黄芪甲苷对MC3T3-E1细胞活性的影响及其机制探讨

Mechanism of Astragaloside on Activity of MC3T3-E1 Cells

目的:探讨黄芪甲苷对小鼠胚胎成骨细胞MC3T3-E1细胞活性的影响及其作用机制。方法:体外培养MC3T3-E1细胞,用不同浓度的黄芪甲苷进行预处理后,MTT法测定细胞增殖情况,碱性磷酸酶法(ALP)测定细胞分化情况,Western blotting分析转化生长因子TGF-β1、成骨细胞信号转导蛋白Smad2/3表达水平;应用小干扰RNA(siRNA)转染法观察TGF-β1 siRNA对MC3T3-E1细胞增殖及活性的影响,加入抗Smad2/3抗体,探讨Smad2/3在黄芪甲苷介导的细胞增殖分化中的作用。结果:黄芪甲苷在一定浓度范围内剂量依赖性促进MC3T3-E1细胞的增殖和分化能力。进一步机制分析表明,黄芪甲苷处理可诱导TGF-β1蛋白表达,TGF-β1siRNA处理后,黄芪甲苷诱导的MC3T3-E1细胞的增殖和分化能力明显下降;此外,黄芪甲苷可显著性诱导Smad2/3表达,经TGF-β1 siRNA处理后,黄芪甲苷诱导的Smad2/3表达显著下降;加入Smad2/3抗体后,黄芪甲苷诱导的MC3T3-E1细胞的增殖和分化能力明显下降。结论:黄芪甲苷可通过刺激TGF-β1-Smad2/3通路的活化促进成骨细胞的增殖和分化,从而有利于骨形成。因此,本研究将为骨折愈合的治疗提供新的研究方向。

Objective： To explore the possible mechanism of astragaloside on MC3T3 -E1 cells activity. Methods： The MC3T3 - E1 cells were cultured and stimulated with various concentrations of astragaloside for 72 h. And then, MTT and ALP assay were used to analyze the proliferation and differentiation of MC3T3 -E1 cells, respectively. Furthermore, the protein expressions of TGF - β1 and Smad2/3 were detected by Western blotting after pretreatment with chemically syn- thesized TGF - β1 siRNA or anti - Smad2/3 antibody. Results ： Astragaloside increased the proliferation and differentia- tion of MC3T3 - E1 cells. Further mechanism analysis showed that astragaloside also promoted the protein expression of TGF - β1, while pretreatment with TGF -β1 siRNA significantly abolished MC3T3 - E1 cells proliferation and differenti- ation induced by astragaloside. Furthermore, astragaloside enhanced the expression of Smad2/3 protein by TGF - β1 pathway, as preconditioning with TGF - β1 siRNA obviously attenuated astragaloside - induced up - regulation of Smad2/ 3. Further analysis confirmed that pretreatment with Smad2/3 antibody dramatically inhibited Smad2/3 expression, concomitant with a significant decrease in MC3T3 -E1 cells proliferation and differentiation. Concision： Astragaloside in- creased MC3T3- E1 ceils proliferation and differentiation through TGF - β1 -Smad2/3 pathway. Consequently, our findings would provide a new research direction for treatment after fracture.