Paeoniflorin-6'-O-benzene sulfonate alleviates collagen-induced arthritis in mice by downregulating BAFF-TRAF2-NF-κB signaling: comparison with biological agents

Abstract

Paeoniflorin-6'-O-benzene sulfonate (CP-25) is a new ester derivative of paeoniflorin with improved lipid solubility and oral bioavailability, as well as better anti-inflammatory activity than its parent compound. In this study we explored whether CP-25 exerted therapeutic effects in collagen-induced arthritis (CIA) mice through regulating B-cell activating factor (BAFF)-BAFF receptors-mediated signaling pathways. CIA mice were given CP-25 or injected with biological agents rituximab or etanercept for 40 days. In CIA mice, we found that T cells and B cells exhibited abnormal proliferation; the percentages of CD19⁺ total B cells, CD19⁺CD27⁺-activated B cells, CD19⁺BAFFR⁺ and CD19⁺TACI⁺ cells were significantly increased in PBMCs and spleen lymphocytes. CP-25 suppressed the indicators of arthritis, alleviated histopathology, accompanied by reduced BAFF and BAFF receptors expressions, inhibited serum immunoglobulin levels, decreased the B-cell subsets percentages, and prevented the expressions of key molecules in NF-κB signaling. Furthermore, we showed that treatment with CP-25 reduced CD19⁺TRAF2⁺ cell expressions stimulated by BAFF and decreased TRAF2 overexpression in HEK293 cells in vitro. Thus, CP-25 restored the abnormal T cells proliferation and B-cell percentages to the normal levels, and normalized the elevated levels of IgA, IgG2a and key proteins in NF-κB signaling. In comparison, rituximab and etanercept displayed stronger anti-inflammatory activities than CP-25; they suppressed the elevated inflammatory indexes to below the normal levels in CIA mice. In summary, our results provide evidence that CP-25 alleviates CIA and regulates the functions of B cells through BAFF-TRAF2-NF-κB signaling. CP-25 would be a soft immunomodulatory drug with anti-inflammatory effect.

Keywords: B cell; BAFF; TRAF2; collagen-induced arthritis; etanercept; paeoniflorin-6′-O-benzene sulfonate (CP-25); rituximab.

Fig. 1

The chemical structures of CP-25

Fig. 2

The AI, SJC and weights were observed in CIA mice and the effect of CP-25. a The arthritis index (AI) of individual CIA mice from day 29 to day 69 after immunization. b The swollen joint counts (SJC) of individual CIA mice from day 29 to day 69 after immunization. c The AI of CIA mice were assessed and the effects of CP-25. d The SJC of CIA mice were observed and the effects of CP-25. e The weights of CIA mice were observed and the effects of CP-25. **P < 0.01 vs. Normal, ^#P < 0.05, ^##P < 0.01 vs. CIA. ^$P < 0.05 vs. CP-25

Fig. 3

Histopathological evaluation of spleen and joint in CIA mice and the effect of CP-25. a In normal mice, → shows red pulp, ↓ shows white pulp. In CIA mice, → shows germinal center, ↓ shows lymphoid follicular hyperplasia, ↙ periarteriolar lymphoid sheaths, ↘ shows red medullary congestion. b The histological appearances of spleen were assessed. ^#P < 0.05 vs. CIA (n = 6). c In normal mice, ↓ shows synovial membrane. In CIA mice, ↑ shows synovium hyperplasia, ↓ shows inflammatory cell infiltration, ← shows vascular pannus, ↖ shows cartilage damage. d The histological appearances of joints were assessed. ^#P < 0.05 vs. CIA (n = 6)

Fig. 4

The effects of CP-25 on thymus/spleen indices and T/B cells proliferations in CIA mice. a Effects of CP-25 on indices of thymus and spleen in CIA mice. **P < 0.01 vs. Normal. ^#P < 0.05 vs. CIA. ^$P < 0.05 vs. CP-25. b Effects of CP-25 on proliferations of T cell and B cell in CIA mice. **P < 0.01 vs. Normal. ^##P < 0.01 vs. CIA. ^$$P < 0.01 vs. CP-25

Fig. 5

The effects of CP-25 on B cells and BAFF receptors in PBMCs of CIA mice by flow cytometry. a CD19⁺ total B-cell percentages in PBMCs were analyzed and the effect of CP-25. b The effect of CP-25 on CD19⁺CD27⁺-activated B-cell percentages in PBMCs. c CD27⁺CD19^–CD138⁺plasma cells percentages in PBMCs were analyzed and the effect of CP-25. d The effect of CP-25 on CD19⁺BAFFR⁺ cell expressions in PBMCs. e The effect of CP-25 on CD19⁺TACI⁺ cell expressions in PBMCs. *P < 0.05, **P < 0.01 vs. Normal. ^#P < 0.05, ^##P < 0.01 vs. CIA. ^$P < 0.05 vs. CP-25

