Matrine prevents bone loss in ovariectomized mice by inhibiting RANKL-induced osteoclastogenesis

Abstract

Osteoporosis is a metabolic bone disease characterized by decreased bone density and strength due to excessive loss of bone protein and mineral content. The imbalance between osteogenesis by osteoblasts and osteoclastogenesis by osteoclasts contributes to the pathogenesis of postmenopausal osteoporosis. Estrogen withdrawal leads to increased levels of proinflammatory cytokines. Overactivated osteoclasts by inflammation play a vital role in the imbalance. Matrine is an alkaloid found in plants from the Sophora genus with various pharmacological effects, including anti-inflammatory activity. Here we demonstrate that matrine significantly prevented ovariectomy-induced bone loss and inhibited osteoclastogenesis in vivo with decreased serum levels of TRAcp5b, TNF-α, and IL-6. In vitro matrine significantly inhibited osteoclast differentiation induced by receptor activator for NF-κB ligand (RANKL) and M-CSF in bone marrow monocytes and RAW264.7 cells as demonstrated by tartrate-resistant acid phosphatase (TRAP) staining and actin-ring formation as well as bone resorption through pit formation assays. For molecular mechanisms, matrine abrogated RANKL-induced activation of NF-κB, AKT, and MAPK pathways and suppressed osteoclastogenesis-related marker expression, including matrix metalloproteinase 9, NFATc1, TRAP, C-Src, and cathepsin K. Our study demonstrates that matrine inhibits osteoclastogenesis through modulation of multiple pathways and that matrine is a promising agent in the treatment of osteoclast-related diseases such as osteoporosis.-Chen, X., Zhi, X., Pan, P., Cui, J., Cao, L., Weng, W., Zhou, Q., Wang, L., Zhai, X. Zhao, Q., Hu, H., Huang, B., Su, J. Matrine prevents bone loss in ovariectomized mice by inhibiting RANKL-induced osteoclastogenesis.

Keywords: NFATc1; osteoclasts; postmenopausal osteoporosis.

Figure 1.

Matrine inhibits osteoclastogenesis in vitro. A) Chemical structure of matrine. B) Formation of TRAP-positive cells from BMMCs and quantification of osteoclasts. C) Formation of TRAP-positive cells from RAW264.7 cells and quantification of osteoclasts. *P < 0.05, **P < 0.01.

Figure 2.

Matrine inhibits osteoclast function in vitro. A) Actin ring structures of osteoclasts and quantification of the actin ring. B) RAW264.7 cells seeded on hydroxyapatite-coated plates. Surfaces were treated similarly and incubated for 7 d, and resorption area was quantified by image analysis. ***P < 0.001.

Figure 3.

Matrine inhibits RANKL-induced NF-κB activation. A) Matrine inhibits RANKL-induced P65 nuclear translocation. B) Phosphorylation of P65, P50, and IκB protein, which were associated with the NF-κB pathway. C) Ratio of the fluorescence intensity at the nuclear site with whole-cell fluorescence intensity. **P < 0.01.

Figure 4.

Matrine suppresses MAPKs and PI3K/AKT pathways in osteoclastogenesis. A) Phosphorylation of ERK, JNK, and P38, which was associated with the MAPK pathway. B) Phosphorylation of C-fos, which was an important downstream transcription factor of the MAPK pathway. C) Phosphorylation of AKT, which was associated with the PI3K/AKT pathways.

Figure 5.

Matrine suppresses osteoclastogenesis-related marker gene expression. Matrine-inhibited protein expression levels of MMP-9, cathepsin K, C-Src, TRAP, and NFATc1 in RAW264.7 cells.

Figure 6.

Matrine inhibits ovariectomy-induced bone loss in vivo. A) Representative H&E staining of femoral sections and difference of trabecular area from each group 6 wk after the operation. B) Representative micro–computed tomography sections of femur from sham-treated, OVX, and matrine-treated OVX (OVX + matrine) mice. C) Trabecular number (Tb.N), BS/TV, BV/TV, and BMD were analyzed. D) Representative TRAP-stained histologic femur sections of the long bone from sham-treated, OVX, and matrine-treated OVX mice. E) IL-6, TNF-α, TRAcp5B, and OCN were examined in serum. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7.

Matrine inhibits osteoclastogenesis through multiple pathways of RANKL signaling.