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. 2022 Aug 25;54(8):1080-1089.
doi: 10.3724/abbs.2022094.

Specnuezhenide suppresses diabetes-induced bone loss by inhibiting RANKL-induced osteoclastogenesis

Xiaoshuang Ye  1 , Juanjuan Jiang  1 , Juan Yang  1 , Wenyan Yan  1 , Luyue Jiang  1 , Yan Chen  1
Affiliations

Specnuezhenide suppresses diabetes-induced bone loss by inhibiting RANKL-induced osteoclastogenesis

Xiaoshuang Ye et al. Acta Biochim Biophys Sin (Shanghai). .

Abstract

Diabetes osteoporosis is a chronic complication of diabetes mellitus (DM) and is associated with osteoclast formation and enhanced bone resorption. Specnuezhenide (SPN) is an active compound with anti-inflammatory and immunomodulatory properties. However, the roles of SPN in diabetic osteoporosis remain unknown. In this study, primary bone marrow macrophages (BMMs) were pretreated with SPN and were stimulated with receptor activator of nuclear factor kappa B ligand (RANKL; 50 ng/mL) to induce osteoclastogenesis. The number of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining. The protein levels of cellular oncogene fos/nuclear factor of activated T cells c1 (c-Fos/NFATc1), nuclear factor kappa-B (NF-κB), and mitogen-activated protein kinases (MAPKs) were evaluated by western blot analysis. NF-κB luciferase assays were used to examine the role of SPN in NF-κB activation. The DM model group received a high-glucose, high-fat diet and was then intraperitoneally injected with streptozotocin (STZ). Micro-CT scanning, serum biochemical analysis, histological analysis were used to assess bone loss. We found that SPN suppressed RANKL-induced osteoclast formation and that SPN inhibited the expression of osteoclast-related genes and c-Fos/ NFATc1. SPN inhibited RANKL-induced activation of NF-κB and MAPKs. In vivo experiments revealed that SPN suppressed diabetes-induced bone loss and the number of osteoclasts. Furthermore, SPN decreased the levels of bone turnover markers and increased the levels of runt-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), calcium (Ca) and phosphorus (P). SPN also regulated diabetes-related markers. This study suggests that SPN suppresses diabetes-induced bone loss by inhibiting RANKL-induced osteoclastogenesis, and provides an experimental basis for the treatment of diabetic osteoporosis.

Keywords: MAPK; NF-κB; RANKL; bone loss; osteoclastogenesis; specnuezhenide.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

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Figure 1
Specnuezhenide inhibits RANKL-induced osteoclastogenesis (A) Chemical structure of SPN. (B) MTT assays were performed after incubation of BMM cells with SPN (0, 10, 50, 200, and 400 μM) for 24 h and 48 h at 37°C in a 5% CO 2 atmosphere. (C) BMMs were plated into 96-well plates (0.32 cm 2) at 1×10 4 cells/well, and pretreated with different concentrations of SPN (0, 10, 50 and 200 μM) for 30 min and incubated with RANKL (50 ng/mL) for 3 days. Osteoclastic formation from BMMs was examined by staining for TRAP activity and is indicated by black arrows. Scale bar= 100 μm. (D) The number of osteoclasts in 96-well plates (0.32 cm 2) was counted. Data are expressed as the mean±SD of triplicate experiments. * P<0.05, *** P<0.001 vs RANKL-treated control.
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Figure 2
Specnuezhenide dose-dependently reduces RANKL-induced gene expression Total RNA was extracted from BMMs treated with SPN (0, 50 and 200 μM) for 30 min and RANKL (50 ng/mL) for 3 days. Relative gene expressions of TRAP, CTSK, MMP-9, DC-STAMP, c-Fos and NFATc1 were analyzed by qRT-PCR. Data are presented as the mean±SD of three independent experiments. * P<0.05, ** P<0.01, *** P<0.001 vs RANKL-treated control.
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Figure 3
Specnuezhenide inhibits the RANKL-mediated expression of c-Fos/NFATc1 BMMs incubated in serum-free medium for 5 h were pretreated with SPN (200 μM) for 30 min prior to RANKL (50 ng/mL) stimulation for 0, 1, and 3 days. (A–C) Total protein was extracted and subjected to western blot analysis using antibodies against NFATc1 and c-Fos. Data are presented as the mean±SD of three independent experiments. * P<0.05, *** P<0.001 vs RANKL-treated control.
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Figure 4
Specnuezhenide suppresses RANKL-induced NF-κB activation (A) BMMs stably transfected with the 3 kB-Luc-SV40 reporter gene were pretreated with SPN (0, 50, and 200 μM) for 30 min, followed by RANKL (50 ng/mL) stimulation for 6 h. Luciferase activity in the lysates was determined after 6 h of RANKL stimulation. (B,C) BMMs were pretreated with SPN (0, 100, and 200 μM) for 30 min, followed by RANKL (50 ng/mL) stimulation for 0, 5, 10, 20, 30, and 60 min. (B) The protein levels of IκB-α were determined by western blot analysis. (C) Statistical results of western blot analysis. Data are presented as the mean±SD of three independent experiments. ** P<0.01, *** P<0.001 vs RANKL‑treated control.
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Figure 5
Specnuezhenide attenuates RANKL-induced MAPK activation BMMs were pretreated with 200 μM SPN, followed by RANKL (50 ng/mL) stimulation for the indicated time periods (0, 5, 10, 30, and 60 min). (A) The protein levels were determined by western blot analysis. (B) Statistical results of western blot analysis assay. Data are presented as the mean±SD of three independent experiments. * P<0.05, ** P<0.01, *** P<0.001 vs RANKL‑treated control.
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Figure 6
Specnuezhenide prevents type 2 diabetes-induced bone loss Rats were sacrificed to isolate the femurs for analysis after 6 weeks of treatment. (A) Three-dimensional reconstructed images of the femurs from the control, DM, DM+ SPN (0.035 mg/kg), and DM+SPN (0.14 mg/kg) groups (n=10) 8 days after treatment. (B) The femurs were subjected to micro-CT for bone evaluation. CT Analyzer software was used to evaluate BV/TV, Tb.N, Tb.Th and Tb.Sp (n=10 per group). (C) H&E staining analysis of the metaphyseal region of femoral bones from the control, DM, DM+SPN (0.035 mg/kg), and DM+SPN (0.14 mg/kg) groups. Scale bar= 200 μm. * P<0.05, ** P<0.01, *** P<0.001 vs RANKL-treated control.
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Grants and funding

This work was supported by the grant from the Jiangsu Commission of Health (No. BJ18026).