Fig. 6

The effects of CP-25 on B cells and BAFF receptors in spleen lymphocytes of CIA mice by flow cytometry. a The percentages of B-cell subsets were observed. b The percentage of CD19⁺ total B cells, CD27⁺CD19^–CD138⁺plasma cells in spleen lymphocytes of CIA mice were analyzed and the effect of CP-25. c CD19⁺CD27⁺-activated B-cell percentages in spleen lymphocytes of CIA mice were analyzed and the effect of CP-25. d The expressions of BAFF receptors were observed. e The percentages of CD19⁺BAFFR⁺ cell and CD19⁺TACI⁺ cell in spleen lymphocytes of CIA mice and the effect of CP-25. *P < 0.05, **P < 0.01 vs. Normal

Fig. 7

The effect of CP-25 on BAFF levels, immunoglobulin expressions. a The effect of CP-25 on BAFF levels in CIA mice. b The effect of CP-25 on IgE and IgG3 levels in CIA mice. c The IgG1 levels in CIA mice and the effect of CP-25. d The levels of serum IgA, IgG2a, and IgG2b in CIA mice and the effect of CP-25. e The levels of serum IgD and IgM in CIA mice and the effect of CP-25. *P < 0.05, **P < 0.01 vs. Normal. ^#P < 0.05, ^##P < 0.01 vs. CIA. ^$P < 0.05, ^$$P < 0.01 vs. CP-25

Fig. 8

The effect of CP-25 on the expressions of CD19⁺TRAF2⁺ cells of mice. a The effect of CP-25 on the expressions of CD19⁺TRAF2⁺ cells of mice by flow cytometry. b The percentages of CD19⁺TRAF2⁺ cells were observed and the effect of CP-25. The expressions of CD19⁺TRAF2⁺ cells were increased after BAFF induction CP-25, rituximab, and etanercept reduced the expressions of CD19⁺TRAF2⁺ cells stimulated by BAFF. **P < 0.01 vs. Control. ^##P < 0.01 vs. BAFF. ^$P < 0.05 vs. CP-25

Fig. 9

The overexpression of TRAF2 in HEK 293 cell and the effect of CP-25. a The figure showed transfection of PIRES2-EGFP-TRAF2 plasmid and the effect of CP-25. b The overexpression of TRAF2 was analyzed by Western blotting. c The effect of CP-25 on the expressions of TRAF2. **P < 0.01 vs. Control. ^##P < 0.01 vs. PIRES2-EGFP. ^&P < 0.05 vs. PIRES2-EGFP-TRAF2

Fig. 10

The effect of CP-25 on the NF-κB signaling molecules expressions. a The expression of TRAF2, MKK3, MKK6, p-P38, and p-NF-κB65 in B cells of mice were analyzed by Western blotting. b The effect of CP-25 on the expressions of key molecules in NF-κB signaling. **P < 0.01 vs. Normal. ^#P < 0.05, ^##P < 0.01 vs. CIA. ^$P < 0.05, ^$$P < 0.01 vs. CP-25

Fig. 11

To summary the effect of CP-25, rituximab and etanercept on BAFF/BAFF receptor-TRAF2-NF-κB signaling in B cell on CIA mice. The whole criteria of CIA mice were increased, the function of B cells was abnormal, and NF-κB signaling pathways mediated by BAFF in B cells of CIA mice were activated. CP-25 reduced the whole criteria, T/B cells proliferations, and downregulated abnormal B-cell function and the key molecules of BAFF-NF-κB signaling pathways. The action intensity of rituximab and etanercept on B cells was strong, which might be one of mechanisms of adverse reaction. AI arthritis index, BAFF B-cell activating factor, BAFFR B-cell activating factor receptor, CIA collagen-induced arthritis, CREB cAMP response element binding protein, MKK3 mitogen-activated protein kinase kinase 3, MKK6 mitogen-activated protein kinase kinase 6, p-P38 phosphorylated p38 mitogen-activated protein kinase, NF-κB nuclear factor of kappa B, IκB inhibit of nuclear factor of kappa B, SJC swollen joint counts, TACI transmembrane activator and calcium modulator and cyclophilin ligand interactor, TRAF2 tumor necrosis factor receptor-associated factors 2