Background Our previous study showed synergistic responses in TNF-alpha (TNF-α) and interleukin-6 with the combination of methotrexate (MTX) and aconite. Modulation of those cytokines has not been applied to rheumatoid arthritis (RA)-mimicked in vivo models.
Objectives To translate in vitro effects of MTX, aconite, and MTX/aconite combination towards anti-arthritic responses in vivo, we investigated arthritis index (AI), histopathologic changes, and levels of TNF-α, immunoglobulin G (IgG) 2a and 2b in a collagen-induced arthritis (CIA) setting.
Methods CIA was induced in five male DBA/OlaHsd mouse per group by intradermal injection of bovine collagen type II and Complete Freund's Adjuvant. In Day21, a bovine collagen type II and Incomplete Freund's Adjuvant were given for booster infection. The mice of arthritis onset were treated daily throughout Day49 with per oral administration of pre-investigated ratios of three to one; MTX (3 mg/kg), aconite (Aconibal®, 1 mg/kg), and MTX/aconite (3 and 1 mg/kg) combination. The AIs were evaluated every week. Histological changes, levels of TNF-α as well as IgG2a and IgG2b in blood using ELISA kit were evaluated at finals. Repeat measure and one-way ANOVA were analysed using SPSS (ver. 18.0 KO for Windows; SPSS Inc., Chicago, IL, USA) to evaluate inter-period and inter-group differences with Tukey's post-hoc tests.
Results The CIA phenotypes adequately presented through three groups' AI reductions (CIA vs. MTX, aconite, or MTX/aconite; p<0.001, for three). There were differences of AI scores in aconite group from MTX one in week 4, 5, and 6 (MTX vs. aconite; p=0.038, p=0.001, p=0.042, respectively). Synergistic responses of AI were not shown any of three groups. The recoveries of synovial tissues were observed in MTX and MTX/aconite groups. The levels of TNF-α were not changed (aconite vs. MTX/aconite; p=0.200 and MTX vs. MTX/aconite; p=0.700). MTX group showed IgG2a reduction (CIA vs. MTX; p<0.001). Interestingly, MTX/aconite combination and aconite group slightly downregulated IgG2b levels as 80.8±5.6% and 90.5±7.4%, respectively (CIA vs. MTX/aconite; p=0.001 and CIA vs. aconite; p=0.010).
Conclusions Synergistic in vitro effects of MTX and aconite combination brought the partial in vivo phenotypic responses: Aconite showed more AI changes than MTX did in 4, 5, and 6 weeks. However, we found the presence of partial FcγRIIB affinity of binding modulation that MTX/aconite could enhance preventing monocyte/macrophage activation via immune complex in RA pathogenesis, as was other benefits of the combination except direct synergies.
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Acknowledgements This study was supported by KIOM (Grant # K17252). The commercial product was donated by the virtue of HanPoong Pharmaceutical Company.
Disclosure of Interest None declared