黄芪多糖对地塞米松诱导的MC-3T3-E1成骨细胞FGF23、Klotho mRNA及蛋白表达的影响

Effects of astragalus polysaccharide on the mRNA and protein expressions of FGF23 and Klotho in MC-3T3-E1 osteoblasts induced by dexamethasone

目的通过研究黄芪多糖对MC-3T3-E1成骨细胞成纤维生长因子-23(FGF23)、Klotho、骨钙素(OC)和碱性磷酸酶(ALP)表达的影响,初步探讨黄芪多糖治疗骨质疏松症(OP)的作用机制。方法实验为6组,分别为正常组、模型组、黄芪多糖低、中、高剂量组、维生素D3组。MTT法检测不同浓度的黄芪多糖在不同时间(24 h,36 h,48 h)对MC-3T3-E1成骨细胞增殖抑制率的影响;比色法检测MC-3T3-E1成骨细胞ALP的活性;ELISA法检测MC-3T3-E1成骨细胞OC的含量;RT-PCR法和Western Blot法检测MC-3T3-E1成骨细胞FGF23、Klotho mRNA和蛋白表达水平。结果MTT法结果显示,与模型组比较,黄芪多糖低、中、高剂量组及维生素D3组MC-3T3-E1成骨细胞增殖抑制率在24 h、36 h、48 h时显著降低(P<0.01),且与黄芪多糖浓度呈负相关;ELISA法结果显示,与模型组比较,黄芪多糖各剂量组与维生素D3组小鼠MC-3T3-E1成骨细胞OC含量均显著升高(P<0.01);比色法结果显示,与模型组比较,黄芪多糖各剂量组与维生素D3组MC-3T3-E1成骨细胞ALP含量均显著升高(P<0.01);RT-PCR法和Western Blot法结果显示,与模型组比较,黄芪多糖各剂量组与维生素D3组均可显著下调FGF23 mRNA和蛋白表达水平(P<0.01),黄芪多糖中、高剂量组与维生素D3组均可显著上调Klotho mRNA和蛋白表达水平(P<0.01)。结论黄芪多糖可防治骨质疏松症,其机制可能与提高MC-3T3-E1成骨细胞活性及调节FGF23、Klotho mRNA和蛋白表达水平有关。

Objective To investigate the effect of astragalus polysaccharide on the expression of fibroblast growth factor-23(FGF23),Klotho,osteocalcin(OC)and alkaline phosphatase(ALP)in MC-3T3-E1 osteoblasts,and to preliminarily ex-plore the treatment of astragalus polysaccharide in the treatment of bone marrow.Mechanisms of action in osteoporosis(OP).Methods The experiment consisted of 6 groups,namely the normal group,the model group,the low,medium and high dose groups of astragalus polysaccharide,and the vitamin D3 group.MTT method was used to detect the effect of different concentra-tions of astragalus polysaccharides on the proliferation inhibition rate of MC-3T3-E1 osteoblasts at different times(24 h,36 h,48 h);colorimetric method was used to detect MC-3T3-E1 osteoblasts ALP activity;ELISA was used to detect the content of OC in MC-3T3-E1 osteoblasts;RT-PCR and Western Blot methods were used to detect the expression levels of FGF23,Klotho mRNA and protein in MC-3T3-E1 osteoblasts.Results MTT assay showed that compared with the model group,the proliferation inhibition rate of MC-3T3-E1 osteoblasts in the low,medium and high dose groups of astragalus polysaccharide and the vitamin D3 group was significantly decreased at 24 h,36 h and 48 h(P<0.01),and was negatively correlated with the concentration of astragalus polysaccharide;ELISA results showed that,compared with the model group,the OC content of MC-3T3-E1 osteoblasts of mice in each dose group of astragalus polysaccharide and vitamin D3 group were significantly increased(P<0.01);the results of colorimetry showed that compared with the model group,the ALP content of MC-3T3-E1 osteo-blasts in each dose group of astragalus polysaccharide and vitamin D3 group were significantly increased(P<0.01);RT-PCR and Western Blot The results showed that compared with the model group,FGF23 mRNA and protein expression levels were sig-nificantly down-regulated in each dose group of astragalus polysaccharide and vitamin D3 group(P<0.01),and Klotho was sig-nificantly up-regulated in the middle and high dose groups of astragalus polysaccharide and vitamin D3 group.mRNA and protein expression levels(P<0.01).Conclusion Astragalus polysaccharide can prevent and treat osteoporosis,and its mechanism may be related to increasing the activity of MC-3T3-E1 osteoblasts and regulating the expression levels of FGF23,Klotho mRNA and protein.