Abstract
BackgroundIcariin, a major active flavonoid glucoside, is widely used for the treatment of bone injury and rebuilding in the clinic because of its roles in suppressing osteoblastogenesis and promoting osteogenesis. The senescence-accelerated mouse SAMP6 was accepted as a useful murine model to reveal the mechanism of senile osteoporosis and the therapeutic mechanism of drug activity. However, little is known about the characteristics of SAMP6 osteoblasts and the associated regulatory roles of icariin.MethodsWe isolated and cultured osteoblasts from SAMP6 or SAMR1 mice and compared their proliferation, migration, and differentiation by performing the CCK-8 assay, cell counting assay, EdU staining, cell cycle analysis, ALP staining and activity measurement, Alizarin red staining, and RT-qPCR analysis to measure the levels of osteoblast markers, including RUNX2, Colla1 and Oc. To assess the effects of icariin on BMP-2-induced osteoblast differentiation, after BMP-2 treatment, osteoblast markers were analyzed by RT-qPCR and semi-quantitative Western blotting. The effects of icariin on connective tissue growth factor (CTGF) were measured by RT-qPCR. shRNA targeting CTGF mRNA was employed to knockdown its expression level in osteoblasts.ResultsThe SAMP6 osteoblasts presented decreased the development and differentiation activity compared with SAMR1 osteoblasts, indicating that they are the potential mechanisms underlying age-associated disease. Moreover, SAMP6 osteoblasts presented upregulated CTGF compared with SAMR1 osteoblasts. Icariin enhanced BMP-2-induced osteoblast differentiation by downregulating CTGF expression, which tightly regulates osteoblast differentiation. By downregulating CTGF, icariin treatment upregulated phosphate-Smad1/5/8, indicating its activating effects on the BMP signaling pathway.ConclusionThese results suggest that decreased osteoblast development and function potentially contributes to age-associated disease. Icariin exerts enhancing effects on BMP-2-mediated osteoblast development via downregulating CTGF.
Highlights
Senescence-accelerated mice (SAM) are a series of inbred stains developed from the AKR/J strain consisting of 4 senescence-resistant strains (SAMR) and 9 senescence-prone strains (SAMP) [1, 2]
The SAMP6 osteoblasts presented decreased the development and differentiation activity compared with SAMR1 osteoblasts, indicating that they are the potential mechanisms underlying age-associated disease
SAMP6 osteoblasts presented upregulated connective tissue growth factor (CTGF) compared with SAMR1 osteoblasts
Summary
Senescence-accelerated mice (SAM) are a series of inbred stains developed from the AKR/J strain consisting of 4 senescence-resistant strains (SAMR) and 9 senescence-prone strains (SAMP) [1, 2]. Compared with the SAMR strains, which present normal senescence, SAM-P6 (SAMP6) strains exhibit accelerated-senescence phenotypes by developing osteoporosis within a few months of birth [3], increased numbers of mature adipocytes (Aps) within the bone marrow [4] or decreased osteoblastogenesis [5], and they have been accepted as an experimental animal model to research age-associated disease [6], including being used as a murine model of senile osteoporosis. Icariin inhibited the formation and activation of osteoblasts [15, 16], a finding that is consistent with the protective effects on bone mass. Zhang et al reported that icariin treatment promoted proliferation and bone formation by upregulating RUNX2, ALP, Colla and Oc after bone morphogenetic protein-2 (BMP-2) stimulation [17]. Little is known about the characteristics of SAMP6 osteoblasts and the associated regulatory roles of icariin.
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