Chinese herbal formula, modified Xianfang Huoming Yin, alleviates the inflammatory proliferation of rat synoviocytes induced by IL-1β through regulating the migration and differentiation of T lymphocytes.

Abstract

Xianfang Huoming Yin (XFH) is a traditional Chinese herbal formula, which has the effect of clearing heat and detoxifying toxins, dispersing swellings, activating blood circulation, and relieving pain. It is usually applied to treat various autoimmune diseases, including Rheumatoid arthritis (RA). The migration of T lymphocytes plays an indispensable role in the pathogenesis of RA. Our previous studies demonstrated that modified Xianfang Huoming Yin (XFHM) could modulate the differentiation of T, B, and NK cells, and contribute to the restoration of immunologic balance. It also could downregulate the production of pro-inflammatory cytokines by regulating the activation of NF-κ B and JAK/STAT signaling pathways in the collagen-induced arthritis mouse model. In this study, we want to investigate whether XFHM has therapeutic effects on the inflammatory proliferation of rat fibroblast-like synovial cells (FLSs) by interfering with the migration of T lymphocytes in vitro experiments. High performance liquid chromatography-electrospray ionization/mass spectrometer system was used to identify the constituents of the XFHM formula. A co-culture system of rat fibroblast-like synovial cells (RSC-364cells) and peripheral blood lymphocytes stimulated by interleukin-1 beta (IL-1β) was used as the cell model. IL-1β inhibitor (IL-1βRA) was used as a positive control medicine, and two concentrations (100μg/mL and 250μg/mL) of freeze-dried XFHM powder were used as intervention measure. The lymphocyte migration levels were analyzed by the Real-time xCELLigence analysis system after 24h and 48h of treatment. The percentage of CD3+CD4+ T cells and CD3+CD8+ T cells, and the apoptosis rate of FLSs were detected by flow cytometry. The morphology of RSC-364cells was observed by hematoxylin-eosin staining. The protein expression of key factors for T cell differentiation and NF-κ B signaling pathway-related proteins in RSC-364cells were examined by western-blot analysis. The migration-related cytokines levels of P-selectin, VCAM-1, and ICAM-1 in the supernatant were measured by enzyme-linked immunosorbent assay. Twenty-one different components in XFHM were identified. The migration CI index of T cells was significantly decreased in treatment with XFHM. XFHM also could significantly downregulate the levels r of CD3+CD4+T cells and CD3+CD8+T cells that migrated to the FLSs layer. Further study found that XFHM suppresses the production of P-selectin, VCAM-1, and ICAM-1. Meanwhile, it downregulated the protein levels of T-bet, ROR γ t, IKKα/β, TRAF2, and NF-κ B p50, upregulated the expression of GATA-3 and alleviated synovial cells inflammation proliferation, contributing to the FLSs apoptosis. XFHM could attenuate the inflammation of synovium by inhibiting T lymphocyte cell migration, regulating differentiation of T cells through modulating the activation of the NF-κ B signaling pathway.