Modified huo-luo-xiao-ling dan suppresses adjuvant arthritis by inhibiting chemokines and matrix-degrading enzymes

Abstract

Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting the joints that can lead to deformities and disability. The prolonged use of conventionally used drugs is associated with severe adverse reactions. Therefore, safer and less expensive therapeutic products are continually being sought. Huo-Luo-Xiao-Ling dan (HLXL), a traditional Chinese herbal mixture, and its modified versions possess anti-arthritic activity. In this paper, we examined the influence of modified HLXL on two of the key mediators of arthritic inflammation and tissue damage, namely, chemokines and matrix-metalloproteinases (MMPs) in the rat adjuvant-induced arthritis (AA) model of RA. We treated arthritic Lewis rats with HLXL (2.3 g/kg) by daily gavage beginning at the onset of AA. The control rats received the vehicle. At the peak phase of AA, rats were sacrificed and their draining lymph node cells (LNC) and spleen adherent cells (SAC) were tested. The HLXL-treated rats showed a significant reduction in the levels of chemokines (RANTES, MCP-1, MIP-1α, and GRO/KC), MMPs (MMP 2 and 9), as well as cytokines (IL-6 and IL-17) that induce them, compared to the control vehicle-treated rats. Thus, HLXL controls arthritis in part by suppressing the mediators of immune pathology, and it might offer a promising alternative/adjunct treatment for RA.

Figure 1

Suppression of chemokines in HLXL-treated arthritic rats. Lymph node cells (LNC) harvested at the peak phase of the disease (d 18) from arthritic rats fed with HLXL or the vehicle (water) were cultured in vitro for 24 h in the presence or absence of sonicated Mtb. Thereafter, the culture supernatants were harvested and tested for chemokines: RANTES (a), MCP-1 (b), MIP-1α (c), and GRO/KC (d). The results were expressed as Δpg/mL. Representative results of one of two independent experiments are shown (***P < 0.005) (Mtb = heat-killed M. tuberculosis H37Ra).

Figure 2

The effect of HLXL on MMP activity in arthritic rats. Spleen adherent cells (SACs) harvested from rats fed with HLXL or the vehicle (water) were stimulated for 24 h with sonicated Mtb. The supernatants obtained from these cultured cells were analyzed for MMP-9 (a) and MMP-2 (b) activity using a zymogram assay. The results are representative of two independent experiments (Mtb = heat-killed M. tuberculosis H37Ra).

Figure 3

Inhibition of proinflammatory cytokines in HLXL-treated arthritic rats. Lymph node cells (LNCs) harvested on d 18 (peak phase of arthritis) from HLXL-fed or water-fed rats were cultured for 24 h with or without sonicated Mtb. The culture supernatants were tested for IL-6 (a) and IL-17 (b) using a multiplex assay. The results were expressed as Δpg/mL. The results are representative of two independent experiments. (Mtb = heat-killed M. tuberculosis H37Ra).

Figure 4

A schematic representation of the diverse mechanisms involved in HLXL-induced suppression of AA. HLXL attenuated arthritis via suppressing the disease-regulating chemokines (regulated upon activation, normal T cell expressed and secreted: RANTES; monocyte chemotactic protein-1: MCP-1; macrophage inflammatory protein-1α: MIP-1α; growth regulated oncogene-keratinocyte chemoattractant: GRO/KC), matrix metalloproteinases (MMP-2 and MMP-9), and cytokines (IL-6 and IL-17). (S; spleen, L; lymph node, IJ; inflamed joint, Mtb; heat-killed Mycobacterium tuberculosis H37Ra).