Acetaminophen changes the RNA m6A levels and m6A-related proteins expression in IL-1β-treated chondrocyte cells.

Background: Acetaminophen is commonly recommended for the early analgesia of osteoarthritis. However, the molecular mechanism by which it acts remains unknown. The aim of this study is to investigate the effect of acetaminophen on inflammation and extracellular matrix degradation in human chondrocytes, and the possible molecular mechanisms involved in its effect.

Methods: The normal chondrocyte cell line C28/I2 was treated with interleukin-1β to mimic the inflammatory state. Acetaminophen and the methylation inhibitor (cycloleucine) were used to treat interleukin-1β-induced C28/I2 cells. The expression of RNA N⁶-methyladenosine -related proteins was detected by RT-qPCR and western blot. The total RNA N⁶-methyladenosine level was measured by dot blot analysis and enzyme linked immunosorbent assay. The levels of interleukin-6, interleukin-8 and anti-tumor necrosis factor-α were measured by enzyme linked immunosorbent assay. The extracellular matrix synthesis and degradation were examined by western blot.

Results: After interleukin-1β stimulated C28/I2 cells, the intracellular RNA N⁶-methyladenosine level increased, and the expression of regulatory proteins also changed, mainly including the increased expression of methyltransferase like 3 and the downregulated expression of AlkB family member 5. The use of cycloleucine inhibited interleukin-1β-induced inflammation and extracellular matrix degradation by inhibiting RNA N⁶-methyladenosine modification. In contrast, acetaminophen treatment counteracted interleukin-1β-induced changes in RNA N⁶-methyladenosine levels and regulatory protein expression. Furthermore, acetaminophen treatment of interleukin-1β-induced C28/I2 cells inhibited the secretion of interleukin-6, interleukin-8 and anti-tumor necrosis factor-α, down-regulated the expression of matrix metalloproteinase-13 and Collagen X, and up-regulated the expression of collagen II and aggrecan. In addition, AlkB family member 5 overexpression activated interleukin-1β-induced chondrocyte viability and suppressed inflammation and extracellular matrix degradation.

Conclusion: Acetaminophen affects inflammatory factors secretion and extracellular matrix synthesis of human chondrocytes by regulating RNA N⁶-methyladenosine level and N⁶-methyladenosine-related protein expression. Stimulation of the normal chondrocyte cell line C28/I2 with the cytokine IL-1β (10 μM) mimics the inflammatory state in vitro. Acetaminophen (Ace, 50 μg/mL) changes the m⁶A related proteins expression and the total RNA m⁶A levels in IL-1β-treated chondrocyte cells. Furthermore, regulation of RNA m⁶A levels (by methylation inhibitor Cyc and/or Ace) affects IL-1β-induced inflammatory cytokines secretion and extracellular matrix synthesis in C28/I2 cells.