Isoliensinine Suppresses Osteoclast Formation Through NF-κB Signaling Pathways and Relieves Ovariectomy-Induced Bone Loss

Abstract

Osteoporosis is among the major contributors of pathologic fracture in postmenopausal women, which is caused by the bone metabolic disorder owing to the over-activation of osteoclasts. Inhibition of osteoclast differentiation and maturation has become a mainstream research interest in the prevention of osteoporosis. Isoliensinine (Iso) is a dibenzyl isoquinoline alkaloid with antioxidant, anti-inflammatory, and anti-cancer activities. However, whether it can be used as a potential treatment for osteoporosis remains undiscovered. Here, we investigated whether Iso might suppress the differentiation of osteoclasts in vitro and in vivo to play an anti-osteoporosis role. Our results showed that Iso inhibits the formation of mature multinuclear osteoclasts induced by RANKL, the bone resorption, and the osteoclast-specific genes expression by blocking the nuclear translocation of NF-κB p65, and the effect was in a dosage-dependent way. Furthermore, we investigated the therapeutic effect of Iso on osteoporosis in ovariectomized (OVX) mice. We found that Iso attenuated bone loss in the OVX mice and significantly promoted BS, Conn. DN, Tb.Th, TB.N, and BV/TV Index. All in all, Iso showed a prominent effect of osteoclast inhibition, with great promise for treating osteoporosis.

Keywords: NF-κB pathway; isoliensinine; osteoclast; osteoporosis; receptor activator of nuclear factor-κB ligand (RANKL).

FIGURE 1

Search for targets of Iso related to osteoporotic disease through network pharmacology. (A) The 3D chemical structure and formula of Iso (B) Venn diagram of Iso prediction targets and targets related to osteoporotic disease (C,D) KEGG enrichment analysis of Iso target genes related to osteoporotic disease. The higher the correlation, the darker the color, the larger the enrichment index, and the larger the bubble.

FIGURE 2

Iso suppresses RANKL-associated osteoclastogenesis in vitro. (A) The cellular cytotoxicity of Iso was evaluated by CCK-8 assay after 48 h of incubation (B) Quantification of TRAP-positive multinucleated cells (nuclei >3) (n = 3 per group). (C) Representative images of TRAP staining showing that Iso inhibited osteoclastogenesis dose-dependently. BMMs were stimulated with RANKL for 5 days in the absence or presence of indicated concentrations of Iso (scale bar = 500 μm). (D) Quantification of TRAP-positive multinucleated cells treated with Iso in different time periods (n = 3 per group). (E) Representative TRAP stained phase-contrast images of the time-dependent effect Iso on osteoclast formation. BMMs stimulated with RANKL and treated with 5 μM Iso on the indicated days were fixed and stained for TRAP activity (scale bar = 500 μm). The above data are expressed as the mean ± SD; n = 3; **p < 0.01 and ***p < 0.001. Iso: Isoliensinine; CCK-8: cell counting kit-8; BMMs: bone marrow macrophages; TRAP: tartrate-resistant acid phosphatase; RANKL: receptor activator of nuclear factor-κB ligand.

FIGURE 3

Iso affects osteoclast fusion, F-actin belt formation and attenuates osteoclastic hydroxyapatite resorption triggered by RANKL. (A) Representative images of osteoclast fusion treated with different concentrations of Iso (2.5 μM and 5 μM). The cell membrane (red) and nucleus (blue) of osteoclasts were stained (scale = 100 μM). (B) Representative images of podosomal belt formation in osteoclasts treated with different concentrations of Iso (2.5 μM and 5 μM). The actin cytoskeleton (red) and nucleus (blue) of osteoclasts were stained (scale = 400 μM). (C) Representative micrographs of TRAP-stained osteoclasts (upper panels) and the assay surface after removal of osteoclasts on hydroxyapatite-coated plates with or without Iso (2.5 μM and 5 μM) (scale = 1000 μM). (D) Process flows of osteoclast fusion assay (E) Double-fluorescence BMMs fusion on day 3 quantified by the membrane merge rate (F) Numbers of nuclei per osteoclast (n = 3). (G) Mean F-actin belt areas (n = 3). (H,I) The TRAP positive cells and resorbed area per well were quantitatively counted with ImageJ (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. All data are expressed as mean ± SD. TRAP: tartrate-resistant acid phosphatase.

