A Novel Prenylflavonoid Icariside I Ameliorates Estrogen Deficiency-Induced Osteoporosis via Simultaneous Regulation of Osteoblast and Osteoclast Differentiation

Abstract

Regulation of osteoblast-mediated bone formation and osteoclast-mediated bone resorption is crucial for bone health. Currently, most clinical drugs for osteoporosis treatment such as bisphosphonates are commonly used to inhibit bone resorption but unable to promote bone formation. In this study, we discovered for the first time that icariside I (GH01), a novel prenylflavonoid isolated from Epimedium, can effectively ameliorate estrogen deficiency-induced osteoporosis with enhancement of trabecular and cortical bone in an ovariectomy (OVX) mouse model. Mechanistically, our in vitro results showed that GH01 repressed osteoclast differentiation and resorption through inhibition of RANKL-induced TRAF6-MAPK-p38-NFATc1 cascade. Simultaneously, we also found that GH01 dose-dependently promoted osteoblast differentiation and formation by inhibiting adipogenesis and accelerating energy metabolism of osteoblasts. In addition, both in vitro and in vivo studies also suggested that GH01 is not only a non-toxic natural small molecule but also beneficial for restoration of liver injury in OVX mice. These results demonstrated that GH01 has great potential for osteoporosis treatment by simultaneous regulation of osteoblast and osteoclast differentiation.

Figure 1

Chemical structures of icariin and its derivatives isolated from Herba Epimedii. (A) Icariin, (B) icariside I, and (C) icaritin.

Figure 2

GH01 inhibits RANKL-induced osteoclast differentiation in vitro. (A) Primary BM macrophages were incubated with the M-CSF (25 ng/mL) and RANKL (50 ng/mL) and treated with GH01 at different doses (0.1, 1.0, 10, and 100 nM). Scale bar = 200 μm. (B, D) Number of TRAP-positive (purple, cytoplasm) multinucleated osteoclasts in each well. The findings showed three independent experiments. (C) Primary BM macrophages were cultured with 100 nM GH01 on day 1–2, 2–4, or 4–6. Mature osteoclasts were assessed via TRAP staining (>3 nuclei). Data are shown as mean ± SD. P values were obtained by one-way ANOVA with multiple comparisons, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3

GH01 represses osteoclast differentiation and resorption in vitro by suppressing MAPK-p38-NAFTc1 cascade. (A) Primary BMMs differentiated on hydroxyapatite-coated wells (in white). Scale bar = 200 μm. (B,D) Western blotting of NFATc1, c-FOS, TRAP, and TRAF6 in osteoclasts treated with GH01 at 0.1 and 100 nM. (C) mRNA expression level of NFATc1, Mmp9, and Ctsk in osteoclasts treated with GH01 at 0.1 and 100 nM. (E) Western blotting of RANKL-induced phosphorylation of p38 involved in the MAPK pathway of osteoclasts treated with GH01 for 0, 5, 15, and 30 min. (F) Immunofluorescence staining for NFATc1 (red), F-actin (green), and DAPI (blue). Scale bar = 50 μm. Data are shown as mean ± SD. P values were obtained by one-way ANOVA with multiple comparisons, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4

GH01 promotes osteoblast differentiation and formation in vitro. (A) Cell viability of primary osteoblasts incubated for 3 and 5 days with different dosages of GH01. (B) ALP activity of osteoblasts assessed with different dosages of GH01 for 7 days. (C) mRNA expression level of Runx2 and Ocn in osteoblasts treated with GH01 at 0.1 and 100 nM. (D) ALP staining and Alizarin red staining for the primary osteoblasts treated with 0.1, 1, 10, 100, and 1000 nM GH01 for 7 and 14 days (scale bar = 500 μm). (E) Western blotting of RUNX2, OPN, and OCN in osteoblasts treated with GH01 at 0.1 and 100 nM at day 14. Data are shown as mean ± SD. P values were obtained by one-way ANOVA with multiple comparisons, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5

GH01 accelerates osteoblastic metabolism. (A,B) OPLS-DA scores (left) and coefficient loading plots (right) from ¹H NMR spectra of osteoblasts in the control, GH01 (0.1 nM), and GH01 (100 nM) groups. Abbreviations: valine (Val); isoleucine (Isoleu); leucine (Leu); alanine (Ala); lysine (Lys); glutamate (Glu); glutamine (Gln); choline (Cho); guanosine triphosphate (GTP); UDP-N-acetylglucosamine (UDP-GlcNAc); adenosine monophosphate (AMP); tyrosine (Tyr); phenylalanine (Phe); and adenosine diphosphate (ADP).

Figure 6

GH01 ameliorates estrogen deficiency-induced osteoporosis in vivo without significant hepatotoxicity. (A) Experimental design for treatment in the study. (B) Body weight changes during the experiment. (C) Liver weight of mice (n = 6). (D,E) Histopathological assessment of H&E-stained and oil-red-O-stained in liver sections, respectively (scale bars = 50 μm). Data are shown as mean ± SD. P values were obtained by one-way ANOVA with multiple comparisons, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 7

GH01 ameliorates OVX-induced bone loss in vivo. (A) μCT imaging and 3D reconstruction of trabecular and cortical bone. Scale bar = 200 μm. (B) Quantitative analyses of 3D parameters for trabecular and cortical bone microarchitecture, including BMD, BV/TV, Tb.N, Tb.Sp, Tb.Th, Ct.Th, and Ct.Ar/Tt. Ar (n = 6). Data are shown as mean ± SD. P values were obtained by one-way ANOVA with multiple comparisons, *p < 0.05, **p < 0.01, and ***p < 0.001.