4-Hexylresorcinol inhibits osteoclastogenesis by suppressing the NF-κB signaling pathway and reverses bone loss in ovariectomized mice

Abstract

4-Hexylresorcinol (4HR) is a small organic compound that is widely used as an antiseptic and antioxidant. In the present study, its role in osteoclastogenesis was investigated. Bone marrow-derived macrophages from mice were used to examine the role of 4HR in osteogenesis. An ovariectomy (OVX) mouse model was constructed to examine the effect of 4HR in vivo, followed by hematoxylin and eosin and tartrate resistant acid phosphatase staining. In the present study, 4HR effectively suppressed receptor activator of NF-κB ligand-induced osteoclastogenesis in a dose-dependent manner. 4HR was also found to significantly suppress the expression of osteoclast (OC)-specific markers, including tartrate-resistant acid phosphatase, cathepsin K, nuclear factor of activated T-cell cytoplasmic 1 and c-Fos in the presence of RANKL in BMMs. Furthermore, 4HR inhibited osteoclastogenesis by inhibiting the activation of the NF-κB signaling pathway in BMMs. Consistent with the in vitro results, 4HR effectively ameliorated OVX-induced bone loss and markedly reduced OC number in the proximal tibia in vivo. In conclusion, the present results suggested that 4HR inhibited osteoclastogenesis in vitro and rescued bone loss in vivo, suggesting that 4HR may serve as a novel therapeutic agent for osteoporosis treatment.

Keywords: 4-Hexylresorcinol; osteoclast; osteoclastogenesis; ovariectomy.

Figure 1

4HR inhibits RANKL-induced osteoclastogenesis in vitro. (A-D) BMMs were seeded into 96-well plates and cultured at 37˚C with complete medium supplemented with 10 ng/ml M-CSF and 100 ng/ml RANKL. (A) BMMs were treated with different concentrations of 4HR (0, 5, 10 and 20 µg/ml) for 3 days in the presence of M-CSF. Cell viability was assessed using a Cell Counting Kit-8. No significant difference was observed among the groups. (B) Representative TRAP staining images on day 5 of OC differentiation following incubation with different concentrations of 4HR (0, 5, 10 and 20 µg/ml). Magnification, x100. (C) The number of TRAP-positive cells with ≥ three nuclei were quantified in each well in (B). ^*P<0.05, ^**P<0.01 and ^***P<0.001. (D) The percentage of the TRAP-positive cell area in each well was measured in (B). ^*P<0.05, ^**P<0.01 and ^***P<0.001. (E-H) BMMs were treated with different concentrations of 4HR (0, 5, 10 and 20 µg/ml) in the presence of 10 ng/ml M-CSF and 100 ng/ml RANKL. mRNA expression levels of osteoclastogenesis markers (E) TRAP, (F) Cathepsin K, (G) NFATc1 and (H) c-Fos on day 3 of OC differentiation are shown for each group. ^*P<0.05, ^**P<0.01 and ^***P<0.001. 4HR, 4-hexylresorcinol; RANKL, receptor activator of nuclear factor kappa B ligand; M-CSF, macrophage colony-stimulating factor; BMMs, bone marrow-derived macrophages; TRAP, tartrate resistant acid phosphatase; Veh, Vehicle; NFATc1, nuclear factor of activated T-cells c1.

Figure 2

4HR suppresses RANKL-induced activation of the NF-κB pathway. (A-E) BMMs cultured at 37˚C with complete medium supplemented with 10 ng/ml M-CSF and 100 ng/ml RANKL. (A) Representative images of western blot analysis showing that 4HR inhibited the RANKL-induced protein expression of TRAP, NFATc1 and Cathepsin K in BMMs after 20 µg/ml 4HR treatment for 3 days in the presence of 10 ng/ml M-CSF and 100 ng/ml RANKL. (B) Quantification of (A). ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. Veh. (C) Luciferase assay of the NF-κB luciferase reporter plasmid in BMMs pretreated with or without 4HR (20 µg/ml) for 30 min in the presence or absence of 10 ng/ml M-CSF and 100 ng/ml RANKL. ^***P<0.001 vs. Veh without 4HR treatment and RANKL stimulation, ^###P<0.001 vs. BMMs without 4HR treatment but with RANKL stimulation. (D) Western blot analysis of the lysate following 100 ng/ml RANKL incubation for the indicated time points (0, 15, 30 and 60 min) in BMMs with or without 4HR preincubation for 30 min (20 µg/ml) in the presence of 10 ng/ml M-CSF. (E) Quantification of (D). ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. Veh without 4HR exposure at the corresponding time points. 4HR, 4-hexylresorcinol; RANKL, receptor activator of NF-κB ligand; TRAP, tartrate resistant acid phosphatase; NFATc1, nuclear factor of activated T-cells cytoplasmic 1; BMMs, bone marrow-derived macrophages; M-CSF, macrophage colony-stimulating factor; Veh, Vehicle; p-IKKβ, phosphorylated-IκB kinase β.

Figure 3

4HR treatment reverses ovariectomy-induced bone loss in vivo. (A) MicroCT images of the proximal tibia 60 days after ovariectomy surgery. Scale bar, 1 mm. (B) BV/TV ratio was higher in the proximal tibia following 4HR administration for 60 days compared with that in the OVX group. (C) Quantification of Tb.N in the proximal tibia showed a significantly higher Tb.N in OVX mice treated with 4HR compared with those in OVX mice. (D) Quantification of Tb.Th showed a significantly higher Tb.Th following 4HR treatment for 60 days after OVX. (E) Quantification of Tb.Sp in 4HR treatment for 60 days after OVX. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. Sham. ^#P<0.05 vs. OVX. n=6 per group.4HR, 4-hexylresorcinol; BV/TV, bone volume/tissue volume; OVX, ovariectomy; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation.

Figure 4

4HR administration increases bone mass and reduces the OC number in vivo. (A) Representative images of H&E staining of the proximal tibia following OVX with or without 4HR administration in mice, arrows showed the trabecular bones. Scale bar, 25 µm. (B) Representative images of TRAP staining in the proximal tibia in each group 60 days after OVX, arrows showed the purple multinucleated osteoclasts. Scale bar, 25 µm. (C) Quantification of TRAP positive cells in the proximal tibia 60 days after OVX. Data were shown as the OC number per bone surface/mm. Serum levels of (D) P1NP and (E) CTX1 were measured. ^**P<0.01 vs. Sham, ^#P<0.05 and ^##P<0.01 vs. OVX. n=6 per group. 4HR, 4-hexylresorcinol; H&E, hematoxylin and eosin; OVX, ovariectomy; TRAP, tartrate resistant acid phosphatase; P1NP, procollagen type 1 amino-terminal propeptide; CTX1, C-terminal telopeptide of type1; NB/S, number per bone surface.