Suppression of autoimmune arthritis by Celastrus-derived Celastrol through modulation of pro-inflammatory chemokines

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the synovial joints, deformities, and disability. The prolonged use of conventional anti-inflammatory drugs is associated with severe adverse effects. Therefore, there is an urgent need for safer and less expensive therapeutic products. Celastrol is a bioactive component of Celastrus, a traditional Chinese medicine, and it possesses anti-arthritic activity. However, the mechanism of action of Celastrol remains to be fully defined. In this study based on the rat adjuvant-induced arthritis (AA) model of RA, we examined the effect of Celastrol on two of the key mediators of arthritic inflammation, namely chemokines and their receptors, and related pro-inflammatory cytokines. We treated arthritic Lewis rats with Celastrol (200μg/rat) or its vehicle by daily intraperitoneal (ip) injection beginning at the onset of AA. At the peak phase of AA, the sera, the draining lymph node cells, spleen adherent cells, and synovial-infiltrating cells of these rats were harvested and tested. Celastrol-treated rats showed a significant reduction in the levels of chemokines (RANTES, MCP-1, MIP-1α, and GRO/KC) as well as cytokines (TNF-α and IL-1β) that induce them, compared to the vehicle-treated rats. However, Celastrol did not have much effect on cellular expression of chemokine receptors except for an increase in CCR1. Further, Celastrol inhibited the migration of spleen adherent cells in vitro. Thus, Celastrol-induced suppression of various chemokines that mediate cellular infiltration into the joints might contribute to its anti-arthritic activity. Our results suggest that Celastrol might offer a promising alternative/adjunct treatment for RA.

Figure 1. Chemokine production by lymph nodes cells (LNC), spleen adherent cells (SAC) and synovial-infiltrating cells (SIC) after Mtb re-stimulation

LNC, SAC and SIC were prepared from Mtb-immunized arthritic rats on d 18 after Mtb immunization. These cells were stimulated in vitro with Mtb (10 μg/ml) for 24 h. Thereafter, the culture supernatants collected from the wells were analyzed for the indicated chemokines and cytokines using a Multiplex assay. The levels shown represent the differences between Mtb re-stimulated and control samples. The results shown are representative of two independent experiments, each done in triplicates.

Figure 2. Effect of Celastrol treatment on chemokine/cytokine levels in SAC and serum

(A) SAC: Mtb-immunized Lewis rats were treated with Celastrol (in DMSO) starting from the onset of the disease. Control rats were treated with vehicle (PBS in DMSO). SAC from these rats were prepared on d 18 after Mtb immunization and then stimulated in vitro with sonicated Mtb (10 μg/ml) for 24 h. Thereafter, the culture supernatants were analyzed for the indicated chemokines/cytokines using a Multiplex assay. The mean ± SD of triplicate assays from 4-5 rats are shown. (B) Serum: Serum was prepared from the blood samples collected from the same rats and then analyzed for various chemokines and cytokines. The results obtained from pooled serum samples from 4 rats tested in triplicate are shown. * = P < 0.05, versus control group.

Figure 3. Inhibition of Mtb-/IL-1β-induced chemokine and cytokine production by Celastrol in rat synovial fibroblasts

Rat synovial fibroblasts plated in a 6-well plate (2 × 10⁵ cells/well) were treated with Celastrol (0.1-0.3 μM) for 30 min, followed by stimulation with sonicated-Mtb (10 μg/ml) (A) or IL-1β (10 ng/ml) (B) for 48 h. Thereafter, the culture supernatants were analyzed for chemokines and cytokines using a Multiplex assay. The graph shows the delta values (Δ= test-medium) of a representative of two independent experiments done in triplicate.

Figure 4. Effect of Celastrol on CCR1 expression in SAC

SAC (1.5-2.0 × 10⁶ cells/well) from Celastrol-treated and PBS-treated rats were restimulated with sonicated Mtb (10 μg/ml) for 24 h (A). Similarly, SAC from an untreated arthritic rat were restimulated with Mtb for 24 h in the presence of different concentrations (0.1 and 0.3 μM) of Celastrol (B). Thereafter, total RNA was isolated from SAC and the mRNA expression for CCR1 was analyzed by qPCR. Results are expressed as “relative message” after normalization to HPRT.

Figure 5. Cell migration assay

Spleen adherent cells (SAC) derived from an arthritic rat were allowed to migrate from the upper to lower surface of the Transwell membrane in response of RANTES (50 ng/ml) in the presence or absence of Celastrol. Cells that migrate to the lower surface of the membrane were stained with DAPI and then observed under Nikon Eclipse TE2000-U microscope with 10× magnification.