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

  1. Fan Xue
  2. Zhenlei Zhao
  3. Yanpei Gu
  4. Jianxin Han
  5. Keqiang Ye  Is a corresponding author
  6. Ying Zhang  Is a corresponding author
  1. Department of Food Science and Nutrition, College of Biosystems Engineering and Food Science, Zhejiang Key Laboratory for Agro-Food Processing; Zhejiang Engineering Center for Food Technology and Equipment, Zhejiang University, China
  2. Department of Pathology and Laboratory Medicine, Emory University School of Medicine, United States
Research Article
Cite this article as: eLife 2021;10:e64872 doi: 10.7554/eLife.64872
9 figures, 2 tables and 2 additional files

Figures

Figure 1 with 1 supplement
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—source data 1

Gate parameters and regions chosen in the Modfit LT software for flow cytometry modeling.

https://cdn.elifesciences.org/articles/64872/elife-64872-fig1-data1-v2.pdf
Figure 1—source data 2

The original files of the full raw unedited micrographs used in Figure 1E.

The folder named ‘40×’ contains the images at a magnification of 40. The folder named ‘100×’ contains the images at a magnification of 100.

https://cdn.elifesciences.org/articles/64872/elife-64872-fig1-data2-v2.zip
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 with 2 supplements
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—source data 1

The original files of the full raw unedited blots and blots with the relevant bands labeled in Figure 2A, F and J.

The folders named ‘Figure 2A’, ‘Figure 2F ’, and ‘Figure 2J ’ contain the original images in Figure 2A, Figure 2F, and Figure 2J, respectively (the individual file name containing ‘(labeled)’ is blot with the relevant bands labeled by a red outline).

https://cdn.elifesciences.org/articles/64872/elife-64872-fig2-data1-v2.zip
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 2—figure supplement 2—source data 1

The original files of the full raw unedited blots and blots with the relevant bands labeled in Figure 2—figure supplement 2A.

The individual file name containing ‘(labeled)’ is blot with the relevant bands labeled by a red outline.

https://cdn.elifesciences.org/articles/64872/elife-64872-fig2-figsupp2-data1-v2.zip
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 3—source data 1

The original files of the full raw unedited blots and blots with the relevant bands labeled in Figure 3C–G.

The folders named ‘Figure 3C’, ‘Figure 3D’, ‘Figure 3E’, ‘Figure 3F’ and ‘Figure 3G’ contain the original images in Figure 3C, Figure 3D, Figure 3E, Figure 3F and Figure 3G, respectively (the individual file name containing ‘(labeled)’ is blot with the relevant bands labeled by a red outline).

https://cdn.elifesciences.org/articles/64872/elife-64872-fig3-data1-v2.zip
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 4—source data 1

Micrographs used for the quantitative analysis in tartrate-resistant acid phosphatase (TRAP) staining.

The folder named ‘7,8-DHF’ contains micrographs of cells with or without 7,8-DHF treatment (individual files are named by the concentration of 7,8-DHF). The folder named ‘BDNF’ contains micrographs of cells with or without BDNF treatment (individual files are named by the concentration of BDNF).

https://cdn.elifesciences.org/articles/64872/elife-64872-fig4-data1-v2.zip
Figure 4—source data 2

The original files of the full raw unedited blots and blots with the relevant bands labeled in Figure 4E.

The individual file name containing ‘(labeled)’ is blot with the relevant bands labeled by a red outline.

https://cdn.elifesciences.org/articles/64872/elife-64872-fig4-data2-v2.zip
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 5—source data 1

Weight record of the experimental rat in each group.

The body weight of the rats was recorded weekly during the experimental period.

https://cdn.elifesciences.org/articles/64872/elife-64872-fig5-data1-v2.xlsx
Figure 5—source data 2

Images of proximal growth plate, trabecular structure, and cortical structure.

The folders named ‘SHAM’, ‘OVX’, ‘OVX-L’, and ‘OVX-H’ contain the original images in Figure 5D.

https://cdn.elifesciences.org/articles/64872/elife-64872-fig5-data2-v2.zip
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 6—source data 1

Micrographs used for the quantitative analysis in tartrate-resistant acid phosphatase (TRAP) staining.

The folders named ‘SHAM’, ‘OVX’, ‘OVX-L’, and ‘OVX-H’ contain micrographs of ‘SHAM’, ‘OVX’, ‘OVX-L’, and ‘OVX-H’ groups, respectively.

https://cdn.elifesciences.org/articles/64872/elife-64872-fig6-data1-v2.zip
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.

