7,8-Dihydroxyflavone modulates bone formation and resorption and ameliorates ovariectomy-induced osteoporosis

Abstract

Imbalances in bone formation and resorption cause osteoporosis. Mounting evidence supports that brain-derived neurotrophic factor (BDNF) implicates in this process. 7,8-Dihydroxyflavone (7,8-DHF), a plant-derived small molecular TrkB agonist, mimics the functions of BDNF. We show that both BDNF and 7,8-DHF promoted the proliferation, osteogenic differentiation, and mineralization of MC3T3-E1 cells. These effects might be attributed to the activation of the Wnt/β-catenin signaling pathway as the expression of cyclin D1, phosphorylated-glycogen synthase kinase-3β (p-GSK3β), β-catenin, Runx2, Osterix, and osteoprotegerin (OPG) was all significantly up-regulated. Knockdown of β-catenin restrained the up-regulation of Runx2 and Osterix stimulated by 7,8-DHF. In particular, blocking TrkB by its specific inhibitor K252a suppressed 7,8-DHF-induced osteoblastic proliferation, differentiation, and expression of osteoblastogenic genes. Moreover, BDNF and 7,8-DHF repressed osteoclastic differentiation of RAW264.7 cells. The transcription factor c-fos and osteoclastic genes such as tartrate-resistant acid phosphatase (TRAP), matrix metalloprotein-9 (MMP-9), Adamts5 were inhibited by 7,8-DHF. More importantly, 7,8-DHF attenuated bone loss, improved trabecular microarchitecture, tibial biomechanical properties, and bone biochemical indexes in an ovariectomy (OVX) rat model. The current work highlights the dual regulatory effects that 7,8-DHF exerts on bone remodeling.

Keywords: 7,8-dihydroxyflavone; bone remodeling; medicine; osteoblasts; osteoclasts; ovariectomy rat model; rat.

Figure 1.. 7,8-Dihydroxyflavone (7,8-DHF) promoted proliferation and differentiation of MC3T3-E1 osteoprogenitor cells.

(A) The effect of brain-derived neurotrophic factor (BDNF) or 7,8-DHF on the proliferation of MC3T3-E1 cells after treatment for 48 hr. (B) The percentage of MC3T3-E1 cells in each cell cycle phase treated with 7,8-DHF for 24 hr. Source file of gate parameters and regions chosen in the Modfit LT software for flow cytometry modeling was available in Figure 1—source data 1. (C) The effect of 7,8-DHF on the mRNA expression level of cyclin D1 was detected by quantitative real-time PCR (qRT-PCR). Results were normalized to the reference gene GAPDH. (D) The effect of BDNF or 7,8-DHF on the alkaline phosphatase (ALP) activity of MC3T3-E1 cells. Results were normalized with total protein quantity. Alizarin red S staining (magnification: 40× or 100×) (E) and quantitative analysis of the extent of mineralization (F) of MC3T3-E1 cells cultured with 7,8-DHF for 21 days. Source files of the full raw unedited micrographs were available in Figure 1—source data 2. All results were expressed as mean ± SD. (A-F: n = 3–4; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ns: not significant, BDNF-treated groups, one-way analysis of variance [ANOVA]; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant, 7,8-DHF-treated groups, one-way ANOVA).

Figure 1—figure supplement 1.. Brain-derived neurotrophic factor (BDNF) promoted mineralization of MC3T3-E1 osteoprogenitor cells.

Alizarin red S staining (A) and quantitative analysis of the extent of mineralization (B) of MC3T3-E1 cells cultured with BDNF for 21 days. All results were expressed as mean ± SD (A, B: n = 3; ^####p < 0.0001, one-way analysis of variance [ANOVA]).

Figure 2.. 7,8-Dihydroxyflavone (7,8-DHF) promoted osteogenesis via osteoblast-related signaling pathways.

