Effect of celastrol on bone structure and mechanics in arthritic rats

Abstract

Objective: Rheumatoid arthritis (RA) is characterised by chronic inflammation leading to articular bone and cartilage damage. Despite recent progress in RA management, adverse effects, lack of efficacy and economic barriers to treatment access still limit therapeutic success. Therefore, safer and less expensive treatments that control inflammation and bone resorption are needed. We have previously shown that celastrol is a candidate for RA treatment. We have observed that it inhibits both interleukin (IL)-1β and tumor necrosis factor (TNF) in vitro, and that it has anti-inflammatory properties and ability to decrease synovial CD68+ macrophages in vivo. Herein our goal was to evaluate the effect of celastrol in local and systemic bone loss.

Methods: Celastrol was administrated intraperitoneally at a dose of 1 µg/g/day to female Wistar adjuvant-induced arthritic rats. Rats were sacrificed after 22 days of disease progression, and blood, femurs, tibiae and paw samples were collected for bone remodelling markers quantification, 3-point bending test, micro-CT analysis, nanoindentation and Fourier transform infrared spectroscopy measurements, and immunohistochemical evaluation.

Results: We have observed that celastrol preserved articular structures and decreased the number of osteoclasts and osteoblasts present in arthritic joints. Moreover, celastrol reduced tartrate-resistant acid phosphatase 5b, procollagen type 1 amino-terminal propeptide and C terminal crosslinked telopeptide of type II collagen serum levels. Importantly, celastrol prevented bone loss and bone microarchitecture degradation. Celastrol also preserved bone nanoproperties and mineral content. Additionally, animals treated with celastrol had less fragile bones, as depicted by an increase in maximum load and yield displacement.

Conclusions: These results suggest that celastrol reduces both bone resorption and cartilage degradation, and preserves bone structural properties.

Keywords: Adjuvant-induced arthritis; Bone loss; Celastrol; Inflammation; Rheumatoid arthritis.

Figure 1

Celastrol reduces the number of bone-related cells in arthritic joints. (A) Representation of the immunohistochemical evaluation performed in left hind paw sections at the ankle joint by day 22 after celastrol treatment. Magnifications of 200× and 400×. (B) Cathepsin K+ cells and osteocalcin positive cells were identified by immunohistochemistry of paw sections. Immunohistochemical analysis was performed using a semiquantitative score. Notice that celastrol treatment significantly reduced both types of cells. Samples were collected at the time of sacrifice. Data are expressed as median score with minimum and maximum interval. H, healthy; A, arthritic; E, celastrol early-treated; L, celastrol late-treated. Healthy, n=16; arthritic, n=10; celastrol early-treated, n=15; and celastrol late-treated, n=15.

Figure 2

Celastrol diminishes bone and cartilage resorption markers. (A) TRACP-5b, (B) P1NP and (C) CTX-II levels were quantified in rat serum samples collected at the time of sacrifice. Celastrol is able to significantly reduce the levels of TRACP-5b, P1NP and CTX-II in comparison with untreated arthritic rats. Data are expressed as median with minimum and maximum interval. H, healthy; A, arthritic; E, celastrol early-treated; L, celastrol late-treated. Healthy, n=13; arthritic, n=18; celastrol early-treated, n=15; and celastrol late-treated, n=15. CTX-II, C terminal crosslinked telopeptide of type II collagen; P1NP, procollagen type 1 amino-terminal propeptide; TRACP-5b, tartrate-resistant acid phosphatase 5b.

Figure 3

Celastrol preserves bone microarchitecture in arthritis. Inflammation-induced bone loss and bone microarchitecture degradation and the protective effect of celastrol are illustrated in representative micro-CT reconstructions (A). Trabecular (B) and cortical (C) bone indices were quantified from micro-CT reconstructions. Notice that tibiae from the celastrol early-treated group have improved trabecular and cortical parameters compared with arthritic rats. Tibiae were collected at the time of sacrifice. Data are expressed as median with minimum and maximum interval. BV/TV, bone volume per tissue volume; H, healthy; A, arthritic; E, celastrol early-treated; L, celastrol late-treated. Healthy, n=30; arthritic, n=30; celastrol early-treated, n=15; and celastrol late-treated n=15.

Figure 4

Bone mechanical properties assessed by nanoindentation in rat tibiae on day 22 post disease induction. (A) Results showed decreased cortical and trabecular hardness in the arthritic group when compared with healthy rats. Of notice, rats treated with celastrol showed increased hardness in cortical bone in comparison with untreated arthritic rats. (B) Illustrative topographic images of the histological features observed in the indentation tissue area. Concentric lamellae are identified in secondary osteons, characteristic of arthritic animals. On the contrary, parallel-lamellae are identified in healthy and celastrol early-treated groups. Magnification 20×. Tibiae were collected at the time of sacrifice. Data are expressed as median with minimum and maximum interval. HViT, Vickers hardness; Os.L/TV, osteocyte lacunae per tissue volume; H, healthy; A, arthritic; E, celastrol early-treated; L, celastrol late-treated. Healthy, n=28; arthritic, n=21; celastrol early-treated, n=14; and celastrol late-treated, n=14.

Figure 5

FTIR measurements from cortical and trabecular bone of rat tibia on day 22 post disease induction. FTIR measurements revealed that arthritic rats had mineral and collagen loss in trabecular bone (A and B). Celastrol-treated groups demonstrated increased mineral content of cortical and trabecular bone (A and C), as well as carbonate cortical content (D). Tibiae were collected at the time of sacrifice. Data are expressed as median with minimum and maximum interval. H, healthy; A, arthritic; E, celastrol early-treated; L, celastrol late-treated. Healthy, n=28; arthritic, n=21; celastrol early-treated, n=14; and celastrol late-treated, n=14. FTIR, Fourier transform infrared spectroscopy.

Figure 6

Celastrol effect on arthritic bone mechanical properties. Maximal load (A), maximal deformation (B), total absorbed energy (C), yield displacement (D), yield load (E), elastic energy (F) and stiffness (G) parameters were obtained by 3-point bending. Celastrol early-treated rats have higher levels of yield point displacement and maximum load compared with untreated arthritic rats. Femurs were collected after 22 days of disease induction. Data are expressed as median with minimum and maximum interval. H, healthy; A, arthritic; E, celastrol early-treated; L, celastrol late-treated. Healthy, n=13; arthritic, n=10; celastrol early-treated, n=15; and celastrol late-treated, n=15.