Korean J Physiol Pharmacol.  2019 Jan;23(1):47-54. 10.4196/kjpp.2019.23.1.47.

SPA0355 prevents ovariectomy-induced bone loss in mice

• Kim SH
• Zhang Z
• Moon YJ
• Park IW
• Cho YG
• Jeon R
• Park BH

• KMID: 2443615
• DOI: http://doi.org/10.4196/kjpp.2019.23.1.47

Abstract

Estrogen withdrawal in post-menopausal women leads to overactivation of osteoclasts, which contributes to the development of osteoporosis. Inflammatory cytokines are known as one of mechanisms of osteoclast activation after estrogen deficiency. SPA0355 is a thiourea derivative that has been investigated for its antioxidant and anti-inflammatory activities. However, its efficacy in bone resorption has not been previously investigated. The aim of this study was to investigate the impact of SPA0355 on the development of osteoporosis and to explore its mode of action. In vitro experiments showed that SPA0355 inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in primary bone marrow-derived macrophages. This effect appears to be independent of estrogen receptor activation as ICI 180,782 failed to abrogate its effects on osteoclasts. Further signaling studies revealed that SPA0355 suppressed activation of the MAPKs, Akt, and NF-κB pathways. SPA0355 also increased osteoblastic differentiation, as evidenced by its effects on alkaline phosphatase activity and mineralization nodule formation. Intraperitoneal administration of SPA0355 to ovariectomized mice prevented bone loss, as verified by three-dimensional images and bone morphometric parameters derived from µCT analysis. Noticeably, SPA0355 did not show hepatotoxicity and nephrotoxicity and also had little effect on hematological parameters. Taken together, the results indicate that SPA0355 may protect against bone loss in ovariectomized mice by stimulation of osteoblast differentiation and by inhibition of osteoclast resorption. Therefore, SPA0355 is a safe and potential candidate for management of postmenopausal osteoporosis.

Keyword

Osteoblastogenesis; Osteoclastogenesis; Osteoporosis; Ovariectomy; SPA0355

MeSH Terms

Alkaline Phosphatase
Animals
Bone Resorption
Cytokines
Estrogens
Female
Humans
Imaging, Three-Dimensional
In Vitro Techniques
Macrophages
Mice*
Miners
Osteoblasts
Osteoclasts
Osteoporosis
Osteoporosis, Postmenopausal
Ovariectomy
Thiourea
Alkaline Phosphatase
Cytokines
Estrogens
Thiourea

Figure

• 

  Fig. 1 Effects of SPA0355 on body weight and uterine weight of OVX mice. OVX mice were treated with vehicle or SPA0355 (10 or 50 mg/kg, i.p.) for five weeks. At the end of the study, body weight (A) and uterine weight (B) were measured. Values are the mean±SEM (n=5). **p<0.01 vs. sham-operated mice. SPA, SPA0355.

• 

  Fig. 2 Preventive effects of SPA0355 on bone loss in OVX mice. (A) Bone marrow density (BMD) was measured using DEXA and expressed as percent change from baseline values (n=5). (B) Representative reconstructed µCT images of femur. (C) Bone morphometric parameters (BV, BV/TV, BS, BS/TV, Tb.Th, and Tb.Sp) were determined (n=5). Values are the mean±SEM. *p<0.05 and **p<0.01 vs. sham-operated mice; #p<0.05 and ##p<0.01 vs. OVX mice. BV, bone volume; TV, total volume; BS, bone surface; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation.

• 

  Fig. 3 Acute and subacute toxicity of SPA0355 on liver and kidney. SPA0355 (50 mg/kg body weight in 100 µl of corn oil) was intraperitoneally injected daily for seven days. (A) Body weight, liver weight per body weight, and uterus weight were determined (n=6). Tissue sections of liver (B) and kidney (C) were prepared and stained by H&E. Bars=250 µm.

• 

  Fig. 4 Effects of SPA0355 on RANKL-induced osteoclast differentiation. (A) BMMs (1×104) were treated with the indicated concentrations of SPA0355 for four days, and cell viability was determined by MTT assay (n=5). (B) BMMs were cultured for four days with M-CSF (10 ng/ml) and sRANKL (30 ng/ml) in the presence or absence of 10 and 20 µM SPA0355. For ICI 182,780 treatment, 100 nM compound was used 2 h prior to SPA0355 treatment. Cells were fixed with 3.7% formalin, permeabilized with 0.1% Triton X-100, and stained with TRAP solution. TRAP-positive multinucleate cells with three or more nuclei were scored as osteoclasts (n=4). ICI, ICI 182,780. (C) The mRNA expression of osteoclastogenesis-related genes was analyzed by real-time RT-PCR (n=10–12). Values are the mean±SEM. *p<0.05 and **p<0.01 vs. untreated control. #p<0.05 and ##p<0.01 vs. RANKL+VEH.

• 

  Fig. 5 Regulation of RANKL-activated signaling pathways by SPA0355. (A) BMMs were treated with M-CSF (10 ng/ml) and sRANKL (30 ng/ml) with or without 10 µM SPA0355 for the indicated periods. Cell lysates were prepared, and the changes of MAPKs, Akt, and NF-κB were evaluated by western blotting. HSP90 and lamin B were used as loading controls for cytosolic and nuclear protein, respectively. (B) BMMs were transiently transfected with an NfkB luciferase construct and then treated with 30 ng/ml RANKL in the presence or absence of 10 µM SPA0355. After 24 h, cells were harvested in reporter lysis buffer, and luciferase activity in cell lysates was assayed (n=4). Values are the mean±SEM. *p<0.05 vs. untreated control. #p<0.05 vs. RANKL+VEH.

• 

  Fig. 6 Stimulation of osteoblast differentiation by SPA0355 in MC3T3-E1 cells. (A) MC3T3-E1 cells (5×104) were treated with SPA0355 in differentiation medium for seven days. Cells were fixed and stained with ALP staining. ALP activity is expressed as p-nitrophenol released (n=3). (B) After 21 days of differentiation, calcium deposits for matrix mineralization were measured by Alizarin red S staining. The Alizarin Red dye was extracted using cetylpyridinium chloride (n=3). Values are the mean±SEM. **p<0.01 vs. vehicle.

• 

  Fig. 7 Proposed summary.

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