Triptolide Prevents Bone Destruction in the Collagen-Induced Arthritis Model of Rheumatoid Arthritis by Targeting RANKL/RANK/OPG Signal Pathway

Abstract

Focal bone destruction within inflamed joints is the most specific hallmark of rheumatoid arthritis (RA). Our previous study indicated that the therapeutic efficiency of triptolide in RA may be due partially to its chondroprotective and anti-inflammatory effects. However, its roles in bone destruction are still unclear. In this study, our data firstly showed the therapeutic effects of triptolide on severity of arthritis and arthritis progression in collagen-induced arthritis (CIA) mice. Then, by micro-CT quantification, triptolide treatment significantly increased bone mineral density, bone volume fraction, and trabecular thickness and decreased trabecular separation of inflamed joints. Interestingly, triptolide treatment could prevent the bone destruction by reducing the number of osteoclasts in inflamed joints, reducing the expression of receptor activator of NF- κ B (RANK) ligand (RANKL) and RANK, increasing the expression of osteoprotegerin (OPG), at both mRNA and protein levels, and decreasing the ratio of RANKL to OPG in sera and inflamed joints of CIA mice, which were further confirmed in the coculture system of human fibroblast-like synovial and peripheral blood mononuclear cells. These findings offer the convincing evidence for the first time that triptolide may attenuate RA partially by preventing the bone destruction and inhibit osteoclast formation by regulating RANKL/RANK/OPG signal pathway.

Figure 1

Effects of triptolide on severity of arthritis and arthritis progression in collagen-induced arthritis (CIA) mice. Mice were orally administered triptolide (Trip, 8, 16, and 32 μg/kg, resp.), methotrexate (MTX, 0.1 mg/kg), or vehicle for 21 days from the first day of the onset of the clinical symptoms of arthritis. At the end of the experiment, the arthritis index, arthritis incidence, the percentage of arthritic limbs, and changes in bodyweight were evaluated. Data are represented as the mean ± SD (n = 12)., ^# P < 0.05 in comparison with the normal control. *P < 0.05,  ⁺ P < 0.01, and *P < 0.001 in comparison with the vehicle control. ^$ P < 0.05 in comparison with methotrexate-treated CIA mice.

Figure 2

Micro-CT scan validates efficiency of triptolide in collagen-induced arthritis (CIA) mice. Mice were orally administered triptolide (Trip, 8, 16, and 32 μg/kg, resp.), methotrexate (MTX, 0.1 mg/kg), or vehicle for 21 days from the first day of the onset of the clinical symptoms of arthritis. At the end of the experiment, Micro-CT scan was performed to validate the efficiency of triptolide in CIA mice. (a) and (b), respectively, showed the three-dimensional reconstructed bones of ankle and knee joints in different groups. Compared with vehicle-treated and methotrexate-treated CIA mice, dose of 32 μg/(kg·day) triptolide markedly reduced the extent of joint destruction in triptolide-treated CIA mice.

Figure 3

Inhibitory effects of triptolide in periarticular bone erosion in knee joints of collagen-induced arthritis (CIA) mice. Mice were orally administered triptolide (Trip, 8, 16, and 32 μg/kg, resp.), methotrexate (MTX, 0.1 mg/kg), or vehicle for 21 days from the first day of the onset of the clinical symptoms of arthritis. At the end of the experiment, Micro-CT scan was performed to validate the efficiency of triptolide in CIA mice. (a) The two-dimensional reconstructed bones of knee joints in different groups. Compared with vehicle-treated CIA mice, dose of 32 μg/(kg·day) triptolide markedly reduced the extent of joint destruction in triptolide-treated CIA mice. (b) The values of four parameters including bone volume, bone mineral density (BMD), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) of proximal end of the tibia facing the articular cavity in different groups. Compared with vehicle-treated and methotrexate-treated CIA mice, triptolide significantly increased BMD, bone volume fraction (BVF), and Tb.Th of inflamed joints and decreased Tb.Sp of inflamed joints. Data are represented as the mean ± SD (n = 12). ^# P < 0.001 in comparison with the normal control. *P < 0.05,  **P < 0.01, and ***P < 0.001 in comparison with the vehicle control. ⁺ P < 0.05,  ⁺⁺ P < 0.01, and ⁺⁺⁺ P < 0.001 in comparison with methotrexate-treated CIA mice.

Figure 4

Triptolide inhibits osteoclast differentiation in CIA mice. Mice were orally administered triptolide (Trip, 8, 16, and 32 μg/kg, resp.), methotrexate (MTX, 0.1 mg/kg), or vehicle for 21 days from the first day of the onset of the clinical symptoms of arthritis. At the end of the experiment, the osteoclast differentiation was evaluated. (a) Tartrate-resistant acid phosphatase (TRAP) stained section from knee joints of vehicle-treated, triptolide-treated, and methotrexate-treated CIA mice. The under graphs is the magnification of the yellow pane in the upper graphs, respectively. (b) The bone destruction score in knee joints of vehicle-treated, triptolide-treated, and methotrexate-treated CIA mice. (c) The number of osteoclasts (multinucleated TRAP positive cells) in knee joints of vehicle-treated, triptolide-treated, and methotrexate-treated CIA mice. Data are represented as the mean ± SD (n = 12). *P < 0.05,  **P < 0.01, and ***P < 0.001 in comparison with the vehicle control. ⁺ P < 0.05  and  ⁺⁺ P < 0.01 in comparison with methotrexate-treated CIA mice.

Figure 5

Triptolide inhibits osteoclast differentiation by targeting RANKL/RANK/OPG signal pathway. Mice were orally administered triptolide (Trip, 8, 16, and 32 μg/kg, resp.), methotrexate (MTX, 0.1 mg/kg), or vehicle for 21 days from the first day of the onset of the clinical symptoms of arthritis. At the end of the experiment, the changes in RANKL, RANK, and OPG expression at both mRNA and protein levels in the ankle joint and serum were, respectively, detected by real-time PCR, immunohistochemistry, and ELISA assay. Triptolide reduces the expression of RANKL and RANK and enhances the expression of OPG and the ratio of RANKL to OPG in the ankle joint at mRNA (a) and protein (b) levels and in serum (c) of CIA mice. Data are represented as the mean ± SD (n = 12). ^# P < 0.001 in comparison with the normal control. *P < 0.05,  **P < 0.01, and ***P < 0.001 in comparison with the vehicle control. ⁺ P < 0.05,  ⁺⁺ P < 0.01, and ⁺⁺⁺ P < 0.001 in comparison with methotrexate-treated CIA mice.

Figure 6

Triptolide inhibits osteoclastogenesis in the coculture system of human fibroblast-like synovial cells (HFLS) and peripheral blood mononuclear cells (PBMCs). (a) Numerous tartrate-resistant acid phosphatase- (TRAPs) positive multinucleated cells considered as osteoclasts were formed in the coculture system of HFLS and PBMCs, while few TRAP-positive multinucleated cells were formed when concentration of 28 nmol/L triptolide were added into this coculture system; (b) The number of osteoclasts per area counted under a light microscope; (c) Expression levels of RANKL and RANK proteins in PBMCs and HFLS measured by Western blot. Soluble OPG concentration in the culture supernatant of HFLS was measured by ELISA. Data are represented as the mean ± SD (n = 3). *P < 0.05,  **P < 0.01, and ***P < 0.001 in comparison with the vehicle control.