FIGURE 4

Iso inhibits osteoclast marker gene expression and attenuates c-fos, CTSK and NFATc1 protein expression. (A) BMMs were treated with M-CSF (25 ng/ml) and RANKL (50 ng/ml) in the presence or absence of the indicated concentrations of Iso. Gene expression was normalized to Hmbs. qPCR analysis of osteoclast-specific genes expression of Mmp9, Nfatc1, Trap, C-fos, Ctsk And Dc-stamp (n = 3 per group). (B) BMMs were treated with or without Iso (5 μM) on day1, day3, day5 and the proteins including c-Fos, NFATc1 and CTSK were measured using western-blot (C–E) The expression of proteins mentioned above was quantitatively analyzed related to β-actin. *p < 0.05, **p < 0.01, ***p < 0.001. BMMs, bone marrow macrophages; Iso, Isoliensinine; PCR, polymerase chain reaction; RANKL, receptor activator of nuclear factor-κB ligand; M-CSF: macrophage colony stimulating factor; Mmp9, matrix metallopeptidase 9; Nfatc1, nuclear factor of activated T cells 1; Trap, tartrate-resistant acid phosphatase; c-fos, Proto-oncogene C-Fos; Ctsk, cathepsin K; Dc-stamp, dendritic cell-specific transmembrane protein.

FIGURE 5

Iso abolishes RANKL-induced activation of NF-κB signaling pathways, but does not suppress MAPK pathway during osteoclastogenesis. (A,B) Molecular docking showed that Iso could bind with P65 and IκBα in NF-κB pathway (C) Immunofluorescence images of p65 nuclear translocation following RANKL stimulation without or with 5 μM Iso treatment (Magnification = ×20, scale = 100 μM). Cell nuclei were counterstained with DAPI. (D,E) Representative western-blot images of protein expression and phosphorylation status of p65, IκBα, ERK, JNK and p38 (F–G) The expression of proteins above was quantitatively analyzed standardized to β-actin (H–J) The expression levels of P38, ERK, JNK. All data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

Iso attenuates bone loss in ovariectomized (OVX) mice. (A) Establishment of an osteoporosis model of OVX mice and an experimental design to evaluate the efficacy of Iso (B–I) Quantitative analysis of bone-related parameters, including Ct. Th, BV/TV Tb. Sp, Tb.N, Tb.Th, Conn. Dn, and BS (n = 5). The above data are expressed as the mean ± SD; *p < 0.05,**p < 0.01, and ***p < 0.001. Iso: Isoliensinine; Vehicle, 1% DMSO in PBS; TRAP, tartrate-resistant acid phosphatase. Ct. Th, cortical bone thickness; BV/TV, bone volume per tissue volume; Tb. Sp, trabecular spacing; Tb.N, trabecular number; Tb.Th, trabecular thickness; Conn. Dn, connectivity density; BS, bone surface.

FIGURE 7

Iso treatment reduces the production of osteoclasts in ovariectomized mice. (A) Representative photographs of H&E and TRAP staining from each treatment group (B–D) Quantitative analysis of bone surface (BS), osteoclast number/bone surface (N.Oc/BS) and osteoclast surface/bone surface (Oc.S/BS). The data are expressed as the means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, n = 5. H&E, hematoxylin and eosin; TRAP, tartrate-resistant acid phosphatase.

FIGURE 8

Iso treatment reduces the production of osteoclasts in ovariectomized mice. A proposed scheme for the inhibition of Iso on osteoclastogenesis. Upon RANKL binding to RANK, NF-κB pathways are activated, leading to the amplification of NFATc1. Several osteoclast-specific genes such as c-Fos, Ctsk, Trap, and Dc-stamp are upregulated as a result. RANKL: receptor activator of nuclear factor-κB ligand. NF-κB, nuclear factor-κB; NFATc1, nuclear factor of activated T cells 1, c-fos, Proto-oncogene, Ctsk, cathepsin K; Trap, tartrate-resistant acid phosphatase; Dc-stamp, dendritic cell-specific transmembrane protein.