MC3T3-E1 cells were treated with or without 7,8-DHF for 3 days. The mRNA level was evaluated by quantitative real-time PCR (qRT-PCR) and the protein level was detected by western blot. GAPDH was used as an internal control. (A) The protein levels of p-GSK3β and GSK3β. (B) Quantification of the p-GSK3β band intensities normalized to total GSK3β band intensities in each case. (C-E) The mRNA levels of β-catenin, Runx2, and Osterix. (F-I) The protein levels of β-catenin, Runx2, and Osterix. The expression levels of target proteins in the 0 μM group were normalized to 1. (J-K) β-Catenin knockdown by siRNA was performed in MC3T3-E1 cells with or without 7,8-DHF treatment. The protein levels of β-catenin, Runx2, and Osterix. The expression levels of target proteins in the 0 μM of negative control group were normalized to 1. Representative images from three independent experiments are shown in (A, F, J). Source files of the full raw unedited blots and blots with the relevant bands labeled were provided in Figure 2—source data 1. (N) The mRNA levels of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL). All results were expressed as mean ± SD (A-N: n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant; A-I, N: one-way analysis of variance [ANOVA]; J-M: two-way ANOVA).

Figure 2—figure supplement 1.. β-Catenin knockdown by siRNA was performed in MC3T3-E1 cells with or without 7,8-dihydroxyflavone (7,8-DHF) treatment.

The mRNA levels of β-catenin (A), Runx2 (B), and Osterix were evaluated by quantitative real-time PCR (qRT-PCR) and GAPDH was used as an internal control. All results were expressed as mean ± SD (A-C: n = 3; *p < 0.05, ns: not significant; two-way analysis of variance [ANOVA]).

Figure 2—figure supplement 2.. 7,8-Dihydroxyflavone (7,8-DHF) had no obvious influence on Smad2 and TAK1.

The protein level was detected by western blot and GAPDH was used as an internal control. (A) Representative images from three independent experiments were shown. (B) Quantification of the p-Smad2 band intensities normalized to total Smad2 band intensities in each case. (C) The protein levels of TAK1. All results were expressed as mean ± SD (A-C: n = 3; one-way analysis of variance [ANOVA]). Source files of the full raw unedited blots and blots with the relevant bands labeled were provided in Figure 2—figure supplement 2—source data 1.

Figure 3.. Chemical inhibition of TrkB blocked 7,8-dihydroxyflavone (7,8-DHF)-mediated osteogenesis.

MC3T3-E1 cells were incubated with or without K252a (100 nM) for 1 hr followed by 7,8-DHF (0.5 μM or 1 μM). (A) K252a suppressed 7,8-DHF-induced proliferation of MC3T3-E1 cells after treatment for 48 hr. (B) K252a suppressed 7,8-DHF-elevated alkaline phosphatase (ALP) activity of MC3T3-E1 cells. (C, D) K252a inhibited 7,8-DHF-induced TrkB phosphorylation in MC3T3-E1 cells. Representative images from three independent experiments are shown in (C). (E-G) K252a inhibited 7,8-DHF-induced activation of Wnt/β-catenin signaling pathway in MC3T3-E1 cells. Representative images from three independent experiments are shown. The expression levels of target proteins in the control group (0 μM 7,8-DHF, without K252a) were normalized to 1. Source files of the full raw unedited blots and blots with the relevant bands labeled were provided in Figure 3—source data 1. All results were expressed as mean ± SD (A-G: n = 3, *p < 0.05, **p < 0.01, ns: not significant, two-way analysis of variance [ANOVA]).

Figure 4.. 7,8-Dihydroxyflavone (7,8-DHF) inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis.

