Isopsoralen Promotes Osteoblast Proliferation and Differentiation via BMP2/Runx2/Osx Signaling Pathway / 世界科学技术-中医药现代化

ABSTRACT

Objective The purpose of this study was to examine effects of Isopsoralen on the osteoblast proliferation and differentiation and find its possible molecular mechanisms for anti-osteoporosis.Methods OCT-1 cells were cultured with common methods.While growing well,cells were cultured with 3 doses(10 μg·mL-1,30u μg·mL-1 and 60 μg·mL-1)of Isopsoralen for 48 h,or with purified bone morphogenetic protein 2(BMP2)protein(50 ng·mL-1).We first determined the effect of Isopsoralen on cell proliferation by MTT assay.The real time RT-PCR was also used to quantify changes in the mRNA levels of several genes,such as BMP2,Runt-related transcription factor 2(Runx2),and Osterix(Osx).We also used the Western blot analysis to evaluate the expression of Runx2 and Osx proteins.At last we used the BMP2loxp/loxp mice to isolate the primary calvaria osteoblasts,cultured with Isopsoralen of the best dose for 48 h after the in vitro conditional gene knockout technology,and tested the gene expressions of Runx2 and Osx.And the alkaline Phosphatase(ALP)staining was also performed.Result Isopsoralen(10 μg·mL-1)can promote osteoblast proliferation obviously.From the real time RT-PCR analysis,Isopsoralen can enhance the BMP2 mRNA levels,the effect of 10 μg·mL-1 was the best,and 30 μg·mL-1 followed.In addition,we found that Isopsoralen(10 μg·mL-1)can enhance the Runx2 mRNA levels significantly.We also found that lower doses of Isopsoralen can enhance the Osx mRNA levels,the effect of 30 μg·mL-1 was the best,and 10 μg·mL-1 followed.From the Western blot analysis,low doses of Isopsoralen(10 μg·mL-1 and 30 μg·mL-1)can stimulate the expression of Runx2 protein.Besides,three doses of Isopsoralen can stimulate the expression of Osx protein,and the effect of 10 μg·mL-1 and 30 μg·mL-1 are better.Finally,the results of in vitro conditional gene knockout experiment showed that the overexpression of Runx2 and Osx genes in osteoblasts,as well as ALP staining,induced by Isopsoralen are BMP2 dependent.Conclusions In this study,we firstly demonstrate that Isopsoralen can stimulate osteoblast proliferation and differentiation by mediating BMP2/Runx2/Osx signaling pathway.