Combination therapy of ginsenoside compound K and methotrexate was efficient in elimination of anaemia and reduction of disease activity in adjuvant-induced arthritis rats

Abstract

Context: Ginsenoside compound K (CK) has anti-inflammatory, immunoregulatory, and myelosuppressive protective effects. Methotrexate (MTX) is widely used in combination therapy for rheumatoid arthritis (RA).

Objective: To evaluate the effects of combination therapy of CK and MTX on anaemia and anti-arthritis in adjuvant-induced arthritis (AA) rats.

Materials and methods: AA was induced in rats by Complete Freund's adjuvant, and divided into five groups (n = 10): normal, AA, CK 80 mg/kg, combination therapy (80 mg/kg CK combined with 0.5 mg/kg MTX), and MTX 0.5 mg/kg. From day 12, CK (once a day for 15 days) or MTX (once every 3 days, five times) were intragastrically administered.

Results: Combination therapy showed increased haemoglobin to 148.5 ± 10.1 g/L compared with AA (129.8 ± 11.7 g/L) and MTX (128.8 ± 18.4 g/L), and decreased reticulocytes in peripheral blood to 4.9 ± 1.1% compared with MTX (9.3 ± 3.3%). In combination therapy group, paw swelling decreased to 5.6 ± 4.3 mL compared with CK (9.4 ± 3.9 mL) and MTX (13.5 ± 7.4 mL), and swollen joint count decreased to 1.4 ± 0.8 compared with CK (2.1 ± 1.0) and MTX (2.4 ± 1.2) at day 24. Combination therapy showed decreased IL-6 to 25.1 ± 17.2 pg/mL compared with MTX (44.9 ± 4.8 pg/mL), and decreased IL-17 to 5.8 ± 3.9 pg/mL compared with MTX (10.7 ± 4.2 pg/mL).

Conclusion: The anti-anaemia effect of CK deserves further study, and CK can be a candidate effective drug for combined treatment in RA with anaemia.

Keywords: Rheumatoid arthritis; anaemia of inflammation; natural products of ginsenosides.

Figure 1.

Combined effects of CK and MTX on arthritis signs in AA rats. (A) global assessment; (B) arthritis index; (C) swollen joint counts; (D) paw swelling; Data are expressed as the mean ± SD, with 10 animals in each group. ^##p < 0.01 vs. normal; *p < 0.05; **p < 0.01 vs. AA; ^▽p < 0.05 vs. MTX; ^●p < 0.05; ^●●p < 0.01 vs. CK.

Figure 2.

Combined effects of CK and MTX on joint erosion and FLS function in AA rats. (A) Photomicrographs of the rat joint (original magnification × 100, haematoxylin and eosin stain). (B) synoviocyte proliferation; (C) inflammatory cellular infiltration; (D) pannus formation; (E) bone erosion. (F) FLS proliferation; (G) the secretion of OPG from FLS; (H) the secretion of RANKL from FLS; (I) the secretion of TNF-α from FLS. Data are expressed as the mean ± SD. ^##p < 0.01 vs. Normal; *p < 0.05; **p < 0.01 vs. AA. Data are expressed as the mean ± SD, with 6 animals in each group.

Figure 3.

Combined effects of CK and MTX on erythrocyte and haemoglobin in AA rats. (A) number of peripheral blood erythrocyte; (B) haemoglobin concentration in peripheral blood; (C) number of reticulocytes in peripheral blood; (D) proportion of reticulocytes in peripheral blood. Data are expressed as the mean ± SD, with 6 animals in each group. ^##p＜0.01 vs. Normal; *p＜0.05; **p＜0.01 vs. AA; ^▽p＜0.05; ^▽▽p＜0.05 vs. MTX.

Figure 4.

Combined effects ofCK and MTX on spleen histopathology and proliferation of T cells and B cells. (A) Photomicrographs of the rat spleen (original magnification × 100, haematoxylin and eosin stain), (B) Lymphoid follicles, (C) Marginal zone, (D) the periarteriolar lymphoid sheaths, (E) T cell proliferation, (F) B cell proliferation. Data are expressed as the mean ± SD, with 6 animals in each group, ^##p < 0.01 vs. Normal; *p < 0.05; **p < 0.01 vs. AA.

Figure 5.

Combined effects of CK and MTX on pro-inflammatory cytokine in AA rats. pro-inflammatory cytokine in serum (A) IL-1β, (B) IL-2, (C) IL-6, (D) IL-17, (E) TNF-α, (F) IFN-γ, (G) PGE-2; (H) the secretion of PGE2 from macrophage; (I) the secretion of IL-17 from macrophage. Data are expressed as the mean ± SD, with 6 animals in each group. ^#p＜0.05; ^##p＜0.01 vs. Normal; *p＜0.05; **p＜0.01 vs. AA; ^▽p＜0.05 vs. MTX.