Guizhi-Shaoyao-Zhimu decoction attenuates bone erosion in rats that have collagen-induced arthritis via modulating NF-κB signalling to suppress osteoclastogenesis

Abstract

Context: Guizhi-Shaoyao-Zhimu decoction (GSZD) is commonly used to treat rheumatoid arthritis (RA), but its mechanism is unclear.

Objective: To investigate the effect of GSZD on bone erosion in type II collagen (CII)-induced arthritis (CIA) in rats and to identify the underlying mechanism.

Materials and methods: The CIA model was prepared in male Wistar rats by two subcutaneous injections of CII, 1 mg/mL. Fifty CIA rats were randomized equally into the control group given saline daily, the positive group given saline daily and methotrexate 0.75 mg/kg once a week, and three GSZD-treated groups gavaged daily with 800, 1600 and 3200 mg/kg of GSZD for 21 days. GSZD effects were assessed by paw volume, arthritic severity index and histopathology. Cytokine levels were determined by ELISA. The effects of GSZD on RAW264.7 cells were evaluated by receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption assay. Expression of IκB-α and p65 was measured by Western blotting. Major components of GSZD were identified by HPLC.

Results: Arthritis index score, paw volume and bone destruction score showed that GSZD improved inflammatory symptoms and reduced joint tissue erosion (p < 0.01). GSZD decreased RANKL, and the number of osteoclasts (OCs) in joint tissues (p < 0.01) and increased osteoprotegerin levels (p < 0.01). GSZD inhibited RANKL-induced RAW264.7 differentiation and reduced bone resorption by OCs. GSZD upregulated IκB (p < 0.01) and p65 (p < 0.01) in the cytoplasm and downregulated p65 (p < 0.01) in the cell nucleus.

Conclusions: Guizhi-Shaoyao-Zhimu decoction has an anti-RA effect, suggesting its possible use as a supplement and alternative drug therapy for RA.

Keywords: RANKL; Rheumatoid arthritis; osteoclast; osteoprotegerin.

Figure 1.

Anti-arthritic activities of GSZD. (A) Overview of methods. (B) Representative images of the joints of CIA rats in different groups. (C) GSZD impact on the changes in paw volume (left) and arthritis score (right) in CIA rats.

Figure 2.

GSZD impact on ankle joint histopathology in CIA model rats visualized by the H&E staining.

Figure 3.

GSZD impact on ankle joint histopathology in CIA model rats visualized by the safranin O-fast green staining.

Figure 4.

Results of the micro-CT imaging of ankle joints (A) and knee joints (B) of CIA rats. Data are the mean ± SD (n = 6), **p < 0.01 vs. control.

Figure 5.

Effects of GSZD on the changes in osteoclasts of the ankle joint of CIA rats detected by TRAP staining (arrows indicate osteoclasts).

Figure 6.

Effects of GSZD on serum levels of pro-inflammatory cytokines. Data are presented as the mean ± SD (n = 10), **p < 0.01 vs. control rats.

Figure 7.

Effects of GSZD on the expression of RANKL (A) and OPG (B) in serum and synovial tissues. Data are the mean ± SD (n = 10), **p < 0.01 vs. control.

Figure 8.

Cytotoxic effect of GSZD on RAW264.7 cells determined by the CCK-8 assay.

Figure 9.

Effects of GSZD on TRAP staining (A) and bone resorption assay (B). Data are presented as the mean ± SD (n = 3), *p < 0.05, **p < 0.01 vs. control.

Figure 10.

Effects of GSZD on the activation of the NF-κB signalling pathway in RANKL-induced OCs. Data are presented as mean ± SD (n = 3), **p < 0.01 vs. control.

Figure 11.

HPLC analysis of the composition of GSZD. Numbers 1–6 represent gallic acid, mangiferin, paeoniflorin, prim-O-glucosylcimifugin, cinnamic acid and glycyrrhizic acid, respectively.

Figure 12.

The mechanism of attenuation of RANKL-induced bone resorption by GSZD. GSZD suppresses the activation of NF-κB signalling during the differentiation of OCs.