Tables

Table 1
The levels of bone biochemical indexes.
GroupS-Ca (mmol/L)U-Ca/CrFSH (mIU/mL)E2 (pg/mL)ALP (U/L)BGP (ng/mL)
SHAM2.63±0.08a0.54±0.07b13.72±0.8957.31±21.2426.74±4.73a2.54±0.27ab
OVX2.49±0.08b1.29±0.49a14.24±0.7050.64±6.4547.03±10.49c2.48±0.22a
OVX-L2.42±0.04b0.42±0.19b13.98±1.5958.28±19.4943.58±4.07bc2.46±0.33a
OVX-H2.47±0.04b0.36±0.13b13.65±2.3461.88±17.3337.57±3.76b2.93±0.28b
  1. S-Ca: serum calcium; U-Ca/Cr: urine calcium/creatinine; FSH: follicle-stimulating hormone; E2: estradiol; ALP: alkaline phosphatase; BGP: bone gal protein. Results were presented as mean ± SD (n=6–7. Data with different letters in each group were significantly different at p<0.05).

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Mus musculus)MC3T3-E1 Subclone 14The Cell Bank of the Chinese Academy of SciencesCat #:GNM15
RRID:CVCL_5437
Cell line (Mus musculus)RAW 264.7The Cell Bank of the Chinese Academy of SciencesCat #:SCSP-5036
RRID:CVCL_0493
Peptide, recombinant proteinBDNFPeproTechCat #:450–02
Peptide, recombinant proteinRANKLR and D SystemsCat #:462-TEC-010
Chemical compound, drug7,8- Dihydroxyflavone; 7,8-DHFTokyo Chemical IndustryCat #:D1916
Chemical compound, drugAscorbic acidAladdinCat #:A103539
Chemical compound, drugβ-Glycerophosphate disodium salt hydrateSigma-AldrichCat #:G9422
Chemical compound, drugDexamethasoneSigma-AldrichCat #:D4902
Chemical compound, drugK252aCell SignalingCat #:12754
AntibodyRabbit monoclonal anti-GSK3βHuabioCat #:ET1607-71WB: 1:1000
AntibodyRabbit monoclonal anti-p-GSK3β (Ser 9)HuabioCat #:ET1607-60WB: 1:1000
AntibodyRabbit monoclonal anti-β-cateninAbcamCat # ab32572WB: 1:5000
AntibodyRabbit monoclonal anti-OsterixAbcamCat #:ab209484WB: 1:1000
AntibodyRabbit monoclonal anti-Runx2Cell SignalingCat #:12556WB: 1:1000
AntibodyRabbit monoclonal anti-Smad2HuabioCat #:ET1604-22WB: 1:1000
AntibodyRabbit monoclonal anti-p-Smad2 (S250)HuabioCat #:ET1612-32WB: 1:1000
AntibodyRabbit monoclonal anti-TAK1HuabioCat #:ET1705-14WB: 1:1000
AntibodyRabbit polyclonal anti-TrkBAffinityCat #:AF6461WB: 1:1000
AntibodyRabbit polyclonal anti-P-TrkB (Tyr706)AffinityCat #:AF3461WB: 1:1000
AntibodyRabbit monoclonal anti-MMP-9AbcamCat #:ab228402WB: 1:1000
AntibodyRabbit polyclonal anti-Adamts5HuabioCat #:1903–32WB: 1:1000
AntibodyRabbit polyclonal anti-GAPDHProteintechCat #:10494–1-APWB: 1:5000
Sequence-based reagentqRT-PCR primersThis paperSee Supplementary file 1
Sequence-based reagentsiRNAThis paperSee Materials and methods, Small interfering RNA transfection section
Commercial assay or kitCell Counting Kit-8; CCK-8BiosharpCat #:BS350A
Commercial assay or kitDNA Content Quantitation Assay (Cell Cycle) kitSolarbioCat #:CA1510
Commercial assay or kitBCA Protein Assay KitSolarbioCat #:PC0020
Commercial assay or kitALP assay kitNanjing JianchengCat #:A059-2
Commercial assay or kitAlizarin Red S solution (1%, pH 4.2)SolarbioCat #:G1452
Commercial assay or kitAcid phosphatase, leukocyte (TRAP) kitSigma-AldrichCat #:387A
Commercial assay or kitTRIzol ReagentInvitrogenCat #:15596026
Commercial assay or kitPrimeScript RT reagent Kit with gDNA EraserTaKaraCat #:RR047A
Commercial assay or kitPowerUp SYBR Green Master MixApplied BiosystemsCat #:A25742
Commercial assay or kitLipofectamine 3000InvitrogenCat #:L3000001
Commercial assay or kitRat FSH ELISA kitCusabioCat #:CSB-E06869r
Commercial assay or kitRat E2 ELISA kitCusabioCat #:CSB-E05110r
Commercial assay or kitRat BGP ELISA kitCusabioCat #:CSB-E05129r
Software, algorithmModFit LTVerity Software House (http://www.vsh.com/products/mflt/index.asp)RRID:SCR_016106
Software, algorithmImageJImageJ (http://imagej.nih.gov/ij/)RRID:SCR_003070
Software, algorithmGraphPad PrismGraphPad (https://www.graphpad-prism.cn/)RRID:SCR_002798Version 9

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