ERK信号通路在檞皮素促大鼠MSCs成骨分化中的作用

目的:观察细胞外信号调节激酶(ERK)信号通路在檞皮素(QUE)促进SD大鼠骨髓间充质干细胞(MSCs)成骨分化过程中的作用。方法:(1)用0.01μmol/L、0.1μmol/L、1μmol/L、10μmol/L和100μmol/LQUE干预MSCs,MTT法检测各浓度QUE对MSCs增殖的影响,碱性磷酸酶(ALP)测定试剂盒检测各浓度QUE对MSCsALP表达的影响;(2)用ERK1/2抑制剂干预后,加入QUE,用ALP测定试剂盒检测ALP的表达,ELISA法检测Ⅰ型胶原(ColⅠ)和骨钙素(BGP)的表达,Westernblotting检测ERK1/2的表达,荧光定量PCR检测转化生长因子β₁(TGF-β₁)mRNA、骨形成蛋白2(BMP-2)mRNA和核心结合因子α1(Cbfα1)mRNA表达。结果:(1)0.1μmol/L、1μmol/L和10μmol/LQUE剂量依赖性地促进MSCsALP的表达,同时能促进MSCs的增殖;(2)与空白组相比,QUE组ALP、BGP和ColⅠ表达均增加(P<0.01),加入ERK1/2抑制剂后,磷酸化的ERK1/2表达减少(P<0.05),同时ALP、BGP和ColⅠ表达降低(P<0.01);(3)与空白组比较,QUE组TGF-β₁mRNA、BMP-2mRNA和Cbfα1mRNA的表达均增加(P<0.05),加入ERK1/2抑制剂后这3个基因的表达都下降(P<0.05)。结论:一定浓度的QUE能促进MSCs的增殖和成骨分化,ERK通路的激活在此过程中起到了重要的作用。

The roles of ERK signaling pathway in osteogenic differentiation of rat MSCs promoted by quercetin

AIM:To study the roles of extracellular signal-regulated kinase(ERK) signal pathway in the process of osteogenic differentiation in rat mesenchymal stem cells(MSCs) promoted by quercetin(QUE).METHODS: The optimal concentration of QUE for promoting osteogenic differentiation of rat MSCs was determined by MTT and alkaline phosphatase(ALP) detection.The activity of ALP was detected by the ALP detection kit.The expression of bone Gla protein(BGP) and collagen typeⅠ(ColⅠ) was observed by ELISA analysis.MSCs were exposed to QUE at optimal concentration with or without ERK1/2 inhibitor PD98059.Non-phosphorylated and phosphorylated expression of ERK1/2 was analyzed by Western blotting.The mRNA expression of transforming growth factor β1(TGF-β1),bone morphogenetic protein 2(BMP-2) and core binding factor α1(Cbfα1) was measured by fluorescence quantitative PCR.RESULTS: QUE at concentrations of 0.1 μmol/L,1 μmol/L and 10 μmol/L induced the expression of ALP in MSCs in a dose-dependent manner,and also promoted MSCs proliferation.The expression levels of ALP,BGP and ColⅠwere higher in QUE group,and was lower in PD89059 group than those in control group.Compared with control group,the level of phosphorylated ERK1/2,and the mRNA expression of TGF-β1,BMP-2 and Cbfα1 increased in QUE group.The mRNA expression of TGF-β1,BMP-2 and Cbfα1 in QUE+PD98059 group decreased as compared with QUE group.CONCLUSION: QUE promotes osteogenic differentiation of MSCs by activating ERK signaling pathway.