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. 2020 Jan 22:10:1587.
doi: 10.3389/fphar.2019.01587. eCollection 2019.

Vindoline Inhibits RANKL-Induced Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss in Mice

Yunfei Zhan  1 , Jiamin Liang  1 , Kun Tian  1 , Zhigang Che  1 , Ziyi Wang  2 , Xue Yang  1 , Yuangang Su  1 , Xixi Lin  1 , Fangming Song  1   2 , Jinmin Zhao  1   3 , Jiake Xu  1   2 , Qian Liu  1   3 , Bo Zhou  1
Affiliations

Vindoline Inhibits RANKL-Induced Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss in Mice

Yunfei Zhan et al. Front Pharmacol. .

Abstract

Osteolytic bone diseases, for example postmenopausal osteoporosis, arise from the imbalances between osteoclasts and osteoblasts in the bone remodeling process, whereby osteoclastic bone resorption greatly exceeds osteoblastic bone formation resulting in severe bone loss and deterioration in bone structure and microarchitecture. Therefore, the identification of agents that can inhibit osteoclast formation and/or function for the treatment of osteolytic bone disease has been the focus of bone and orthopedic research. Vindoline (Vin), an indole alkaloid extracted from the medicinal plant Catharanthus roseus, has been shown to possess extensive biological and pharmacological benefits, but its effects on bone metabolism remains to be documented. Our study demonstrated for the first time, that Vin could inhibit osteoclast differentiation from bone marrow macrophages (BMMs) precursor cells as well as mature osteoclastic bone resorption. We further determined that the underlying molecular mechanism of action of Vin is in part due to its inhibitory effect against the activation of MAPK including p38, JNK, and ERK and intracellular reactive oxygen species (ROS) production. This effect ultimately suppressed the induction of c-Fos and NFATc1, which consequently downregulated the expression of the genes required for osteoclast formation and bone resorption. Consistent with our in vitro findings, in vivo administration of Vin protected mice against ovariectomy (OVX)-induced bone loss and trabecular bone deterioration. These results provided promising evidence for the potential therapeutic application of Vin as a novel treatment option against osteolytic diseases.

Keywords: MAPK; NFATc1; osteoclast; osteoporosis; vindoline.