(A) Representative images of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts after the treatment with brain-derived neurotrophic factor (BDNF) or 7,8-DHF for 5 days (magnification: 100×, scale bar: 200 μm). Source files of micrographs used for the quantitative analysis are available in Figure 4—source data 1. (B) The average number of TRAP-positive multinucleated (nuclei ≥ 3) cells per cell. (C) The effects of BDNF or 7,8-DHF on the cytoactive of RAW264.7 cells. (D) The mRNA level of c-fos. Results were normalized to the reference gene GAPDH. (E–G) The protein levels of matrix metalloprotein-9 (MMP-9) and Adamts5. GAPDH was used as an internal control. The expression levels of target proteins in the 0 μM group were normalized to 1. Representative images from three independent experiments are shown in (E). Source files of the full raw unedited blots and blots with the relevant bands labeled were provided in Figure 4—source data 2. All results were expressed as mean ± SD (B, D, F, G: n = 3, C: n=4; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001, ns: not significant, BDNF-treated groups, one-way analysis of variance [ANOVA]; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant, 7,8-DHF-treated groups, one-way ANOVA).

Figure 5.. 7,8-Dihydroxyflavone (7,8-DHF) alleviated osteoporosis phenotypes and enhanced mechanical properties in ovariectomy (OVX) rats.

(A) The body weights of all rats were recorded weekly during the experimental period. Source file of the weight record was available in Figure 5—source data 1. (B) Uteruses were isolated and weighed after euthanized. The uterine index was represented as uterus weight divided by body weight. Results were expressed as mean ± SD. (C) The bone mineral density (BMD) in left femur of rats by dual-energy X-ray absorptiometry (DXA). Results were expressed as mean ± SD (A-C: n = 6–7, *p < 0.05, **p < 0.01, ****p < 0.0001, ns: not significant, one-way analysis of variance [ANOVA]). (D) Representative micro-CT images from each group: three-dimensional (3D) architecture of trabecular bone within the distal metaphyseal femur region. Source files of the raw unedited images of proximal growth plate, trabecular structure, and cortical structure were available in Figure 5—source data 2. (E–G) Right femurs were isolated and subjected to a compression test for biomechanical property analysis. The maximum load (E), the fracture deflection (F) and the fracture strain (G) were evaluated for each group. Results were expressed as mean ± SD. All results were expressed as mean ± SD (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant, one-way ANOVA).

Figure 6.. 7,8-Dihydroxyflavone (7,8-DHF) improved bone remodeling in ovariectomy (OVX) rat.

(A) Representative images of left femurs sections stained with H&E. (B) Representative images of tartrate-resistant acid phosphatase (TRAP)-stained decalcified left femurs sections. Source files of micrographs used for the quantitative analysis are available in Figure 6—source data 1. (C) Quantitative statistics of osteoclast number per bone surface (N.Oc/BS). Results were expressed as mean ± SD (n = 5, *p < 0.05, **p < 0.01, ns: not significant, one-way analysis of variance [ANOVA]).

Figure 7.. 7,8-Dihydroxyflavone (7,8-DHF) exerts dual regulation of bone remodeling.

7,8-DHF promotes osteoblastic proliferation and differentiation through the TrkB-Wnt/β-catenin signaling pathway and inhibits osteoclastogenesis at the same time in vitro. Furthermore, 7,8-DHF improves bone mass, trabecular microarchitecture, tibial biomechanical properties, and bone biochemical indexes in vivo as indicated in an ovariectomy (OVX)-induced osteoporosis rat model.

Author response image 1.. 7,8-DHF promoted osteogenic differentiation of BMSCs and inhibited osteoclastic differentiation of BMMs.

(A) The effect of 7,8-DHF on the proliferation of BMSCs after treatment for 24, 48 and 72 h. (B) The effect of 7,8-DHF on the ALP activity of BMSCs. Results were normalized with total protein quantity. (C) Representative images of TRAP-positive multinucleated osteoclasts after the treatment with 7,8-DHF for 5 d. (magnification: 100×, scale bar: 200 μm) (D)The average number of TRAP-positive multinucleated (nuclei ≥ 3) cells per cell. (D) The effects of 7,8-DHF on the cytoactive of BMMs. All results were expressed as mean ± SD. (A, E: n = 4; B-D: n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant, one-way ANOVA).

Author response image 2.. Representative samples were reconstructed in 3D to generate visual representations of trabecular and cortical structure.