基于铁死亡通路研究淫羊藿苷对大鼠成骨细胞增殖分化的影响

Effect of icariin on proliferation and differentiation of rat osteoblasts based on Ferroptosis pathway

目的探讨铁死亡对成骨细胞增殖分化的作用机制及淫羊藿苷与其相关的作用。方法以成骨细胞为研究对象,构建FPN1过表达质粒转染成骨细胞,并用淫羊藿苷干预转染后成骨细胞。实验分为三组:(1)对照组;(2)FPN1过表达组;(3)淫羊藿苷组(FPN1过表达+淫羊藿苷)。测定各组成骨细胞增殖分化情况;免疫荧光检测细胞内活性氧及FPN1情况;Western blot检测FPN1和谷胱甘肽过氧化物酶(Gpx4)及成骨细胞标记物Rux2与碱性磷酸酶(ALP)蛋白表达。结果与对照组比较,FPN1过表达组成骨细胞矿化能力明显降低,成骨细胞内活性氧含量显著升高,但是淫羊藿苷可以显著逆转这种作用。免疫荧光结果显示淫羊藿苷可以使成骨细胞内FPN1荧光强度下降;Western blot结果显示淫羊藿苷组相对FPN1过表达组可显著降低FPN1表达,提高成骨细胞标记物及Gpx4蛋白表达。结论淫羊藿苷能够抑制FPN1表达调节铁死亡通路最终促进成骨细胞增殖及分化。

Objective To investigate the mechanism of ferroptosis on proliferation and differentiation of osteoblasts and the effect of icariin. Methods The osteoblasts were transfected with FPN1 expression plasmid, and the transfected osteoblasts were interfered with icariin. The experiment was divided into three groups: 1)control group, 2)FPN1 overexpression group, and 3)icariin group( FPN1 overexpression + icariin). The proliferation and differentiation of osteoblasts in each group were tested. And the intracellular reactive oxygen species and FPN1 were detected by immunofluorescence. Then, the protein expression of FPN1, glutathione peroxidase(Gpx4), osteoblast markers Rux2 and alkaline phosphatase(ALP) was detected by Western blot. Results Compared with the control group, the mineralization ability of osteoblast in the FPN1 overexpression group was significantly reduced, and the reactive oxygen species content in the osteoblasts of FPN1 overexpression was significantly increased, but icariin could significantly reverse this effect. Immunofluorescence results showed that icariin could decrease the FPN1 fluorescence intensity in osteoblasts. Western blot results showed that the icariin group could significantly reduce FPN1 expression and increase the expression of osteoblast markers and Gpx4 protein compared with the FPN1 overexpression group. Conclusion Icariin can inhibit the expression of FPN1, regulate Ferroptosis pathway and finally increase the proliferation and differentiation of osteoblasts.