Suppression of ongoing experimental arthritis by a chinese herbal formula (huo-luo-xiao-ling dan) involves changes in antigen-induced immunological and biochemical mediators of inflammation

Abstract

Rheumatoid arthritis (RA) is one of the major autoimmune diseases of global prevalence. The use of the anti-inflammatory drugs for the treatment of RA is associated with severe adverse reactions and toxicity. This limitation has necessitated the search for novel therapeutic products. We report here a traditional Chinese medicine-based herbal formula, Huo luo xiao ling dan (HLXL), which has potent antiarthritic activity as validated in the rat adjuvant-induced arthritis (AA) model. HLXL (2.3 g/Kg) was fed to Lewis (RT.1(1)) rats daily by gavage beginning at the onset of arthritis and then continued through the observation period. HLXL inhibited the severity of ongoing AA. This suppression of arthritis was associated with significant alterations in the T cell proliferative and cytokine responses as well as the antibody response against the disease-related antigen, mycobacterial heat-shock protein 65 (Bhsp65). There was a reduction in the level of the proinflammatory cytokines IL-17 and IL-1β but enhancement of the anti-inflammatory cytokine IL-10 level. In addition, there was inhibition of both the anti-Bhsp65 antibody response and the serum level of nitric oxide. Thus, HLXL is a promising CAM modality for further testing in RA patients.

Figure 1

HLXL feeding reduced the progression and severity of ongoing AA in the LEW rat. Arthritic LEW rats were fed by gavage daily with HLXL (2.3 g/kg body weight, experimental group), water (negative control group), or MTX (0.5 mg/kg, positive control group), from day 12 to day 23 after Mtb immunization. Rats were observed regularly throughout the course of AA. Arthritic scores (mean ± SEM) (a) of a representative experiment (n = 4 per group) are shown. The days of significant difference in the arthritic scores of HLXL/MTX group versus the control are marked (*, P < .05; **, P < .01). Also shown are representative hind paw histopathology sections of water-fed control (b) and HLXL-fed (c) rats on day 18.

Figure 2

Alteration of the T cell proliferative and cytokine responses of HLXL-treated arthritic rats. LNCs of arthritic rats harvested on d 7 after initiation of the daily feeding of HLXL or water were tested for their T cell proliferative (a) and cytokine response (b)–(d) to antigenic restimulation with Bhsp65 in vitro. Ova served as the control antigen. The results of the proliferation assay were expressed as mean stimulation index (SI) ± SEM. The cpm value for PPD was 9,240 (the control group) and 5,577 (HLXL group). For cytokines, the level of mRNA for IL-17 (b), IFN-γ (c), and IL-10 (d) was measured by qRT-PCR, normalized to HPRT expression, and compared with that of cells in medium. The results of a representative experiment (n = 3 per group) are shown. The data are expressed as “fold relative to cells in the medium”. (*, P < .05; **, P < .01).

Figure 3

HLXL inhibited the antibody response to Bhsp65 of LEW rats with AA. Blood samples were collected from experimental (HLXL-fed) and control (water-fed) groups of rats (n = 3 per group) at the indicated time points, and then the sera were tested at a dilution of 1 : 100 each by ELISA for total Ig (a) and IgG2a (b). The results were expressed as optical density (O.D.) at 450 nm (mean ± SEM). (*P < .05).

Figure 4

Treatment with HLXL suppressed IL-1β production by the synovium-infiltrating cells of arthritic rats. The levels of IL-1β (a) and TNF-α (b) in SIC harvested from the water-fed and HLXL-treated rats (n = 3 per group) on d 19 after Mtb injection were determined by ELISA. The results were expressed as pg/mg. (*P < .05).

Figure 5

The influence of HLXL on the level of serum NO in arthritic LEW rats. Sera were obtained from HLXL-fed and water-fed rats (n = 3 per group) on the indicated days as described under Materials and Methods. The level of NO in these samples was determined by a colorimetric assay. The results were presented as mM (mean ± SEM). (*P < .05).

Figure 6

A schematic representation of the diverse mechanisms involved in HLXL-induced immunomodulation of adjuvant arthritis. HLXL suppressed arthritis via the induction of disease-regulating cytokines coupled with the inhibition of pathogenic events that facilitated the initiation and propagation of arthritis. (*IFN-γ has a dual role in autoimmunity.)