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Asarinin is one of the main active chemical components isolated from Xixin, a Chinese medicine. To investigate the role of asarinin in rheumatoid arthritis (RA), the present study investigated the effect of an asarinin-medicated serum on human fibroblast-like synoviocytes in vitro. An asarinin-medicated serum was generated and analyzed by high-performance liquid chromatography. Fibroblast-like synoviocytes were isolated from patients with osteoarthritis and RA. The third generation of the rheumatoid synoviocytes was used in the experimental research and the third generation of osteoarthritic synoviocytes was used as control cells. Trypan blue staining was performed to detect the viability of RA synovial fibroblasts (RASFs). ELISA, reverse transcription-quantitative (RT-q) PCR and western blotting were also performed to detect the expression of various cytokines. Additionally, RT-qPCR was employed to detect Toll-like receptor (TLR) 2 and TLR4. The results revealed that medicated asarinin serum inhibited the viability of RASFs in a dose- and time-dependent manner. The serum also suppressed the expression of interleukin (IL)-17A, tumor necrosis factor-α, interferon-γ, IL-6, TLR2 and TLR4. The inhibitory effect of asarinin drug serum on RASFs may be achieved by inhibition of T helper cell (Th)1/Th17 cytokines through suppression of TLR2 and TLR4. [ABSTRACT FROM AUTHOR]