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Figures

Figure 1
Figure 1
Vin attenuated RANKL‐induced osteoclast formation in vitro. (A) The chemical structure and formula of Vin. (B) Effect of Vin on BMM cell viability. M-CSF-dependent BMMs were treated with indicated concentrations Vin for 48 h and cell viability assessed using the CCK-8 assay (n = 3). (C) Representative light micrographs of the dose-dependent effect of Vin on RANKL‐induced osteoclast formation. M-CSF-dependent BMMs stimulated with 100 ng/ml RANKL without or with indicated of Vin for 5 days were fixed and stained for TRAP activity. BMMs without RANKL stimulation and Vin treatment served as untreated mock controls. (D and E) The number and size (as % area of total well area) of TRAP-positive osteoclasts with 3 or more nuclei were quantified. Data are presented as the mean ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001, relative to RANKL (+) controls. Scale bar, 200 μm.
Figure 2
Figure 2
Vin decreased intracellular ROS generation in BMMs. (A) Representative fluorescence micrograph of the effect of Vin on RANKL-induced intracellular ROS production. M-CSF-dependent BMMs stimulated with RANKL without or with 5 or 10 μM of Vin for 48 h were incubated with DCFH-DA for 40 min and the oxidative conversion of non-fluorescent DCFH-DA to highly fluorescent DCF was detected under fluorescence microscopy. (B) The mean fluorescence intensity of DCF were quantified for each treatment. (C) BMMs per well were counted. All data were confirmed in three independent experiments. Data are presented as the mean ± SD; **p < 0.01, and ***p < 0.001, relative to RANKL (+) controls. Scale bar, 200 μm.
Figure 3
Figure 3
Vin inhibited mature osteoclast bone resorption in vitro. (A) Representative fluorescence micrographs for the effect of Vin on podosomal F-actin belt formation. BMM-derived osteoclasts stimulated with 100 ng/ml RANKL in the absence or presence of Vin (5 or 10 μM) for 5 days were fixed and actin cytoskeleton stained with Rhodamine-conjugated Phalloidin (red). Nuclei were counterstained with DAPI (blue). (B) Quantification of the osteoclasts treated with the indicated concentrations of Vin. (C) Quantification of the nuclei number per osteoclast. (D) Representative light micrographs of the effect of Vin on the resorptive activity of osteoclasts cultured on hydroxyapatite-coated culture plates. Equal number of pre-osteoclasts stimulated with 100 ng/ml RANKL for 3 days were seeded onto OsteoAssay plates and then treated without or with Vin (5 or 10 μM) for further 48 h. Cells were then removed and resorption pits visualized under light microscopy. (E) The percentage of resorption pit area relative to total well area was quantified for each experimental condition; (n = 3). Data are presented as the mean ± SD. **p < 0.01, and ***p < 0.001, relative to RANKL (+) or ‘0' control. Scale bar, 200 μm.
Figure 4
Figure 4
Vin blocked RANKL‐induced activation of MAPK signaling and subsequent NFATc1 induction. (A) Representative immunoblots for the effect of Vin on RANKL-induced phosphorylation of ERK, JNK, and p38 MAPKs. Total cellular proteins from BMM pretreated without or with 10 μM Vin for 1 h, and then stimulated with 100 ng/ml RANKL for 0, 5, 10, 20, 30, or 60 min were subjected to western blot analyses using specific antibodies against total and phosphorylated forms of ERK, p38, and JNK. (B) Relative changes in the phosphorylation status of ERK, JNK, and p38 relative to their respective total protein counterpart were quantified by densitometry; (n = 3). (C) Representative immunoblots for the effect of Vin on RANKL-induced degradation of IκBα and phosphorylation of NF-κB p65. Total cellular proteins from experimental conditions above were analyzed by western blot using specific antibodies against IκBα, and total and phosphorylated forms of p65. (D) Relative changes in the phosphorylation status of p65 relative to total p65, and total protein levels of IκBα relative to β-actin were quantified by densitometry; (n = 3). (E) Representative immunoblots for the effect of Vin on RANKL-induced expression of c-Fos and NFATc1. Total cellular proteins from BMM stimulated with RANKL without or with 10 μM Vin for 0, 1, 3, or 5 days were subjected to western blot analyses using specific antibodies against c-Fos and NFATc1. (F) Relative expression of c-Fos and NFATc1 relative to β-actin were quantified by densitometry; (n = 3). β-actin was used as internal loading control for all experimental conditions. Data are presented as the mean ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001.
Figure 5
Figure 5
Vin reduced the expression of osteoclast marker genes. (A–D) Real-time qPCR was performed on RNA extracted from cells stimulated with 100 ng/ml RANKL without or with indicated concentrations of Vin for 5 days. The expression levels of CTSK, MMP9, NFATc1, and TRAP were normalized to GAPDH and then compared to RANKL (+) or “0” control to obtain relative fold change; (n = 3). Data are presented as the mean ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001.
Figure 6
Figure 6
Vin lessened the deleterious effect of OVX-induced bone loss in mice. (A) Representative 3D reconstructions of micro‐CT scans of the tibia from Sham, OVX, OVX + low-dose Vin (5 mg/kg body weight), and OVX + high-dose Vin (10 mg/kg body weight). (B–E) Quantitative analyses of morphometric bone parameters of bone volume to tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp) and trabecular thickness (Tb. Th) of each experimental condition. (F) Representative images of decalcified tibial bone tissue from mice in each experimental groups stained for TRAP activity. (G–H) Quantitative analyses of N.Oc/BS and Oc.S/B. Data are expressed as means ± SD. ns: no significance, *p < 0.05, **p < 0.01, and ***p < 0.001 relative to respective control group.
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