Icariin induced proliferation and differentiation of MC3T3-E1 osteoblasts via ERK and JNK signaling coupled with estrogen receptor / 中华内分泌代谢杂志

ABSTRACT

Objective To explore the detailed underlying molecular and signaling mechanisms in the effects of icariin on bone formation by an in vitro cell model. Methods The proliferation of MC3T3-E1 osteoblast-like cells was evaluated by MTT, and gene expression of cell cycle related proteins in MC3T3-E1 cells after icariin treatment was detected by real-time PCR. The phosphorylation of MAPK signals, including ERK, P38, and JNK was determined by Western blot, and then the inhibitors of MAPK signals were used to treat cells with icariin alone or together to determine the role of MAPKs in the process of icariin treatment on MC3T3-E1 cell proliferation. Alkaline phosphatase and Alizarin red staining were used to detect the formation of mineralization nodules, and gene expressions of alkaline phosphatase, type Ⅰ collagen, and osteocalcin in osteoblasts after being treated by icariin were evaluated by real-time PCR. ICI182780, and nilutamide was used to decide the participation of estrogen and androgen receptor signals in the process of icariin treatment on the differentiation and mineralization of MC3T3-E1 cells. Results Treatment with icariin promoted MC3T3-E1 cell growth in a time- and dose-dependent manner. This treatment also revealed that icariin increased the expression of mRNAs encoding both cyclin E and PCNA, positive regulators of cell growth, but decreased levels of mRNAs encoding Cdkn2b, a negative regulator of cell cycle progression. When MC3T3-E1 cells were cultured in a differentiated condition, icariin enhanced mineralized nodule formation and increased the expression of mRNAs encoding alkaline phosphatase, type Ⅰ collagen, and osteocalcin. Treatment with icariin significantly induced phosphorylation of both ERK and JNK and this phosphorylated effect occurred very rapidly within 5 minutes and reached peak at 15 minutes. Furthermore, the stimulated effects of icariin on proliferation and gene expression of cyclin E, PCNA, and Cdkn2b in MC3T3-E1 cells were dramatically attenuated by treatment with both U0126 and SP600125, inhibitors of MAPKs. Interestingly, such stimulating effects of icariin were at least partly reduced by treatment with ICI182780, an inhibitor of estrogen receptor. Icariin induced mineralized nodule formation and gene expression of alkaline phosphatase, type Ⅰ collagen, and osteocalcin in MC3T3-E1 cells were also partly reduced when the cells were treated with ICI182780. Conclusions Our findings indicate that the anabolic effect of icariin on bone formation is, at least partly, mediated through the MAPK signaling pathway in order to modulate osteoblast proliferation and differentiation.