The study of mechanisms of protective effect of Rg1 against arthritis by inhibiting osteoclast differentiation and maturation in CIA mice

Abstract

Ginsenoside Rg1 is a natural product extracted from Panax ginseng C.A. Although Rg1 protects tissue structure and functions by inhibiting local inflammatory reaction, the mechanism remains poorly understood. In vitro, Rg1 dose-dependently inhibited TRAP activity in receptor activator of nuclear factor-κB ligand- (RANKL-) induced osteoclasts and decreased the number of osteoclasts and osteoclast resorption area. Rg1 also significantly inhibited the RANK signaling pathway, including suppressing the expression of Trap, cathepsin K, matrix metalloproteinase 9 (MMP9), and calcitonin receptor (CTR). In vivo, Rg1 dramatically decreased arthritis scores in CIA mice and effectively controlled symptoms of inflammatory arthritis. Pathologic analysis demonstrated that Rg1 significantly attenuated pathological changes in CIA mice. Pronounced reduction in synovial hyperplasia and inflammatory cell invasion were observed in CIA mice after Rg1 therapy. Alcian blue staining results illustrated that mice treated with Rg1 had significantly reduced destruction in the articular cartilage. TRAP and cathepsin K staining results demonstrated a significant reduction of numbers of OCs in the articular cartilage in proximal interphalangeal joints and ankle joints in Rg1-treated mice. In summary, this study revealed that Rg1 reduced the inflammatory destruction of periarticular bone by inhibiting differentiation and maturation of osteoclasts in CIA mice.

Figure 1

Effect of Rg1 on cell viability and apoptosis. (a) CCK8 assay was performed after incubation of RAW 264.7 cells (2 × 10⁴ cells/mL) with Rg1 (1, 10, and 100 μg/mL) for 24, 48, 72, and 96 h in 96-well plates. (b) RAW 264.7 cells (1.0 × 10⁵ cells/mL) were stimulated with or without the presence of Rg1 (1, 10, and 100 μg/mL) for 96 h in a 6 cm culture plate. Cells were collected and stained with Annexin V and PI and then examined by flow cytometry. (c) FACS histogram of the percentage of early and late apoptosis cells. Values are expressed as the mean ± SD of triplicate experiments. All data are representative of three different experiments.

Figure 2

Effects of Rg1 on osteoclast differentiation and resorption pit formation in RANKL-stimulated RAW 264.7 cells. (a) RAW 264.7 cells (2 × 10⁴ cells/mL) were stimulated with RANKL (100 ng/mL) for 96 h with or without the presence of Rg1 (1, 10, and 100 μg/mL). Cells were fixed with 3.7% formalin, permeabilized with 0.1% Triton X-100, and stained with TRAP solution. (b) RAW 264.7 cells (5 × 10³ cells/mL) were cultured on bone slices with various concentrations of Rg1 in the presence of RANKL (50 ng/mL). After culturing for 10 days, the dentine slice was recovered from the culture and subjected to visualization of resorption pits. (c) The cells were stained for TRAP, and TRAP-positive multinuclear cells containing more than three nuclei were counted as multinucleated osteoclasts (mOCs). (d) The percentages of resorbed area were determined using Image-J software. Values are expressed as the mean ± SD of triplicate experiments. ∗P > 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 3

Effect of Rg1 on the mRNA expression of osteoclastic marker genes in RANKL-stimulated RAW 264.7 cells. RAW 264.7 cells (2 × 10⁴ cells/mL) were stimulated with RANKL (50 ng/mL) in the presence of Rg1 (1, 10, and 100 μg/mL) for 96 h in 12-well plates. mRNA expressions of osteoclastogenic marker genes were determined by RT-PCR. Values are expressed as the mean ± SD of triplicate experiments. ∗P > 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 4

Effect of Rg1 on the RANKL-induced expression of c-Fos, c-Jun, and NFATc1 in RAW 264.7 cells. (a) RAW 264.7 cells (1 × 10⁵ cells/mL) were plated in a 6 cm culture plate and cultured with or without RANKL (50 ng/mL) and/or Rg1 (100 μg/mL) for indicated periods. The cell lysates were subjected to Western blot analysis with an antibody to c-Fos, c-Jun, and NFATc1. (b) RAW 264.7 cells (1 × 10⁵ cells/mL) were plated in a 6 cm culture plate, and RAW264.7 cells were cultured with RANKL (50 ng/mL) and/or increasing concentrations of Rg1 for 48 h. The cells were subjected to Western blot analysis. (c) RAW264.7 cells (2 × 10⁴ cells/mL) were cultured with or without RANKL (50 ng/mL) and/or Rg1 (1, 10, and 100 μg/mL) for 48 h in 12-well plates. The total cellular RNA was extracted and used for RT-PCR analysis to detect c-Fos, c-Jun, and NFATc1 mRNA. All data are representative of three different experiments.

Figure 5

Effects of Rg1 on RANKL-induced IκB, NF-κB, and MAP kinase activation. IκB, NF-κB, and MAP kinase activation was represented by the levels of protein phosphorylation. RAW 264.7 cells (1 × 10⁵ cells/mL) were cultured with or without Rg1 (100 μg/mL) in the presence of RANKL for indicated times. Western blot analysis was performed with whole cell lysates (10 mg). Blots were probed with antibodies specific for IκB, NF-κB, and MAP kinase. The densities of phosphorylated protein (p-) levels (upper panels) were normalized to the density of total protein levels (lower panels). GAPDH was used as the loading control.

Figure 6

Effects of Rg1 on incidence, arthritis scores, and paw swelling with prolonged time in CIA mice. Mice were intraperitoneally injected with placebo and Rg1 for 14 days after 28 days since the first immunization. Paw thickness (a), incidence (b), and arthritis scores (c) were measured at 1-day intervals. Rg1 markedly reduced the mean arthritis scores and paw swelling. Data are the mean ± SD of eight mice. ∗P < 0.05, compared with relevant controls.

Figure 7

Rg1 resulted in reduced histological inflammation, cartilage loss, and OC formation in CIA mice. (a) Histological evaluations of antiarthritis effects of Rg1 were observed in joint slides stained by HE (original magnification, 200x). Histologic scores in the sections shown in (a). (b) Representative histopathologies of proximal interphalangeal joint, and ankle joint stained with Alcian blue in CIA (original magnification, 200x). Histologic analysis of cartilage in CIA mice after Rg1 therapy. Sections were scored in a blinded manner on a four-point scale as described in Materials and Methods section. A significantly less loss of Alcian blue staining was observed in animals treated with Rg1 than compared to controls, indicating inhibition of cartilage destruction. (c) Trap staining in ankle joints and proximal interphalangeal joint isolated from normal, CIA, and Rg1-treated mice. The expression of Trap significantly decreased in Rg1-treated mice compared with CIA mice. (d) Immunohistochemical staining for cathepsin K in ankle joints and proximal interphalangeal joint isolated from normal, CIA, and Rg1-treated mice. The expression of cathepsin K significantly decreased in Rg1-treated mice compared with CIA mice. Data are the mean ± SD, n = 8 mice. ∗P < 0.05 versus CIA treated with vehicle.