Celastrus aculeatus Merr. suppresses the induction and progression of autoimmune arthritis by modulating immune response to heat-shock protein 65

Abstract

Complementary and alternative medicine products are increasingly being used for the treatment of autoimmune diseases. However, the mechanisms of action of these agents are not fully defined. Using the rat adjuvant arthritis (AA) model of human rheumatoid arthritis, we determined whether the ethanol extract of Celastrus aculeatus Merr. (Celastrus), a Chinese herb, can down-modulate the severity of AA, and also examined the Celastrus-induced changes in immune responses to the disease-related antigen mycobacterial heat-shock protein 65 (Bhsp65). AA was induced in the Lewis (LEW; RT.1l) rat by immunization subcutaneously with heat-killed M. tuberculosis H37Ra (Mtb). Celastrus was fed to LEW rats by gavage daily, beginning either before Mtb challenge (preventive regimen) or after the onset of AA (therapeutic regimen). An additional group of rats was given methotrexate for comparison. All rats were graded regularly for the signs of arthritis. In parallel, the draining lymph node cells of Celastrus-treated rats were tested for proliferative and cytokine responses, whereas their sera were tested for the inflammatory mediator nitric oxide. Celastrus feeding suppressed both the induction as well as the progression of AA, and the latter effect was comparable to that of methotrexate. Celastrus treatment induced relative deviation of the cytokine response to anti-inflammatory type and enhanced the production of anti-Bhsp65 antibodies, which are known to be protective against AA. Celastrus feeding also reduced the levels of nitric oxide. On the basis of our results, we suggest further systematic exploration of Celastrus as an adjunct therapeutic modality for rheumatoid arthritis.

Figure 1

Feeding of Celastrus suppresses the induction of adjuvant arthritis (AA) in the Lewis (LEW) rat. (a) LEW rats (n = 7 per group) were fed by gavage daily either Celastrus (triangles; 3 g/kg body weight, experimental group) or water (circles; control group) starting on day 4 prior to M. tuberculosis H37Ra (Mtb) immunization (1 mg/rat) and then continuing throughout the observation period. Following Mtb injection, these rats were scored regularly for signs of arthritis. The difference in the mean arthritic scores of the Celastrus-fed and Water-fed rats during the course of AA was significant (*p < 0.05 by Wilcoxon rank sum test). (b) The results of an independent repeat experiment including two groups of Celastrus-treated rats are shown in this section. The difference in arthritic scores of Celastrus-fed versus Water-fed rats was significant (*p < 0.05) for each of the groups tested (triangles; 1.5 g/kg, n = 4; squares, 3 g/kg, n = 4). (c) The photograph shows the hind paw of a representative LEW rat from the water-fed (left) and Celastrus-fed (middle) groups on day 16 after Mtb immunization. The hind paw of a naive LEW rat (right) is also shown for comparison; each unit on the scale equals 1 mm, with 10 units between numbered marks.

Figure 2

The cytokine response to mycobacterial hsp65 (Bhsp65) of lymph node cells (LNCs) of Celastrus-fed versus water-fed Lewis (LEW) rats. Two groups of LEW rats (n = 6 to 9) were fed either Celastrus (3 g/kg) or water as described in the legend to Figure 1. A sub-group of these LEW rats was euthanized on day 12 after M. tuberculosis H37Ra immunization, and the draining LNCs of these rats were cultured in a 96-well plate in the presence of the indicated recall antigens (HEL, hen eggwhite lysozyme; B177, synthetic peptide 177–191 of Bhsp65). The supernatant was collected after 72 h of cell culture and tested in ELISA for (a) IFN-γ and (b) IL-10. The results are expressed as pg/ml (mean + standard error of the mean). The difference in the level of IL-10 but not of IFN-γ in response to Bhsp65 in Celastrus-fed versus water-fed rats is statistically significant (*p < 0.05). The mean IFN-γ/IL-10 ratio in response to Bhsp65 of Celastrus-treated (8.79) rats was significantly reduced compared to that of the water-fed (20.76) rats.

Figure 3

Antibody response to mycobacterial hsp65 (Bhsp65) of Celastrus-fed Lewis (LEW) rats. LEW rats (n = 4 to 6) were fed either Celastrus (3 g/kg) or water as described in the legend to Figure 1. Blood samples were collected from LEW rats immediately before (preimmune serum; day 0) challenge with M. tuberculosis H37Ra (Mtb; 1 mg/rat) as well as at different time points thereafter (days 10, 18 and 24). These sera were tested separately at different dilutions (1:50 to 1:8,100) by ELISA for total immunoglobulin against Bhsp65. The results are expressed as optical density (O.D.) at 540 nm (mean + standard error of the mean). At one representative concentration of sera (for example, 1:100 dilution), the level of antibody response to Bhsp65 in Celastrus-fed rats was significantly higher than that of water-fed rats on days 18 and 24 (**p < 0.01 each).

Figure 4

Levels of nitric oxide (NO) in lymph node cell (LNC) culture supernatant and serum of Celastrus-treated Lewis (LEW) rats. (a) LNC culture supernatant and (b) sera were obtained from Celastrus-fed and Water-fed rats as described in Materials and methods. The level of NO in these samples was determined by a colorimetric assay. The results are presented as μM (mean + standard error of the mean). The level of NO secreted into the culture supernate following mycobacterial hsp65 (Bhsp65) restimulation of LNCs of Celastrus-fed rats was significantly (**p < 0.005, *p < 0.05) lower than that of water-fed rats on days 16 and 24 following M. tuberculosis H37Ra (Mtb) immunization (a). The levels of NO in sera of Celastrus-fed rats was significantly (**p < 0.01, *p < 0.05) decreased at days 16 and 24 compared to those of water-fed rats (b). However, the levels of NO in sera of both these Mtb-immunized groups of rats were higher (++p < 0.01, +p < 0.05) compared to those of naïve sera. In each section, some of the error bars are too small to be detected.

Figure 5

Celastrus induces therapeutic down-modulation of adjuvant arthritis (AA) that is comparable to that when using methotrexate (MTX). A cohort of Lewis (LEW) rats was immunized subcutaneously with M. tuberculosis H37Ra (Mtb; 1 mg/rat) at the base of the tail and then split into different groups (n = 4 per group). Beginning day 9 thereafter, coinciding with the onset of clinical signs of arthritis in the hind paws, these rats were fed daily by gavage either Celastrus (experimental group) or water (negative control group). (a) Experimental rats were fed with 3 g/kg of Celastrus, or (b) with either 3 or 1.5 g/kg of Celastrus. An additional group of experimental rats shown in (b) received MTX (0.5 mg/kg; positive control). All these rats were observed and scored regularly for the severity of AA. In both (a) and (b) the difference in the mean arthritic score of each of the Celastrus-fed versus water-fed group of rats was significant (*p < 0.05 by Wilcoxon rank sum test). Similarly, in (b), a significant (*p < 0.05) difference in arthritic scores was observed between MTX-fed and water-fed rats, whereas comparable (p > 0.05) arthritic scores were observed for Celastrus-fed versus MTX-fed rats.