Foxf1 gene increases the risk of osteoporosis in rats by inhibiting osteoblast formation and promoting osteoclast differentiation through the upregulation of NF-κB pathway

Abstract

Objectives: a) To explore the expression of Foxf1 and NF-κB in bone tissue of ovariectomized rats with osteoporosis and b) to investigate the role and mechanism of NF-κB pathway regulated by Foxf1 gene in the differentiation and formation of rat osteoclasts and osteoblasts with cell experiments.

Methods: Ovariectomized rat model of osteoporosis was established with 3-month-old female SD rats. The rats were divided into sham group (n=10) and osteoporosis group (n=10). Real time fluorescent quantitative PCR and Western blot were used to detect the expression levels of Foxf1 and NF-κB genes and proteins in the femur tissues of rats and analyze their correlation.

Results: Both Foxf1 and NF- κB were highly expressed in the femur tissues. Upon the overexpression of Foxf1 gene in osteoblasts and osteoclasts in vitro, the gene and protein expression of NF-κB were also upregulated, significantly reducing the gene and protein expression levels of osteogenic factors, including ATF4, OCN, ALP and Runx2.

Conclusions: Foxf1 gene could inhibit osteoblast formation and promote osteoclast differentiation by NF-κB pathway, which may increase the risk of osteoporosis in rats.

Keywords: Foxf1 Gene; NF-κB Pathway; Osteoblasts; Osteoclasts; Osteoporosis.

Figure 1

Concentration values of bone metabolism indexes in peripheral blood of rats in each group were detected using ELISA. A: BGP. B: Ca. C: P.

Figure 2

Expressions of Foxf1 and NF-κB genes and proteins in femur tissues of rats in each group were detected using qRT-PCR and WB, respectively. A shows the detection of Foxf1 and NF-κB gene expression in femur tissues of rats in each group using qRT-PCR; B and C show the expression bands of Foxf1 and NF-κB proteins in the femur tissues of rats in each group detected by WB and the statistical chart of quantitative analysis.

Figure 3

Expression of related genes and proteins of osteoblasts and osteoclasts derived from the rat bone marrow mesenchymal stem cell and macrophages transfected with Foxf1 gene overexpressed plasmid were detected by qRT-PCR and WB. A and B show the expression bands of ATF4, OCN, ALP and Runx2 proteins induced osteogenesis after the transfection of rat bone marrow mesenchymal stem cells with Foxf1 overexpressed plasmid by WB, and the statistical analysis of the expression levels. C shows the expression levels of ATF4, OCN, ALP and Runx2 genes induced osteogenesis after the transfection of rat bone marrow mesenchymal stem cells with Foxf1 overexpressed plasmid by qRT-PCR. D and E show the expression bands of TRAP, MMP9, CLC7 and Cathepsin-K proteins related to the function of osteoclasts after osteoclasts were induced by the transfection of macrophages with rat-deprived Foxf1 overexpressed plasmid by WB, and the statistical analysis of the expression levels. F shows the expression levels of TRAP, MMP9, CLC7 and cathepsin-K genes related to the function of osteoclasts after osteoclasts were induced by the transfection of macrophages with rat-deprived Foxf1 overexpressed plasmid by qRT-PCR.

Figure 4

Expression of the Foxf1, NF-κB genes and proteins of osteoblasts and osteoclasts derived from the rat bone marrow mesenchymal stem cell and macrophages transfected with Foxf1 gene overexpressed plasmid were detected by qRT-PCR and WB. A and B show the expression bands of Foxf1x and NF-κB proteins induced osteogenesis after the transfection of rat bone marrow mesenchymal stem cells with Foxf1 overexpressed plasmid by WB, and the statistical analysis of the expression levels. C shows the expression of Foxf1x and NF-κB proteins induced osteogenesis after the transfection of rat bone marrow mesenchymal stem cells with rat-deprived Foxf1 overexpressed plasmid by qRT-PCR. D and E show the expression bands of Foxf1x and NF-κB proteins after osteoclasts were induced by the transfection of macrophages with rat-deprived Foxf1 overexpressed plasmid by WB, and the statistical analysis of the expression levels. F shows the expression levels of Foxf1x and NF-κB proteins after osteoclasts were induced by the transfection of macrophages with rat-deprived Foxf1 overexpressed plasmid by qRT-PCR.