTORC1信号通路中mTOR、S6K mRNA水平及蛋白磷酸化表达上调,4E/BP1 mRNA水平及蛋白磷酸化表达下调;在加入pp242抑制mTORC1信号表达后,mTOR、S6K mRNA水平及蛋白磷酸化表达下调,4E/BP1 mRNA水平及蛋白磷酸化表达上调,BMMCs成骨分化效应显著被抑制,Runx2、Osterix的mRNA水平和蛋白表达显著下调,ALP及OCN表达下调,细胞内钙离子含量增高。结论Kae通过mTORC1信号通路促进牵张力下小鼠BMMCs成骨分化。

Objective To investigate the activation of the mammalian target of rapamycin complex 1(mTORC1)sig⁃naling pathway molecules during the process by which kaempferol(Kae)promotes osteogenic differentiation of mouse bone marrow mesenchymal cells(BMMCs)under cyclic and uniaxial tension.Methods BMMCs isolated and cultured in vitro were subjected to uniaxial dynamic tension with a 10%shape variable.The appropriate concentration of Kae was selected by cytotoxicity testing.The endogenous mTOR signal was inhibited by pp242.Four hours after traction,alkaline phosphatase(ALP)and osteocalcin(OCN)were detected by chemical colorimetry and ELISA,and the relative concentration of intracellular calcium was detected by flow cytometry.Phosphorylation of mTOR,4E/BP1,and ribosomal protein S6 kinases(S6K),which are the main molecules of the endogenous mTORC1 signaling pathway,and expression of osteogenic transcription factors(Runx2 and Osterix)were detected by western blotting(WB),and mRNA expression levels of the above factors were detected by qRT⁃PCR.Results The cytotoxicity test showed that 10μmol/L Kae had little inhibitory effect on cell proliferation but had the strongest osteogenic ability.Four hours after stretching,Kae effectively promoted the osteogenic differentiation of BMMCs.The expression of ALP was(153.04±18.72)U/mg,the expression of OCN was(1.64±0.25)U.The mRNA and protein levels of Runx2 and Osterix were upregulated,and the intracellular calcium content was decreased.The mRNA and protein phosphorylation of mTOR and S6K was upregulat⁃ed,and the opposite effect was observed with 4E/BP1.After pp242 was added to inhibit mTOR signaling,mTOR and S6K mRNA and protein phosphorylation were downregulated,but 4E/BP1 mRNA and protein phosphorylation was upregulated.The osteogenic differentiation of BMMCs was also significantly inhibited,mRNA and protein expression of Runx2 and Osterix were significantly downregulated,ALP and OCN expression were downregulated,and intracellular calcium content was increased.Conclusion Kae promotes osteogenic differentiation of mouse BMMCs under uniaxial dynamic tension through the mTORC1 signaling pathway.